We performed a systematic review and meta-analysis by means of an electronic search of the published literature, with no language restrictions, until October 2008. We included studies on chronically infected patients of any age who were in the indeterminate phase or had visceral involvement and for whom treatment with benznidazole was compared with placebo or no treatment. The primary endpoint was response to therapy (whether serological, parasitological or clinical), as it was measured in each of the studies included. Clinical response to therapy was also analysed.

We identified 696 studies, from which we chose 9: 3 clinical trials and 6 observational studies. Compared with placebo or no treatment, benznidazole increases 18-fold the probability of a response to therapy [global odds ratio (OR), 18.8; 95% confidence interval (CI), 5.2–68.3]. This effect was mainly observed in clinical trials (OR, 70.8; 95% CI, 16–314), whereas in observational studies it was much less marked (OR, 7.8; 95% CI, 2.1–28.9), and even less so when only observational studies in adults were considered (OR, 6.3; 95% CI, 1.6–24.7). Patients treated with benznidazole had a significantly lower risk of clinical events (OR, 0.29; 95% CI, 0.16–0.53). Up to 18% of patients discontinued treatment due to toxicity (cutaneous reactions followed by gastrointestinal disturbances); this was less common in children than in adults.

Analysis of available information reveals that the efficacy of treatment in late chronic infection is doubtful. Although data generally point to a beneficial effect, this could be marginal. This uncertainty is largely the result of differences in the study populations, endpoints and follow-up periods, and the fact that almost all of the information on treatment in the late chronic phase comes from non-randomized studies.

Fifty-one staphylococci from Washington State beaches were characterized using antimicrobial susceptibility testing, carriage of acquired tetracycline and/or macrolide resistance genes, staphylococcal cassette chromosome mec (SCCmec) typing, the BBL Crystal^TM Gram-Positive ID System and/or 16S rRNA sequencing, coagulase test and multilocus sequence typing (MLST) for MRSA.

Five multidrug-resistant MRSA SCCmec type I, of which three were MLST type ST45, one ST59 and one a new MLST type, ST1405, plus one susceptible non-typeable (NT) MRSA ST30 were characterized. Thirty-three MRCoNS isolates, representing 21 strains from 9 Staphylococcus spp., carried a range of SCCmec types [I (2), II (6), III (3), V (2), I/II (1) and NT (7)] and varied in their antibiotic susceptibility to other antibiotic classes and carriage of acquired tetracycline/macrolide resistance gene(s). MRSA and MRCoNS donors co-transferred tet(M) and erm(A) genes to an Enterococcus faecalis recipient at a frequency of 10^–8.

This is the first report of MRSA and MRCoNS isolated from marine water and intertidal beach sand. The MLST types and antibiotic carriage of five MRSA isolates were similar to hospital MRSA isolates rather than US community-acquired MRSA isolates. Our results suggest that public marine beaches may be a reservoir for transmission of MRSA to beach visitors as well as an ecosystem for exchange of antibiotic resistance genes among staphylococci and related genera.

MICs of 30 antimicrobial agents were determined by broth microdilution. Resistance and virulence genes were detected via a diagnostic DNA microarray and specific PCRs. The genomic relationships were determined by ApaI-PFGE, spa typing and SCCmec typing.

Twenty-two distinct resistance patterns were observed. All 54 isolates were tetracycline resistant, mediated by tet(M), tet(K) and/or tet(L), with 14 isolates being only resistant to β-lactam antibiotics and tetracyclines. Trimethoprim resistance, seen in 28 isolates, was mostly due to the gene dfrK or dfrG. Among the 24 macrolide/lincosamide-resistant isolates, the genes erm(A), erm(B) and/or erm(C) were detected. The two chloramphenicol/florfenicol-resistant isolates harboured the gene fexA. The eight gentamicin-resistant isolates carried the gene aacA/aphD. Fifty-three isolates harboured SCCmec type V elements while the remaining one carried mecA and ugpQ, but no recombinase genes. All isolates were PVL negative, but one and three isolates, respectively, were positive for the enterotoxin B and enterotoxin K and Q genes. Eight different spa types were identified with t011 being the most predominant. Six ApaI-PFGE clusters with up to nine individual patterns were detected.

MRSA ST398 isolates varied slightly in their virulence properties and spa types but differed distinctly in their antimicrobial resistance pheno- and genotypes as well as their ApaI-PFGE patterns. These data underline the ability of ST398 to acquire genetic material that might increase antimicrobial resistance and virulence.

Erythromycin resistance determinants were screened by PCR. The population structure, including multilocus sequence typing, was analysed by using quantitative clonal diversity (diversity ratio, Simpson, Selander–Levin and Shannon mathematical indexes).

The leading resistance gene was erm(B) (74.3% of the isolates), followed by the erm(B) plus mef(A) combination (17.9%) and mef(A) alone (7.7%). The most frequent serotypes were 14 (18%), 19A (15.4%) and 6B (11.5%). A polyclonal structure was detected in resistant strains, including the Spain^9V-3, Spain^6B-2 and Denmark¹⁴-32 international clones. Both genetic diversity and genetic distribution were high, particularly among clones containing erm(B) and erm(B) plus mef(A) determinants.

The resistance determinants erm(B) and the combination of erm(B) plus mef(A) were observed within multiple S. pneumoniae bacteraemic clones. The preservation of a polyclonal structure might provide a suitable background for further evolution of antibiotic resistance.

A total of 308 CNSPA isolates collected at a medical centre from 2000 to 2005 and 26 XDRPA collected from six medical centres in different regions of Taiwan in 2003 were included. MBL genes and Tn6001 were detected by PCR. Clonal relatedness was determined by PFGE.

Of the 308 CNSPA isolates, 30 (10%) were XDRPA, including 27 (9%) colistin-only-susceptible (COS) and 3 (1%) colistin-only-intermediate (COI) P. aeruginosa. bla_VIM-3 was found in 16 (53%) isolates of the XDRPA (n = 30), whereas only 72 (26%) of the non-XDRPA (n = 278) carried the gene. In450 was higher in COS P. aeruginosa (12/27; 44%) than in non-XDRPA isolates (53/278; 19%). Tn6001 was highest in COS P. aeruginosa (11/27; 41%), followed by COI P. aeruginosa (1/3; 33%), and lowest in non-XDRPA (46/278; 17%). Of 26 XDRPA from six medical centres, higher prevalences of bla_VIM-3 (16/26; 62%), In450 (16/26; 62%) and Tn6001 (12/26; 46%) were found. Genotyping by PFGE revealed 60 pulsotypes. Hybridization of a bla_VIM-3-specific probe following PFGE suggested that the mobile element Tn6001 might have transferred horizontally.

Tn6001 and In450 play an important role in the dissemination of CNSPA and XDRPA. The prevalence of these genetic constituents was higher in XDRPA than in non-XDRPA isolates, suggesting that the mobile element Tn6001 might have transferred horizontally.

Five Salmonella field isolates and mutants, in which efflux pump inhibitor tests previously pointed towards an up-regulation of efflux, plus one negative control were included in the study. MIC values were determined of antibiotics that were indicative of AcrAB overexpression. The regulatory regions acrRA, soxRS, marORAB, acrSE and ramRA of original strains and mutants were sequenced and compared. The gene expression of acrA, tolC, ramA and soxS was assessed by quantitative real-time PCR. Conjugation experiments and tet gene PCRs were performed to explain unexpected variations in MIC values of tetracycline.

In four mutant strains, changes in the ramRA regulatory region, causing up-regulation of ramA, were detected. These changes comprised point mutations and deletions of 10 or 15 bp within the ramR gene and a single bp exchange located in the binding site of the RamR protein in Salmonella Infantis, Paratyphi and Livingstone mutants. An insertion of 49 bp within the soxR gene was involved in soxS up-regulation and enhanced efflux activity in the fifth mutant from Salmonella Virchow. The loss of tetracycline resistance in one Salmonella Paratyphi mutant could be explained by the loss of a plasmid carrying a tet(A) gene.

Changes in the ramR-ramA region as well as in the soxR gene occur in mutants of Salmonella serovars other than Typhimurium and seem to be involved in the up-regulation of efflux activity.

Twenty-seven isolates were analysed by PCR and sequencing to identify the genes responsible for the β-lactamase resistance phenotypes. The transferability of the phenotypes was tested by conjugation to Escherichia coli K12J53, plasmid detection with S1-PFGE, hybridization and PCRs of the transconjugants. The genetic relationship was determined by PFGE.

We found bla_CTX-M-9 and bla_CTX-M-10 in Salmonella Virchow PT19. bla_CTX-M-14 was detected in Salmonella (IV) 44:z₄,z₂₃:-, Salmonella Enteritidis PT6a, Salmonella Typhimurium DT193 and Salmonella Typhimurium DT104B. bla_CTX-M-1 was found in Salmonella Litchfield. bla_CTX-M-15 and bla_CTX-M-32 were found in Salmonella Enteritidis PT1. bla_SHV-12 was found in Salmonella Blockley, Salmonella Hadar PT2, Salmonella Enteritidis PT21, Salmonella Enteritidis PT1 and Salmonella Bredeney. bla_SHV-2 was found in Salmonella Livingstone. bla_CMY-2 was detected in Salmonella Bredeney, Salmonella Newport, Salmonella Enteritidis PT5b and Salmonella Heidelberg. bla_DHA-1 was detected for the first time in Spain in Salmonella Newport. One strain of Salmonella Senftenberg harboured two extended-spectrum β-lactamases, bla_SHV-12 and bla_CTX-M-9. We have found a large variety of β-lactamase families as well as several members of major relevance, such as CTX-M-15, CTX-M-32, CMY-2 and DHA-1. XbaI-PFGE, conjugation assays and S1-PFGE hybridization showed that all these β-lactamases were mediated by plasmids.

This study demonstrates the emergence of a public health risk related to resistance to β-lactams in Salmonella. The resistance trends need to be monitored carefully.

GusA, a well-established reporter gene in other systems, was cloned into the vector pPacVInteg allowing stable expression in G. lamblia under control of the promoter from the glutamate dehydrogenase (gdh) gene. The resulting transgenic strain was compared with the wild-type strain in a vitality assay, characterized with respect to susceptibility to nitazoxanide, metronidazole and—as assessed in a 96-well plate format—to a panel of 15 other compounds to be tested for anti-giardial activity.

GusA was stably expressed in G. lamblia. Using a simple glucuronidase assay protocol, drug efficacy tests yielded results similar to those from cell counting.

G. lamblia WB C6 GusA is a suitable tool for high-throughput anti-giardial drug screening.

The gp120-interacting plant lectin HHA (‘Hippeastrum hybrid agglutinin’), the non-nucleoside reverse transcriptase inhibitor KRV2110 and the fusion inhibitor enfuvirtide (T20) were combined in 12 drug associations by using the Ray combination design method. Their activity against HIV-1_BaL was assessed by the lymphocyte infectivity reduction assay and by the single-cycle BaL pseudovirus (PV) assay. In addition, their cell tolerance was evaluated for HEC-1 and HeLa epithelial cell lines, both originating from genital tissue.

All evaluated combinations showed synergistic activity in both lymphocyte infectivity reduction and single-cycle BaL PV assays. The combination HHA + KRV2110 resulted in the highest cell viability, whereas the combinations including T20 exhibited a dose-dependent decrease in cell viability, demonstrating the differential tolerance of epithelial cell lines to the combinations.

These observations provide a rational basis for in vitro testing of microbicide candidate molecule combinations, including anti-HIV-1 and cytotoxic cellular assays.

Mutants defective in genes encoding oxidative stress defence activities were tested by time–kill curves for their contribution to antibiotic tolerance in comparison with the E. faecalis JH2-2 wild-type (WT).

In killing assays, WT cultures lost 0.2 ± 0.1 and 1.3 ± 0.2 log₁₀ cfu/mL after 24 h of vancomycin or penicillin exposure, respectively. A deletion mutant of the superoxide dismutase gene (sodA) exhibited a lack of tolerance as cultures lost 4.1 ± 0.5 and 4.8 ± 0.7 log₁₀ cfu/mL after 24 h of exposure to the same drugs. Complementation of sodA re-established the tolerant phenotype. Bacterial killing was an oxygen-dependent process and a model is presented implicating the superoxide anion as the mediator of this killing. As predicted from the model, a mutant defective in peroxidase activities excreted hydrogen peroxide at an elevated rate.

SodA is central to the intrinsic ability of E. faecalis to withstand drug-induced killing, and the superoxide anion seems to be the key effector of bacterial death.

Adjuvant activity of plant phenolics was tested using growth inhibition assays in combination with subinhibitory concentrations of novobiocin. The antibacterial susceptibilities of susceptible and MDR A. baumannii to a variety of antibiotics were determined in the absence and presence of ellagic and tannic acids. The effect of the adjuvants on bacterial outer membrane function was examined by using the fluorescence dye 1-N-phenylnaphthylamine (NPN). The efflux pump inhibition was measured by the intracellular accumulation of ethidium bromide (EtBr) and pyronin Y.

At 40 µM, ellagic and tannic acids enhanced the activity of novobiocin, coumermycin, chlorobiocin, rifampicin and fusidic acid against A. baumannii. There were no increases in the uptake of NPN or in the accumulation of EtBr after strains were treated with these adjuvants; however, the intracellular accumulation of pyronin Y by the treated cells was significantly increased, suggesting that ellagic and tannic acids act as efflux pump inhibitors.

Susceptibility of MDR A. baumannii to a variety of antibiotics was enhanced in the presence of ellagic and tannic acids. The use of such plant compounds might provide effective treatments for resistant Gram-negative bacterial infections.

A lytic bacteriophage in combination with ciprofloxacin was used for the treatment of K. pneumoniae B5055 biofilm. The efficacy and the frequency of resistant variant formation were estimated after respective treatments. The resistant variants were characterized for their virulence potential.

Bacteriophage alone was able to eradicate the biofilm effectively and no significant difference was observed in its ability to eradicate biofilm in combination with ciprofloxacin. However, combination treatment using ciprofloxacin and bacteriophage significantly arrested the emergence of resistant variants. The small number of variants that developed had a lower propensity to form biofilms, produced small amounts of cell-associated capsular polysaccharide and demonstrated increased susceptibility to mouse peritoneal macrophages. Altered morphology and changed pattern of the outer membrane proteins of bacterial isolates were also observed.

The combination treatment not only killed the bacteria, but also restricted the formation of resistant variants significantly as compared with individual treatments. Hence, a combination of bacteriophage and ciprofloxacin offers an effective strategy to combat the emergence of treatment-associated resistance.

MICs and minimum bactericidal/fungicidal concentrations were determined for each microorganism grown in suspension and biofilm using microbroth dilution and ATP bioluminescence, respectively. Chequerboard assays were used to determine synergistic, indifferent or antagonistic interactions between CHG and EO or 1,8-cineole.

Antimicrobial activity was demonstrated by CHG, EO and 1,8-cineole; however, CHG was significantly more active against microorganisms in both planktonic and biofilm modes of growth (P < 0.05). Crude EO was significantly more efficacious against microorganisms grown in suspension compared with 1,8-cineole (P < 0.05). Synergistic activity was demonstrated between CHG and both EO and 1,8-cineole against suspensions of Staphylococcus aureus, methicillin-resistant S. aureus (MRSA), Escherichia coli and Candida albicans, and biofilm cultures of MRSA and Pseudomonas aeruginosa.

In conclusion, CHG may be combined with either crude EO or its major component 1,8-cineole for enhanced, synergistic antimicrobial activity against a wide range of microorganisms in planktonic and biofilm modes of growth; however, the superior antimicrobial efficacy associated with crude EO alone, compared with 1,8-cineole, favours its combination with CHG.

MICs of nemonoxacin were determined for 798 recently collected (2005–07) and non-duplicate isolates of Gram-positive cocci by the agar dilution method. These isolates included: methicillin-susceptible Staphylococcus aureus (MSSA; n = 100); methicillin-resistant S. aureus (MRSA), including ciprofloxacin-susceptible (n = 50), ciprofloxacin-resistant (n = 100), vancomycin-intermediate (n = 50) and daptomycin-non-susceptible (DNS-MRSA; n = 5) isolates, and community-acquired MRSA (CA-MRSA; n = 101); invasive Streptococcus pneumoniae isolates (n = 150); levofloxacin-non-susceptible (MICs of 4–64 mg/L) S. pneumoniae isolates (n = 30); and enterococci (n = 212), including vancomycin-resistant enterococci (VRE; n = 112).

Nemonoxacin had potent activity against MSSA (MIC₉₀ of ≤0.03 mg/L), ciprofloxacin-susceptible MRSA (MIC₉₀ of ≤0.03 mg/L) and CA-MRSA (MIC₉₀ of 0.06 mg/L). For all invasive S. pneumoniae isolates, the activity of nemonoxacin (MIC₉₀ of 0.06 mg/L) was similar to that of gemifloxacin and much better than that of levofloxacin (MIC₉₀ of 2 mg/L) and moxifloxacin (MIC₉₀ of 0.25 mg/L). Nemonoxacin had a 32- to 64-fold higher activity than levofloxacin against levofloxacin-non-susceptible isolates. Nemonoxacin exerted limited activity against ciprofloxacin-resistant MRSA (MIC₉₀ of 1 mg/L), vancomycin-intermediate MRSA (MIC₉₀ of 2 mg/L), DNS-MRSA (MIC₉₀ of 1 mg/L), vancomycin-susceptible enterococci (MIC₉₀ of 2 mg/L for Enterococcus faecalis and 4 mg/L for Enterococcus faecium) and VRE (MIC₉₀ of 4 mg/L for E. faecalis and 16 mg/L for E. faecium).

Our findings point to a potentially useful role for nemonoxacin in the treatment of infections caused by MSSA, ciprofloxacin-susceptible MRSA and S. pneumoniae with various resistance phenotypes.

Killing curves (final inoculum: 1.0–5.0 x 10⁷ cfu/mL) were performed with 0.88 mg/L final concentrations (serum C_max after a 100 mg 1 h infusion) in Mueller–Hinton broth supplemented with Ca²⁺ and Mg²⁺ (MH) and in MH with 4 g/dL human albumin. Controls were curves in MH with concentrations similar to the free fraction (fC_max = 0.17 mg/L) calculated using protein binding. Activity was measured as log₁₀ initial inoculum reduction (log₁₀ initial inoculum–log₁₀ at 12 h/24 h). Target strains (tigecycline MIC/MBC; mg/L) were: methicillin-resistant Staphylococcus aureus heteroresistant to vancomycin (0.12/0.25); Enterococcus faecium (0.12/0.25); Escherichia coli producing extended-spectrum β-lactamase (0.12/0.25); and Acinetobacter baumannii (0.25/1).

At 24 h the fC_max produced mean decreases of ≤0.1 cfu/mL for all strains, in contrast to the bactericidal activity (mean >3 log₁₀ reduction) provided by C_max concentrations in the presence or absence of albumin for E. coli and E. faecium, and an activity nearly bactericidal for S. aureus (mean ~2.8 log₁₀ reduction). In the case of the A. baumannii isolate the C_max in the presence or absence of albumin produced a mean reduction of 2.56 log₁₀ cfu/mL at 12 h (time of one dosing interval), with a bacteriostatic profile when considering 24 h colony counts (similar counts at 0 and 24 h).

Correcting the total concentration for the reported literature binding values is unreliable since tigecycline antibacterial activity was greater than that suggested by the free fraction of the drug.

The efficacy of the combination, evaluated by measuring lesion size and parasite burden in the skin and spleen, was assessed in BALB/c mice infected by L. (L.) major. Miltefosine was administered orally at 25 mg/kg/day for 10 days, while 10% paromomycin gel was applied topically twice a day for 10 days.

Treatment of the experimentally infected animals with topical paromomycin + oral miltefosine combination induced a statistically significant reduction in lesion size and parasite burden in the skin, with complete healing of ulcers, as compared with those treated with oral miltefosine or placebo. Furthermore, topical paromomycin + oral miltefosine combination was as effective as topical paromomycin alone to reduce the lesion size and parasite load in lesions. However, the efficacy of the combination was significantly higher than that observed for the other treatments, including topical paromomycin alone, in reducing the parasite burden in spleen.

The combination of topical paromomycin gel and oral miltefosine provides an enhanced efficacy in the treatment of L. (L.) major-infected mice, thus presenting a significantly higher activity than that observed for the monotherapeutic regimens.

BALB/c mice were challenged with alginate-embedded mucoid clinical isolates isolated in 1988, 1997 or 2003. Mice were euthanized on day 1, 2 or 3 post-infection for estimation of quantitative bacteriology, histopathology, and measurement of granulocyte colony-stimulating factor (G-CSF) and macrophage inflammatory protein 2 (MIP-2).

There was a significant reduction of bacteria when comparing treatment initiated 1 h post-infection with treatment initiated after 24 h for isolates 1997 and 2003. Treatment initiated 1 h post-infection also resulted in a reduction of the pulmonary cytokines G-CSF, for all three isolates, and MIP-2, for isolates 1997 and 2003. Histological evaluation showed a shift from the acute-type inflammatory immune response to a chronic-type in mice infected with isolate 2003.

A significant reduction in the number of bacteria was observed when initiating treatment 1 h post-infection compared with initiating treatment after 24 h, although the latest isolate avoided complete clearance. Early antibiotic treatment directed at the mucoid phenotype in mice also reduced the inflammation and, thereby, the lung tissue damage.

Children were recruited in weight-based dosage bands and nutritional status classified according to weight for height. Total and unbound plasma nevirapine concentrations were measured over a full dosing interval. Multivariate linear and logistic regression analyses were performed to determine the effects of malnutrition, age, dose and other factors on nevirapine exposure and likelihood of achieving therapeutic nevirapine trough concentrations.

Forty-three children were recruited (37 included for analysis). Mild to moderate malnutrition was present in 12 (32%) children; 25 (68%) were of normal nutritional status. There was no effect of malnutrition on any measure of total drug exposure or on the unbound fraction of nevirapine. Nevirapine exposure was strongly related to dose administered (P = 0.039) and to age (for every yearly increase in age there was an ~88% increase in the odds of achieving a therapeutic nevirapine concentration; P = 0.056, 95% confidence interval 0.983–3.585).

Use of divided adult Triomune^®30 tablets in treating young children results in significant underdosing. No independent effect of malnutrition on total and unbound nevirapine exposures was observed. These data support the use of bespoke paediatric antiretroviral formulations.

Patients successfully treated with 1000 mg saquinavir boosted with 100 mg ritonavir twice daily together with two nucleoside or nucleotide reverse transcriptase inhibitors [N(t)RTIs] who were switched to 2000 mg saquinavir with 100 mg ritonavir once daily with unchanged N(t)RTI therapy were analysed. CD4 cells, HIV-RNA PCR and metabolic parameters were compared between baseline and 3, 6, 9 and 12 months after the switch. Saquinavir and ritonavir drug levels were measured before and a median of 3 weeks after switching from twice to once daily at 0, 1, 2, 4, 6, 9, 12 and 24 h after intake of the medication. The area under the serum concentration–time curve from 0 to 24 h (AUC_0–24) was calculated using the trapezoidal rule.

Eighteen patients (16 males, median age of 41 years) with a median CD4 cell count of 464 cells/mm³ were analysed. HIV-RNA PCR remained <500 copies/mL for all patients. After switching from 100 mg twice daily to 100 mg once daily, the AUC_0–24 for ritonavir decreased significantly [21 874 to 10 267 ng·h/mL, geometric mean ratio (GMR) = 0.47; P < 0.001], whereas the AUC_0–24 for saquinavir decreased only marginally from 35 000 to 34 490 ng·h/mL (GMR = 0.99; P = 0.426). The CD4 cell count and the fasting metabolic parameters remained unchanged.

Once-daily treatment with ritonavir-boosted saquinavir was well tolerated and resulted in similar saquinavir drug exposure despite much lower ritonavir concentrations when compared with a twice-daily dosing schedule.

Plasma and DNA samples were obtained from 330 HIV-infected Thai women who received intra-partum single-dose nevirapine in the PHPT-2 clinical trial to prevent perinatal HIV transmission. Nine SNPs within CYP2B6, CYP3A4 and ABCB1 were genotyped by real-time PCR. Nevirapine plasma concentrations were determined by HPLC and used in a population pharmacokinetic analysis.

Higher nevirapine exposure was observed in women carrying the CYP2B6 516G>T polymorphism, but this did not reach statistical significance (P = 0.054). The TGATC CYP2B6 haplotype (g.3003T, 516G, 785A, g.18492T and g.21563C) was associated with increased nevirapine clearance and lower exposure (P = 0.0029). The median time for nevirapine concentrations to reach 10 ng/mL post-partum (nevirapine IC₅₀ for HIV-1) was 14 days [interquartile range (IQR, 14–18)] for TGATC homozygotes, 16 days (14–20) for TGATC heterozygotes and 18 days (14–20) for non-TGATC homozygotes (P = 0.020).

The CYP2B6 516G>T impact on nevirapine concentrations was less pronounced after intra-partum single-dose nevirapine than reported under steady-state conditions, perhaps due to lack of enzyme auto-induction at the time of dosing. Although the TGATC CYP2B6 haplotype may shorten the persistence of nevirapine post-partum, its practical implications for the prevention of HIV transmission or selection of resistance mutations are likely limited.

Adults with proven or probable IA, defined strictly according to EORTC-MSG criteria, were eligible. Those with possible IA were enrolled, but were not evaluable for efficacy unless upgraded to proven/probable disease within 7 days of registration based on investigations performed within 48 h after enrolment. Caspofungin dosage was 70 mg (day 1) followed by 50 mg/day. The primary endpoint was the proportion of patients with complete or partial response at the end of caspofungin therapy in the modified intention to treat (MITT) group; secondary endpoints were response and survival at day 84 and safety.

In the MITT group (n = 61), 75% of patients had cancer not in remission (relapsing or refractory), 85% were neutropenic at enrolment and 49% had a Karnofsky score of ≤50. At end of treatment, 1 and 19 patients had complete and partial response, respectively [success rate 33% (20/61)], 9 (15%) achieved stabilization and 31 (51%) had disease progression. One patient was not evaluable. The 6 and 12 week survival rates were 66% (40/61) and 53% (32/60), respectively. Baseline characteristics associated with survival at day 84 were an underlying disease in remission (not relapsing or refractory) and Karnofsky score. Recovery from neutropenia at the end of treatment was also significantly associated with survival. No serious drug-related adverse events or discontinuations due to drug-related adverse events were observed.

Caspofungin provided an observed response rate compatible with the null hypothesis of a true response rate of ≤35%. Underlying disease-related factors had a major impact on results.

A case note review of 1122 patients with 1188 episodes of HIV-associated PCP from three observational cohorts in Copenhagen, London and Milan, between 1989 and 2004, was conducted.

Trimethoprim/sulfamethoxazole (962 PCP episodes, 81%) was the most frequently used first-line therapy, followed by intravenous pentamidine (87 episodes, 7%), clindamycin/primaquine (72 episodes, 6%) and ‘other’ (atovaquone, dapsone/pyrimethamine, trimetrexate or inhaled pentamidine; 67 episodes, 6%). Rates of unchanged therapy were trimethoprim/sulfamethoxazole = 79%, clindamycin/primaquine = 65% and pentamidine = 60% (P < 0.001). First-line therapy was changed because of failure in 82 (7%) episodes and because of toxicity in 198 (17%) episodes. Three month survival rates were trimethoprim/sulfamethoxazole = 85%, clindamycin/primaquine = 81% and pentamidine = 76% (P = 0.09). After adjustment for possible confounders, pentamidine was associated with a significantly greater risk of death at 3 months [hazard ratio (HR) = 2.0, 95% confidence interval (CI) = 1.2–3.4]. Second-line therapy survival rates differed: trimethoprim/sulfamethoxazole = 85%; clindamycin/primaquine = 87%; and pentamidine = 60% (P = 0.01). Multivariable time-updated Cox regression analysis showed a greater risk of death associated with pentamidine (HR = 3.3, 95% CI = 2.2–5.0), but not for clindamycin/primaquine, when both were compared with trimethoprim/sulfamethoxazole.

Pentamidine was associated with a greater risk of death when used as first- and second-line therapy for HIV-associated PCP, and was associated with more treatment changes. Clindamycin/primaquine appeared superior to pentamidine as second-line therapy for PCP in patients failing or developing toxicity with trimethoprim/sulfamethoxazole. In patients failing first-line treatment with non-trimethoprim/sulfamethoxazole regimens, second-line therapy should be trimethoprim/sulfamethoxazole.

A retrospective analysis of microbiology data from two centres in Maryland and Chicago was performed. Adult hospital inpatients with positive clinical cultures for specific antimicrobial-resistant bacterial pathogens between 1999 and 2005 (55 427 isolates) were included. The proportions of isolates not susceptible to specific antimicrobial agents were compared between patients ≥65 and <65 years. Additional analyses examined temporal trends in the frequency of resistance and the frequency of resistance among the oldest patients (≥80 years), in bacteria isolated from blood cultures and in bacteria obtained from intensive care unit patients.

Heterogeneity was observed in the frequency of resistance among different bacteria between older and younger patients, between the two centres and over the study period. Staphylococcus aureus isolates were more likely to be resistant to methicillin when obtained from older patients at Chicago (50.9% versus 40.9%; P < 0.001). In contrast, younger patients yielded a greater proportion of enterococci resistant to vancomycin at Maryland (19.4% versus 16.5%; P = 0.009). Results were variable when resistance to fluoroquinolones, cephalosporins and imipenem were compared for Pseudomonas aeruginosa, Escherichia coli and Klebsiella spp.

Overall, advanced patient age was not uniformly associated with a greater likelihood of antimicrobial resistance among all bacterial pathogens. Moreover, the frequency of resistance in older and younger patients varied considerably at the two sites over the study period. Variability in the frequency of resistance precludes simplistic conclusions regarding the relationship between age and resistance.

A random stratified, cross-sectional prevalence survey was conducted in NH residents who were screened for MRSA carriage by multisite enriched culture. Characteristics of NHs and residents were collected by a questionnaire survey and analysed by two-stage logistic regression modelling. MRSA isolates were genotyped by PFGE, staphylococcal cassette chromosome mec (SCCmec) typing, multilocus sequence typing (MLST) and resistance genes.

Of 2953 residents screened in 60 NHs, 587 (19.9%) were MRSA carriers. Risk factors included hospital contact, antibiotic exposure, impaired mobility and skin lesions at the resident level, and lack of MRSA surveillance, lack of antibiotic therapeutic formulary and the combination of less-developed infection control activities and a high ratio of physicians to residents at the institution level. MRSA isolates showed eight major types, three of which were predominant: B2-ST45-SCCmec IV (49%; where ST stands for sequence type); A21-ST8-SCCmec IV (13%); and A20-ST8-SCCmec IV (10%). Each was recovered in 55, 21 and 25 NHs, respectively. The geographical distribution of NH genotypes paralleled that of acute-care hospitals.

A high prevalence of MRSA carriage in NH residents was associated with hospital care, co-morbidities and less-developed coordination of institutional care. The predominant MRSA strains from NH residents and hospitalized patients of the same area were identical. Strengthening and coordination of MRSA surveillance and control activities are warranted within and between NHs and hospitals.

Data were collected during a 7 year surveillance period (2001–07) from volunteer surgery wards participating in the INCISO Surveillance Network in Northern France. Main SAP practices, i.e. antibiotic choice, timing of first dose and total SAP duration, were evaluated and compliance checked based on French recommendations. The study focused on selected procedures in digestive, orthopaedic, gynaecological and cardiovascular surgery, for which standard SAP is recommended. Multilevel logistic regression analysis (a two-level random effect model) was carried out to identify SAP-, patient- and procedure-specific factors associated with SSI.

Of 8029 patients who underwent the selected surgeries, 91.3% received SAP and 2.5% developed SSI. Among those receiving SAP, 83.3% received appropriate antibiotic agents and 76.6% had an optimal timing of administration. SAP duration was considered to be appropriate in 35.0%, too long (SAP unnecessarily prolonged) in 45.2% and too short (lack of intra-operative redosing when recommended) in 19.8%. In the multivariate analysis, a too-short SAP duration remained the only inappropriate practice associated with higher SSI risk (odds ratio = 1.8, 95% confidence interval: 1.14–2.81), after adjustment for surgery procedure group, the National Nosocomial Infections Surveillance System risk index, age and infection risk variability among hospitals. No significant relationships were observed between SSI and the other SAP parameters.

A too-short SAP duration was the most important SAP malpractice associated with an increased risk of SSI. Information directed at practitioners should be reinforced based on standard recommendations.

Data on clinical activity and outcomes were collected prospectively on 334 episodes of treatment administered by the Sheffield OPAT service between January 2006 and January 2008. Cost-effectiveness was calculated by comparing real costs of OPAT with estimated inpatient costs for these patient episodes incorporating two additional sensitivity analyses.

Of the OPAT episodes, 87% resulted in cure or improvement on completion of intravenous therapy. The readmission rate was 6.3%, and patient satisfaction was high. OPAT cost 41% of equivalent inpatient costs for an Infectious Diseases Unit, 47% of equivalent inpatient costs using national average costs and 61% of inpatient costs using minimum inpatient costs for each diagnosis.

Using this service model, OPAT is safe and clinically effective, with low rates of complications/readmissions and high levels of patient satisfaction. OPAT is cost-effective when compared with equivalent inpatient care in the UK healthcare setting.

An ideal dataset was established of the parameters that should be reported when calculating a drug dosing regimen from first principles. A Medline search was performed of relevant literature producing 64 citations from which completeness of the specified criteria was examined.

None of the studies analysed presented the full dataset that we established as necessary. Of concern, basic pharmacokinetic parameters such as volume of distribution (V_d) and clearance (CL) were specified in only 79% and 81% of studies, respectively.

A large proportion of current studies do not report key information necessary to devise a rational dosing regimen for patients with acute renal failure receiving CRRT, and we hope this dataset will be a useful guide when reporting future pharmacokinetic data.

HIV-1 molecular clones carrying subtype B or C proteases had these coding regions subjected to site-directed mutagenesis to include T74S alone or in combination with four known protease inhibitor (PI) primary drug resistance mutations. All clones were used in a phenotypic assay to evaluate their susceptibility to most commercially available PIs. The impact of T74S on virus fitness was also assessed for all viruses through head-to-head competitions and oligonucleotide ligation assays to measure the proportion of each virus in culture.

Viruses of both subtypes carrying T74S did not have their susceptibility altered to any tested PI. Viruses with the four resistance mutations showed strong resistance to most PIs with fold changes ranging from 5 to 300 times compared with their wild-type counterparts. Surprisingly, the addition of T74S to the multiresistant clones restored their susceptibilities to indinavir and ritonavir and partially to lopinavir, close to those of wild-type viruses. Most 74S-containing viruses were more fit than their 74T counterparts.

Our results suggest that T74S is not a major drug resistance mutation, but it resensitizes multiresistant viruses to certain PIs. T74S is a bona fide accessory mutation, restoring fitness of multidrug-resistant viruses in both subtypes B and C. T74S should be further studied in clinical settings and considered in drug resistance interpretation algorithms.

Partial NS5B sequences from HCV-positive women were amplified by RT–PCR. Additionally, subcloning was performed to evaluate intrapatient variability in selected samples.

HCV NS5B genotypes were 45 genotype 1a (57.0%), 11 genotype 1b (13.9%), 5 genotype 2a (6.3%), 3 genotype 2b (3.8%), 9 genotype 3a (11.4%) and 6 genotype 4a (7.6%). One HCV genotype 1a-infected patient was found to have the C316Y mutation (1.3%). Clonal analysis further revealed that all NS5B sequences from this individual—representing three serum samples collected 4 years apart—contained the C316Y mutation. In contrast, the S282T resistance mutation was not found in any samples.

The C316Y polymerase resistance mutation was found in 1.3% of samples from HCV-infected women. The presence of this mutation over time suggests significant replicative fitness of this variant and has implications for development of new specifically targeted antiviral therapies against HCV (STAT-C) targeting this region.

DNase I footprinting was used to identify high-affinity DNA binding sites. Microarrays and gel electrophoresis were used to assess the ELB-21-induced phenotype.

High-affinity interstrand binding sites in which guanine residues were separated by 4 bp, and also some intrastrand cross-linking sites of variable length were identified. Exposure of EMRSA-16 to 0.015 mg/L ELB-21 elicited a 2-fold or greater up-regulation of 168 genes in logarithmic phase and 181 genes in stationary phase; the majority of genes affected were associated with resident prophages Sa2 and Sa3, pathogenicity island SaPI4 and DNA damage repair. ELB-21 induced a marked increase in the number of viable phage particles in culture supernatants. The expression of only a limited number of genes showed a >50% reduction. Sixteen extracellular and four intracellular proteins were differentially expressed during logarithmic and stationary phases, including RecA, proteins associated with staphylococcal pathogenesis (IsaA, CspA), cell division and wall synthesis.

ELB-21 kills S. aureus by forming multiple interstrand and intrastrand DNA cross-links, resulting in induction of the DNA damage response, derepression of resident prophages and modulation of a limited number of genes involved with cell wall synthesis.

K. pneumoniae clinical isolates were analysed for the presence of IS26 insertions characteristic of plasmid-borne bla_SHV, and differences in their bla_SHV promoter sequences and expression levels. A high resolution melting (HRM)-based method for rapid promoter analysis was developed.

An IS26 insertion characteristic of the plasmid-borne bla_SHV-1/bla_SHV-2/bla_SHV-5 family was 100% linked to a promoter mutated in the –10 region, a mutation previously only found on the chromosome. The mutation was shown by real-time reverse transcriptase PCR to be associated with increased bla_SHV expression.

Plasmid-borne bla_SHV is associated with strong promoters. It is likely that an SHV-dependent ESBL-positive phenotype requires both a strong promoter and a coding sequence mutation. An HRM assay can indicate bla_SHV expression.

The acrA gene was inactivated in Salmonella Typhimurium SL1344 by insertion of the aph gene and this mutant complemented with pWKS30acrA. The antimicrobial susceptibility of the mutant to six antibiotics as well as various dyes and detergents was determined. In addition, efflux activity was quantified. The ability of the mutant to adhere to, and invade, tissue culture cells in vitro was measured.

Following disruption of acrA, RT–PCR and western blotting confirmed that acrB/AcrB was still expressed when acrA was disrupted. The acrA mutant was hypersusceptible to antibiotics, dyes and detergents. In some cases, lower MICs were seen than for the acrB or tolC mutants. Efflux of the fluorescent dye Hoechst H33342 was less than in wild-type following disruption of acrA. acrA was also required for adherence to, and invasion of, tissue culture cells.

Inactivation of acrA conferred a phenotype distinct to that of acrB::aph and tolC::aph. These data indicate a role for AcrA distinct to that of other protein partners in both efflux of substrates and virulence.

The transcriptomes of Salmonella enterica serovar Typhimurium SL1344 and Escherichia coli K-12 MG1655 after exposure to the MIC of triclosan (0.12 mg/L) were determined in microarray experiments. Phenotypic validation of the transcriptomic data included RT–PCR, ability to form a biofilm and motility assays.

Despite important differences in the triclosan-dependent transcriptomes of the two species, increased expression of efflux pump component genes was seen in both. Increased expression of soxS was observed in Salmonella Typhimurium, however, within E. coli, decreased expression was seen. Expression of fabBAGI in Salmonella Typhimurium was decreased, whereas in E. coli expression of fabABFH was increased. Increased expression of ompR and genes within this regulon (e.g. ompC, csgD and ssrA) was seen in the transcriptome of Salmonella Typhimurium. An unexpected response of E. coli was the differential expression of genes within operons involved in iron homeostasis; these included fhu, fep and ent.

These data indicate that whilst a core response to triclosan exposure exists, the differential transcriptome of each species was different. This suggests that E. coli K-12 should not be considered the paradigm for the Enterobacteriaceae when exploring the effects of antimicrobial agents.

Urine samples positive for Gram-negative bacilli as revealed by Gram staining were collected over a 3 month period at Bicêtre hospital, France. Aliquots of these urine samples were frozen for subsequent molecular analysis, and the bacteria were cultured and identified by standard bacteriological techniques (biochemical tests, disc diffusion antibiogram and synergy testing). LC-PCR and standard PCR followed by sequencing was performed on all ESBL-positive and on 70 randomly chosen ESBL-negative urine samples.

Over the study period, 810 urine samples were collected from 655 patients. Thirty-six ESBL-producing Enterobacteriaceae, mostly Escherichia coli (77%), were identified from 29 patients, of which half were outpatients. Twenty-five urine samples (19 patients) were found to be positive for bla_CTX-M genes using the LC-PCR assay. The bla_CTX-M genes belonged to the bla_CTX-M-1, bla_CTX-M-9 and bla_CTX-M-2 groups (68%, 24% and 8%, respectively). Standard PCR and sequencing of the entire bla_CTX-M genes confirmed the LC-PCR results; 17 CTX-M-15, 6 CTX-M-9 and 2 CTX-M-2. Among the remaining ESBLs, eight were of the TEM type and three of the SHV type.

The LC-PCR assay represents a powerful tool for rapid identification of CTX-M producers in urine samples.

A linezolid-resistant strain of S. epidermidis was selected by serial passages with increasing concentrations of linezolid. The MICs of linezolid, ciprofloxacin, tetracycline, rifampicin, vancomycin, gentamicin, tobramycin, chloramphenicol and oxacillin were determined. The 23S rRNA gene was amplified and sequenced, to search for mutations conferring linezolid resistance. The MIC of linezolid was also determined in the presence of reserpine. Finally, the accumulation of linezolid was measured and quantified by HPLC/UV.

The obtained resistant strain had an MIC of linezolid of 64 mg/L and was stable after several passages on blood agar. The MIC measured in the presence of 25 mg/L reserpine, an efflux pump inhibitor, was not altered (MIC of 64 mg/L). The sequence of the 23S rRNA gene showed that the mutation G2576T (Escherichia coli numbering) was not present and no other mutation was found. An analysis of the accumulation of linezolid was performed, comparing the uptake of the resistant strain with that of the susceptible one. This showed that the resistant strain had significantly lower levels of linezolid accumulation than its susceptible parental strain.

The mechanism of resistance to linezolid, in this resistant strain, may be related to a decrease in the antimicrobial uptake. This new mechanism of resistance was also related to a little loss of fitness.

Plasma membranes of L. donovani promastigotes were depleted of sterol using methyl-β-cyclodextrin (MCD) and cholesterol oxidase (CH-OX). Sterols were quantified and HePC susceptibility was assessed using the MTT test. A biomimetic model of the outer leaflet of a Leishmania plasma membrane was used to decipher the HePC–lipid interactions.

CH-OX, which is known to act more specifically on condensed membranes, therefore at the level of lipid rafts, gave a better extraction yield in HePC-resistant parasites, confirming the more rigid structure of their membranes than those of wild-type parasites. Sterol depletion was responsible for a 40% decrease in HePC susceptibility in both wild-type and HePC-resistant parasites. Sterol repletion of the sterol-depleted parasites restored HePC susceptibility. The biomimetic model of the outer leaflet of a Leishmania plasma membrane confirmed that condensed microdomains were able to incorporate higher quantities of HePC than fluid ones and this result was amplified when the sterol concentration was increased.

Sterol and lipid rafts probably play a significant role as an HePC reservoir providing a constant supply to the previously described transporter. In addition, ¹H NMR experiments suggested that HePC stimulated lipid trafficking in parasites.

The expression of P-gp, MRP1, MRP2, SLCO1A2, 1B1, 1B3, 2B1, 3A1 and 4A1 was assessed by PCR. Inhibitors of P-gp (XR9576, GF120918, dipyridamole) and MRP (MK571, frusemide, dipyridamole), and SLCO substrate or inhibitor (estrone-3-sulphate or montelukast, respectively) were used to study the role of drug transporters in the accumulation of [¹⁴C]efavirenz and [³H]nevirapine. Lipophilicity was measured by the octanol/saline partition coefficient.

CEM cells, MT4 cells and PBMCs express various SLCO isoforms, with SLCO3A1 detected in all of the cells. XR9576, dipyridamole and GF120918 had no effects on the accumulation of [¹⁴C]efavirenz, while MK571 and frusemide produced variable effects in the cells. The accumulation of [¹⁴C]efavirenz was significantly decreased in all the cells by montelukast and estrone-3-sulphate.

P-gp expression had no effect on the accumulation of [¹⁴C]efavirenz and [³H]nevirapine. MRP1/2 expression, lipophilicity and SLCO-like transporters (possibly SLCO3A1) may have greater influence on the accumulation of [¹⁴C]efavirenz than [³H]nevirapine.

LTB₄ release and uptake of Ca²⁺ by the cells were measured using an enzyme immunoassay and fura-2/AM-based spectrofluorimetric procedures, respectively.

Treatment of neutrophils with posaconazole resulted in dose-related attenuation of PAF-activated release of LTB₄ and influx of Ca²⁺, which attained statistical significance at 1 µM of the antimycotic.

Although primarily an antimycotic, posaconazole possesses secondary anti-inflammatory activities, which may contribute to the therapeutic efficacy of this agent in patients with sepsis.

In this study, we compare the ability of linezolid, DA-7157 and DA-7218 to inhibit intracellular growth of Nocardia brasiliensis within the human monocyte cell line THP-1.

The addition of oxazolidinones to the infected macrophage monolayer at concentrations 0.25x, 1x, 4x and 16x the MIC for N. brasiliensis resulted in an inhibitory effect on bacterial growth as follows DA-7157 ≥ DA-7218 > linezolid.

The excellent intracellular antimicrobial activity detected suggests that these compounds could be effective in the treatment of actinomycetoma. However, more studies are needed both in vitro and in vivo, including clinical trials, to confirm this issue.

The drug interaction effects on nine human isolates of MAP were determined by the chequerboard method adapted for the BACTEC^TMMGIT^TM960 culture system and by calculation of the fractional inhibitory concentration index (FICI) for drug combinations.

Synergism (FICI ≤ 0.5) was observed between 6-mercaptopurine and azithromycin (seven isolates), clarithromycin, rifampicin, rifabutin (four isolates each) and ethambutol (two isolates). 6-Mercaptopurine was not antagonistic with any of the antibacterial agents tested. Among the combinations of two and three antibacterials tested, the clarithromycin/rifampicin combination was synergistic against four isolates, while all other combinations showed no interaction.

This in vitro study suggests that 6-mercaptopurine may be synergistic with macrolides and rifamycin derivatives against MAP. The activity of clarithromycin against MAP seems to be enhanced by rifampicin.

To evaluate the occurrence of heterogeneous vancomycin-intermediate S. aureus (hVISA) among MRSA strains tolerant to vancomycin and/or with increased vancomycin or daptomycin MIC values. The accuracy of the macro Etest method (MET) compared with population analysis profiling (PAP) for the detection of hVISA was also assessed.

A total of 1800 MRSA strains were collected from bloodstream infections at the nine sites (40 strains per year, per medical centre during the 2002–06 study period). Vancomycin and daptomycin MIC testing was performed by reference broth microdilution (all strains) and MBC tests on 50% of strains (randomly selected). A subset of isolates (n = 268) having an increased vancomycin MBC (≥16 mg/L), an increased vancomycin MIC (≥1 mg/L) and/or an increased daptomycin MIC (>0.5 mg/L) were tested for susceptibility to vancomycin and teicoplanin by MET.

Overall, 181 of 900 (20.1%) MRSA tested exhibited vancomycin tolerance, varying from 10% to 43% among the medical centres evaluated, and from 11.7% in 2004 to 27.8% in 2005. No resistance trend was observed in any medical centre or in the overall study data. Daptomycin showed bactericidal activity against all strains tested. The accuracy of MET for identifying hVISA strains varied significantly with the criteria applied for positivity.

The most frequently used criteria to define hVISA, i.e. MET reading values ≥8 mg/L for both vancomycin and teicoplanin or ≥12 mg/L for teicoplanin only, detected 20 of 36 PAP-positive strains (55.6% sensitivity), indicating that the prevalence of hVISA could be higher than currently appreciated. Daptomycin was bactericidal against hVISA strains.

From 1996 to 2008, 1146 consecutive S. aureus isolates from ICU patients in 14 large referral hospitals were collected. The susceptibility to relevant antibiotics was determined by microbroth dilution according to CLSI guidelines.

Resistance to flucloxacillin was only found in 12 isolates (1%). The resistance to clarithromycin, ciprofloxacin and moxifloxacin showed a significant trend over time, from 4.2% to 10.3%, from 1.0% to ~10% and from 0.0% to ~5.0%, respectively (P < 0.05). The resistance to penicillin, clindamycin and doxycycline increased over time, from 74% to 75%, from ~3.0% in 1996 to 3.2% in 2008 and from 2.2% in 1996 to 8.2% in 2008, respectively (P > 0.05). Resistance to cephalosporins, carbapenems, rifampicin and gentamicin was sporadically observed. No resistance was found to vancomycin, teicoplanin and linezolid.

The empirical choice of flucloxacillin in the case of putative S. aureus infections in patients admitted to ICUs in the Netherlands is still justified.

Measurement of torezolid cell content and antibacterial activity in comparison with linezolid using human macrophages (THP-1) and human endothelial cells [human umbilical vein endothelial cells (HUVECs)], applying models allowing for the quantitative evaluation of the pharmacodynamics of antibiotics towards intracellular bacteria.

Torezolid accumulated rapidly in THP-1 macrophages, reaching a stable intracellular to extracellular ratio of ~10 (compared with ~1–2 for linezolid) within 15 min. On a weight concentration basis (mg/L), torezolid was ~5- to 10-fold more potent intracellularly (lower concentration needed to achieve a bacteriostatic effect) than linezolid against phagocytosed S. aureus, L. monocytogenes and L. pneumophila, with no change in maximal efficacy (~1 log₁₀ reduction of the original, post-phagocytosis inoculum). When drugs were compared at equipotent concentrations (multiples of the MIC), no difference was seen between linezolid and torezolid, but the higher potency of torezolid allowed control of intracellular infections caused by linezolid-resistant S. aureus.

Torezolid exerts intracellular activity at lower extracellular concentrations than linezolid because of its greater potency independent of its greater intracellular accumulation. This may confer an advantage to torezolid in vivo if the drug can be used at dosages creating serum concentrations similar to those achieved with linezolid.

Five clinical MRSA isolates held at Southmead Hospital were used (SMH 15841, SMH 40289, SMH 40275, SMH 33922 and SMH 33024) together with a VRSA isolate (SMH 19898); inocula of 10⁶ and 10⁸ cfu/mL were used. Daptomycin (6 mg/kg once daily), vancomycin (1 g twice daily) and teicoplanin (400 mg once daily) regimens were simulated. ABEs were measured using the 24 h area-under-the-bacterial kill curve (AUBKC) and log change in viable count at 24 h (24). For daptomycin, dose escalation was used to determine the relationship between ABE and AUC/MIC.

Daptomycin was bactericidal against the MRSA strains. Daptomycin and vancomycin were active against the VRSA strain; teicoplanin had a static effect. The higher inoculum reduced the ABEs. Analysis of variance (ANOVA) indicated that daptomycin had a superior ABE to teicoplanin and vancomycin. Daptomycin fAUC/MIC was related to AUBKC and 24; the fAUC/MIC ratios for a static effect and 1 log and 3 log drop were 37.2 ± 16.5, 40.6 ± 17.8 and 49.8 ± 19.2, respectively.

These data define the fAUC/MIC sizes for daptomycin for bacteriostatic and bactericidal ABEs and indicate that a 6 mg/kg dose of daptomycin is superior to vancomycin and teicoplanin against MRSA and VRSA strains.

SBPT04 was delivered by intraperitoneal (ip) and oral (po) routes, and mice were monitored for morbidity, mortality and relapse of disease. Pharmacokinetic studies were performed to evaluate bioavailability. Phase I and Phase II metabolism of SBPT04 was assessed in mouse and human microsomes.

SBPT04, a potent inhibitor of the enoyl-ACP reductase enzyme ftuFabI, has efficacy against F. tularensis in the murine model of infection when delivered by both ip and po routes. SBPT04 delivered ip cleared infection by day 4 of treatment, and SBPT04 delivered po resulted in delayed dissemination. Importantly, SBPT04 delivered ip or po demonstrated efficacy with no signs of relapse of disease. Pharmacokinetic studies show increased serum concentrations following ip delivery compared with po delivery, which correlates with the observed survival rate of 100%.

In addition to being a potent lead, this work substantiates substituted diphenyl ethers as a platform for the development of novel broad-spectrum chemotherapeutics to other bacterial agents in addition to F. tularensis.

Using this model, animals were treated for 7 days with 100 mg/kg/day levofloxacin or 200 mg/kg/12 h cloxacillin, or were left untreated. Antibiotic efficacy was evaluated by means of bacterial counts from tissue-cage fluid (TCF); these counts were derived separately in total, intracellular and extracellular bacteria. The presence of intracellular bacteria was checked by electron microscopy. Population analysis was performed with surviving bacteria recovered at the end of levofloxacin therapy.

Among a total number of bacteria (mean log cfu/mL ± SD) from TCF of 6.86 ± 0.6, we identified 6.38 ± 0.8 intracellular bacteria and 5.57 ± 0.5 extracellular bacteria. Levofloxacin was more efficient than cloxacillin (P < 0.05) against both intracellular and extracellular bacteria. The killing activity of levofloxacin against the intracellular population was higher than against the extracellular bacteria (P = 0.1). The frequency of levofloxacin-resistant mutants among surviving bacteria at the end of levofloxacin therapy was similar to that for the wild-type strain.

Intracellular bacteria accounted for the largest proportion of the total inoculum in this model of foreign-body infection. The intracellular activity of an antibiotic seems to be an additional relevant factor in the antibiotic response to these infections.

Sprague-Dawley rats received a single intravenous bolus of 1.0 mg/kg NAB 7061 or NAB 739. Plasma concentrations of NAB 7061 or NAB 739 were determined by HPLC or liquid chromatography–mass spectrometry. The pharmacokinetic parameters of NAB 7061 and NAB 739 were calculated using non-compartmental analysis.

Corresponding total body clearance, volume of distribution at steady state and terminal half-life of NAB 7061 and NAB 739 averaged 3.84 and 2.63 mL/min/kg, 339 and 222 mL/kg, and 66.2 and 69.0 min, respectively. Approximately 7.16% and 19.4% of the dose was eliminated in an unchanged form via the urine in 24 h for NAB 7061 and NAB 739, respectively.

While both compounds had generally similar pharmacokinetics to colistin, even minor alterations in the chemical structures appear to have an impact on their pharmacokinetics, especially on their clearance by the kidney. There are also substantial differences in relation to the relative contributions of renal and non-renal clearance to overall elimination from the body.

HIV-negative adult volunteers were pre-screened for CYP3A5 *3, *6 and *7 polymorphisms and enrolment was balanced for CYP3A5 expressor status, gender and race (African-American versus non-African-American). Participants received atazanavir 400 mg daily for 7 days followed by atazanavir/ritonavir 300 mg/100 mg daily for 7 days with pharmacokinetic studies on days 7 and 14. Other measures collected were bilirubin, UGT1A1 *28, and ABCB1 1236C > T, 2677G > T/A and 3435C > T genotypes. Data analyses utilized least squares regression.

Fifteen CYP3A5 expressors and 16 non-expressors participated. The day 7 atazanavir oral clearance (CL/F) was 1.39-fold faster (0.25 versus 0.18 L/h/kg; P = 0.045) and the C_min was half (87 versus 171 ng/mL; P = 0.044) in CYP3A5 expressors versus non-expressors. Non-African-American CYP3A5 expressor males had 2.1-fold faster CL/F (P = 0.003) and <20% the C_min (P = 0.0001) compared with non-African-American non-expressor males. No overall CYP3A5 expressor effects were observed during the ritonavir phase. One or two copies of wild-type ABCB1 haplotype (1236C/2677G/3435C) was predictive of slower atazanavir and ritonavir CL/F compared with zero copies (P < 0.06). Indirect bilirubin increased 1.6- to 2.8-fold more in subjects with UGT1A1 *28/*28 versus *1/*28 or *1/*1.

CYP3A5 expressors had faster atazanavir CL/F and lower C_min than non-expressors. The effect was most pronounced in non-African-American men. Ritonavir lessened CYP3A5 expressor effects. The wild-type ABCB1 CGC haplotype was associated with slower CL/F and the UGT1A1 *28 genotype was associated with increased bilirubin. Thus, CYP3A5, ABCB1 and UGT1A1 polymorphisms are associated with atazanavir pharmacokinetics and pharmacodynamics in vivo.

An open label study in 24 HIV-infected children between the age of 2 and 18 years, naive to PIs, randomized to receive either the WHO-recommended dose of lopinavir/ritonavir or a low dose (70% of the standard dose) twice daily in combination with zidovudine and lamivudine. A 12 h pharmacokinetic study was done at 4–6 weeks after starting treatment. Treatment outcomes were evaluated at week 48. The clinical trial number of the study is NCT00887120.

The medians [interquartile ranges (IQRs)] of age, body surface area, percentage CD4 and plasma HIV RNA were 9.5 years (7.0–12.3), 0.9 m² (0.8–1.1), 17% (11%–24%) and 4.6 log₁₀ copies/mL (4.1–4.9), respectively. The median (IQR) lopinavir dose was 279 mg/m²/dose (263–294) and 194 mg/m²/dose (176–206) in the standard and low-dose arms, respectively. Median (IQR) AUC_0–12 and C_trough of lopinavir were 117.6 mg·h/L (74.0–128.5) and 4.9 mg/L (2.7–8.0) for the standard arm and 83.8 mg·h/L (56.0–112.9) and 3.4 mg/L (2.7–5.4) for the low-dose arm. One child in the low-dose arm had a lopinavir pre-dose level of <1.0 mg/L. At week 48, the median percentage CD4 was 22% (15%–28%) and 27% (21%–31%) in the standard and low-dose arms, respectively, while 50% and 83% of children had HIV RNA <50 copies/mL, respectively (P = 0.19).

Low-dose lopinavir displayed adequate pharmacokinetic parameters and good efficacy as compared with standard-dose lopinavir in Thai children. A larger study to investigate the efficacy of low-dose lopinavir is warranted.

This retrospective observational study reports the changes in the viral load (VL) after the withdrawal of raltegravir from patients carrying virus with resistance mutations. We selected patients under stable treatment and with stable VL during at least the previous 2 months before the withdrawal.

Five patients (A–E) were selected. The median changes in VL and CD4 counts at the end of the raltegravir interruption were –0.04 log copies/mL (range, –0.20 to +0.19) and +58 cells/mm³ (range, –56 to +252), respectively.

All VL changes were well below the clinically relevant variation of 0.5 log copies/mL at the end of the interruption. Thus, this study indicates that, for viruses harbouring one of the two main resistance pathways described for raltegravir, no relevant antiviral activity seems to persist in vivo. Even if further observations would be useful to reinforce this conclusion, the cost/benefit and risk/benefit of maintaining raltegravir as part of a salvage regimen in the presence of raltegravir mutations seem debatable, especially in the absence of relevant antiretroviral activity in this context.

Hospitalized patients with acute cholecystitis received a single, 1 h infusion of 400 mg of moxifloxacin before cholecystectomy. Serum and gallbladder wall tissue samples were collected during surgery, and the moxifloxacin concentrations were measured by HPLC.

Sixteen patients (eight men and eight women) were included between January 2007 and April 2008. The time between start of infusion and gallbladder removal ranged from 50 min to 21 h 10 min. The serum concentration at the time of cholecystectomy was between 0.39 and 4.37 mg/L, and the tissue concentration between 1.73 and 17.08 mg/kg. The tissue-to-serum concentration ratio ranged from 1.72 to 6.33.

The results show that moxifloxacin penetrates well into gallbladder tissue and is therefore a therapeutic option for biliary tract infection. The highest concentrations in serum and gallbladder tissue were measured shortly after the end of a 1 h infusion. As perioperative prophylaxis, moxifloxacin should therefore be administered 30–60 min before the first surgical incision.

Four hundred children admitted to our hospital were studied prospectively. The patients were randomized to ODD or MDD groups alternately. The primary outcomes were: (i) a good clinical outcome, as defined; and (ii) occurrence of side effects, if any. Clinical efficacy was determined by comparing the proportion of patients with a favourable response between the two groups, while pharmacokinetic profile was assessed by comparing the peak and trough concentrations of the drug in a subgroup of patients. Safety of the two regimens was compared, besides recording any symptoms due to side effects of the drug, with the help of serum creatinine and brainstem-evoked response audiometry (in a subgroup of the patients).

We found ODD of gentamicin in hospitalized Indian children to be efficacious and safe. A favourable clinical response was achieved in 167 of the 188 patients (89%) in the ODD group and in 161 of the 212 patients (76%) in the MDD group. Similarly, a higher number of patients in the ODD group showed favourable gentamicin peak concentrations as compared with the MDD group (100% versus 87%). The MDD group showed a higher number of trough concentrations in the undesirable range as compared with the ODD group (17% versus 0%).

The study supports extended-interval (single daily) dosing in hospitalized Indian children due to its efficacy and safety with the added advantage of needing fewer injections.

During 21 March to 20 April 2008, 241 K. pneumoniae isolates detected at Integrated Regional Laboratories (Ft. Lauderdale, FL) for which the ertapenem MICs were ≥4 mg/L were studied. PCR, cloning and sequence analysis were used to detect bla_KPC and to characterize the β-lactamase and outer membrane proteins (Omps). The expression level of KPC enzymes was studied by immunoblotting. Genetic relatedness of isolates was investigated with rep-PCR and PFGE. Clinical records of patients were investigated.

Seven KPC-Kp strains were isolated from different patients located at a single LTACH, with a further three isolates being recovered from patients at different hospitals. All KPC-Kp isolates in patients from the LTACH and from one hospital patient were genetically related and shared PFGE patterns that clustered with known sequence type (ST) 258 strains. These strains were highly resistant to carbapenems (MICs ≥ 32 mg/L) due to an increased level of KPC expression and loss of Omps. Rectal colonization was documented in all LTACH patients with KPC-Kp isolates. Treatment failures were common (crude mortality rate of 69%). Active surveillance and enhanced infection control practices terminated the KPC-Kp outbreak.

The detection of KPC-Kp in an LTACH represents a serious infection control and therapeutic challenge in a new clinical setting. The speed at which the epidemic of KPC-Kp is spreading in our healthcare system mandates urgent action.

A multisite retrospective analysis of survival of patients with MRSA bacteraemia, which included patients from March 1995 to December 2008. Periods before and after the publication of UK guidelines were compared.

Data were analysed for 1675 patients with a mean age of 69.8 years. Survival for the period up to and including 2003 was 64.3%, and was 62.8% for both 2004–2005 and 2006–2008.

No significant difference in survival in relation to local practice review or the publication of national guidelines was detected.

Nasal swabs were collected from animals and farm workers in four Chinese provinces. MRSA isolates were recovered and characterized by PFGE, Panton–Valentine leucocidin PCR, staphylococcal chromosomal cassette (SCC) mec typing, spa typing, multilocus sequence typing, antimicrobial susceptibility testing and testing for inducible clindamycin resistance.

A total of 60 MRSA isolates were recovered from swine and swine workers. Two predominant multidrug resistance profiles were identified: ciprofloxacin/clindamycin/erythromycin/cefoxitin/gentamicin/tetracycline/chloramphenicol and ciprofloxacin/clindamycin/erythromycin/cefoxitin/gentamicin/tetracycline. All isolates were determined to be spa type t899, contained the group III SCCmec element and were Panton–Valentine leucocidin negative. Multilocus sequence type ST9 (n = 46) was identified as the dominant sequence type. One dominant PFGE cluster and a dominant strain type were identified.

MRSA from Chinese pigs and farm workers (ST9) differed from the European pig-associated clone (ST398) with regard to clonal type, SCCmec content and resistance profile.

Antibiogram analysis, contour-clamped homogeneous electric field electrophoresis, multilocus sequence typing, Panton–Valentine leucocidin determinant detection and accessory genetic regulator typing were performed to characterize the isolates. MRSA was further characterized by staphylococcal cassette chromosome mec typing.

The S. aureus population consisted of 13 clonal complexes and two Singleton lineages together with 56 sporadic isolates. Five lineages contained MRSA; however, these were not the predominant methicillin-susceptible S. aureus (MSSA) lineages. There was greater diversity amongst the MSSA while the MRSA appeared to have emerged clonally following acquisition of the staphylococcal cassette chromosome mec. Three MRSA lineages were considered to have been endemic in the communities and have subsequently become predominant lineages of CA-MRSA in the wider WA community. People colonized with MSSA tended to harbour clones of a different genetic lineage at each anatomical site while people colonized with MRSA tended to harbour clones of the same lineage at each site. Overall, the isolates were resistant to few antimicrobials.

Although the evidence suggests that in WA CA-MRSA strains arose in remote communities and have now disseminated into the wider community, there is no evidence that they arose from the predominant MSSA clones in these communities.

Ninety-six isoniazid-resistant M. tuberculosis isolates, including 29 multidrug-resistant isolates, were analysed for mutations in the rpoB, katG, inhA, oxyR and ahpC genes.

Mutations in the rpoB gene were detected in 25 (86.2%) of the 29 rifampicin-resistant isolates. Of the 96 isoniazid-resistant isolates, 61 (63.5%) had mutations in codon 315 of the catalase–peroxidase-encoding gene (katG). Mutations in codon 315 were observed at a higher frequency in the multidrug-resistant isolates than in the isoniazid-resistant isolates (86.2% versus 53.7%, respectively, P = 0.003). Mutations in the oxyR-ahpC promoter region and in the inhA gene were observed in 14.6% and 2.1% of the isolates, respectively. Genotyping performed on the 96 M. tuberculosis isolates revealed a total of 94 different genotyping patterns. A distinct genotypic pattern was found in 92 isolates, whereas 4 isolates belonged to two clusters with identical genotypes, suggesting that the majority of the isolates were not from an outbreak of a single drug-resistant clone.

This study provides the first molecular characterization of isoniazid- and rifampicin-resistant M. tuberculosis isolates from Myanmar and gives information on the molecular basis for rifampicin and isoniazid drug resistance in M. tuberculosis. The study generates useful information for the development of potential rapid molecular drug susceptibility tests.

Four poultry flocks naturally colonized with Campylobacter were treated with amoxicillin and monitored before, during and up to 4 weeks post-treatment. The numbers of Campylobacter were determined and the isolates speciated and typed by flaA short variable region (SVR) sequence analysis and PFGE. The susceptibility of the isolates to antibiotics, presence of the Cj0299 gene encoding a β-lactamase and β-lactamase production (nitrocefin hydrolysis) were also determined.

Amoxicillin-resistant Campylobacter were isolated from Flock 1 before and during treatment, but Campylobacter were not detected afterwards. Flock 2 was colonized by amoxicillin-susceptible strains throughout sampling. No amoxicillin-resistant isolates arose during or after treatment. Flock 3 contained amoxicillin-susceptible and -resistant types pre-treatment. Resistant isolates were detected during treatment, while antibiotic-susceptible isolates re-emerged at 3 weeks post-treatment. All Campylobacter isolates from Flock 4 were amoxicillin resistant, irrespective of sampling time. All but one of the 82 amoxicillin-resistant (MICs 16 to >128 mg/L) Campylobacter jejuni and Campylobacter coli tested for the presence of Cj0299 carried the gene and all of these produced β-lactamase. Co-amoxiclav remained active against amoxicillin-resistant isolates.

Amoxicillin therapy had little effect on the numbers of amoxicillin-resistant commensal Campylobacter except for one flock where amoxicillin-resistant Campylobacter temporarily dominated. Amoxicillin therapy did not select amoxicillin-resistant isolates from a previous susceptible strain. Co-amoxiclav remained active against amoxicillin-resistant isolates.

In 2007–08, an unexpected increase in fosfomycin resistance in ESBL-producing urinary E. coli was observed. Laboratory records were reviewed and a prospective surveillance study was initiated on all urinary tract infections caused by ESBL-producing, fosfomycin-resistant E. coli. bla_ESBL types, phylogroups, genetic environment and afa/dra operon were determined by PCR and sequencing. Molecular epidemiology was analysed by PFGE and multilocus sequence typing. To elucidate possible mechanisms of fosfomycin resistance, uhpT, glpT, uhpA, ptsI, cyaA and murA genes were analysed. Fosfomycin consumption was determined as recommended by WHO.

From 2004 to 2008, fosfomycin consumption increased by 50%, while fosfomycin resistance in ESBL producers increased from 2.2% to 21.7%. Of 26 isolates studied, 24 produced CTX-M-15 and belonged to the O25b-ST131-phylogroup B2 clonal strain. PFGE revealed two clusters. Cluster I included 18 isolates, 16 of them indistinguishable from strains producing CTX-M-15 previously described in Madrid. The five isolates of Cluster II had the IS26 linked to bla_CTX-M-15 and the afa/dra operon. In Cluster I isolates, no mutations in glpT, uhpT, uhpA, ptsI, cyaA and murA were detected. Cluster II isolates showed a 15 bp deletion (A¹⁶⁹–C¹⁸³) in uhpA.

Fosfomycin resistance in urinary E. coli has increased due to the acquisition of this resistance by a previously circulating CTX-M-15-producing E. coli O25b-ST131-phylogroup B2 strain. This happened during a period when the use of fosfomycin increased by 50%.

Forty-seven KPC-P Kpn isolates were studied. Antibiotic susceptibilities were determined by Vitek 2, Etest or agar dilution. β-Lactamases and PMQR determinants were detected by PCR. For plasmid characterization, transformation, transconjugation, restriction mapping and Southern blot analysis were performed.

Six out of 47 (13%) KPC-P isolates carried aac(6')-Ib-cr. Acquisition of aac(6')-Ib-cr-encoding plasmids increased the MIC of ciprofloxacin by 2-fold. In five of six KPC-P isolates, aac(6')-Ib-cr and bla_KPC-2 were encoded on the same plasmid.

The most prevalent PMQR gene in the studied KPC-P K. pneumoniae isolates is aac(6')-Ib-cr. The co-existence of PMQR genes with KPC on the same plasmid poses a serious epidemiological, clinical and public-health threat.

Cefoxitin-resistant isolates, defined as having an MIC ≥ 32 mg/L, were screened for the ampC classes DHA, FOX, ENT and CIT in a multiplex PCR followed by sequence analysis. Plasmid analysis by restriction fragment length polymorphism (RFLP) and replicon typing was performed on a convenience sample of cefoxitin-resistant Salmonella.

In 2005, 5.3% (181/3442) and in 2006, 3.1% (102/3250) of Salmonella isolates collected from all provinces across Canada displayed cefoxitin resistance. Seventy-one out of 283 (25.1%) were multidrug resistant (MDR), as defined by resistance to at least three different antibiotic classes. The bla_CMY-2 gene was harboured by 96.8% (274/283) of the cefoxitin-resistant isolates. Analysis of CMY-2 plasmids revealed that 19.7% contained genes conferring resistance to multiple antimicrobials. Replicon typing of transformant CMY-2 plasmid DNA revealed the predominance of I1-I and A/C. Of the MDR CMY-2 plasmids, 75% contained replicon type A/C. RFLP patterns of CMY-2 plasmids revealed clusters corresponding to the I1-I and A/C replicon types.

Incompatibility group I1-I is the most prevalent of the Salmonella CMY-2 plasmids, while A/C is associated with MDR CMY-2 plasmids.

SmrA was identified in the S. maltophilia genome based on the detection of ABC transporter conserved motifs and alignment with experimentally proven MDR ABC transporters. The smrA gene was cloned and expressed in the hypersusceptible acrAB mutant Escherichia coli strain SM1411. The resistance to several antimicrobial agents was tested using Stokes' disc diffusion and broth microdilution MIC methods. Norfloxacin accumulation and efflux assays were performed using a fluorescence method with and without the efflux pump inhibitors sodium O-vanadate and reserpine.

Cloning and expression of smrA in Escherichia coli conferred increased resistance to structurally unrelated compounds, including fluoroquinolones, tetracycline, doxorubicin and multiple dyes. Moreover, the expression of smrA in E. coli reduced norfloxacin uptake and enhanced its efflux, features that could be inhibited by the ABC efflux pump inhibitors.

SmrA is a member of the ABC multidrug efflux pump family. The findings warrant further study of the role of this molecule in S. maltophilia isolates, to estimate the potential impact of this system in antimicrobial resistance.

The effects of XF-73 on the growth and survival of S. aureus SH1000 were investigated by viable count and culture absorbance techniques. Inhibition of macromolecular synthesis and disruption of membrane integrity after exposure to XF-73 were examined by radiolabelling experiments, the BacLight fluorescent dye assay and measurement of K⁺ and ATP leakage from the cell. The effect of XF-73 on a staphylococcal coupled transcription–translation system was also investigated.

XF-73 was rapidly bactericidal against S. aureus SH1000 and demonstrated more rapid killing kinetics than all other comparator agents when tested at an equivalent multiple (4x) of the MIC. Exposure of S. aureus to XF-73 for 10 min completely inhibited DNA, RNA and protein synthesis. XF-73 had no effect on transcription and translation in vitro. Cells exposed to XF-73 gave a positive response in the BacLight assay, which detects membrane damage. The drug also caused substantial loss of K⁺ and ATP from the cell, but did not promote bacterial lysis.

XF-73 exhibited rapid membrane-perturbing activity, which is likely to be responsible for inhibition of macromolecular synthesis and the death of staphylococci exposed to the drug.

A strong anion exchange HPLC separation method with UV detection was used to quantify T-705 RTP and GTP levels in Madin–Darby canine kidney cells. Antiviral activity was determined by virus yield reduction assay.

Accumulation of T-705 RTP in uninfected cells increased linearly from 3 to 320 pmol/10⁶ cells in cells exposed to 1–1000 µM extracellular T-705 for 24 h, approaching maximum levels by 9 h. Virus infection did not result in greater T-705 RTP accumulation compared with uninfected cells. Catabolism of T-705 RTP occurred after removal of T-705 from the extracellular medium, with a half-life of decay of 5.6 ± 0.6 h. Based upon these results, short-term incubation of T-705 with H5N1 virus-infected cells was predicted to provide an antiviral benefit. Indeed, 4–8 h 10–100 µM T-705 treatment of cells resulted in virus yield reductions, but less than continuous exposure. A 100-fold higher extracellular concentration of T-705 was required to inhibit intracellular GTP levels compared with ribavirin, which helps explain ribavirin's greater toxicity.

The favourable intracellular metabolic properties of T-705 combined with its reduced cell-inhibitory properties make this compound an attractive candidate for treating human influenza virus infections.

The trypanocidal effect upon trypomastigotes and amastigotes was evaluated by light microscopy through the determination of the IC₅₀ values. The cytotoxicity was determined by the MTT colorimetric assay against mouse cardiomyocytes. For the subcellular localization, transmission electron microscopy and fluorescence approaches were used. The fluorescence intensity within the kinetoplast DNA (kDNA) and nuclear DNA (nDNA) of treated parasites was determined using the Image J program.

Compounds 2, 5 and 7 showed the lowest IC₅₀ values (micromolar range) against intracellular amastigotes and trypomastigotes. In the presence of blood, all the tested ADs exhibited a reduction of their activity. The compounds did not exhibit toxicity to cardiac cells and the highest selectivity index (SI) was achieved by compound 5 with an SI of >137 for trypomastigotes and compound 7 with an SI of >107 for intracellular parasites. The subcellular effects upon bloodstream forms treated with compounds 5 and 7 were mainly on kDNA, leading to its disorganization. The higher accumulation in the kDNA observed for all tested ADs was not directly related to their efficacy.

Our results show the high activity of this new series of ADs against both trypomastigote and amastigote forms, with excellent SIs, especially compound 7, which merits further in vivo evaluation.

A head-to-head comparison of a standard radiolabelled thymidine incorporation assay and the SYBR Green I-based fluorescence assay for determination of in vitro inhibition by pyrvinium and metronidazole was performed.

The 50% inhibitory concentration (IC₅₀) for treatment of E. histolytica with pyrvinium was 4–5 µM for both assays compared with 1–2 µM for metronidazole. For pyrvinium treatment of G. intestinalis, an IC₅₀ of ~12 µM was determined by the radiolabelled thymidine assay alone, with maximum inhibition around 60%. In contrast, the IC₅₀ for metronidazole treatment using this assay was ~2 µM.

Pyrvinium is a potential gut lumen agent for treatment of intestinal amoebiasis, but possibly not for giardiasis. SYBR Green I is an alternative screening method for E. histolytica, but not G. intestinalis.

The inhibitory effect of a library of small chemical compounds was examined in vitro using the conditional A. nidulans PKC strain and a panel of pathogenic fungal isolates. Microscopy was used to assess alterations to fungal ultrastructure following treatment.

Three ‘hit’ compounds affecting cell wall integrity were identified from a screen of 5000 small chemical compounds. The most potent compound, CW-11, was further characterized and shown to specifically affect cell wall integrity. In clinical isolates of Aspergillus fumigatus, CW-11 induces morphological changes characteristic of damage to the cell wall, including wall thickening and rupturing. Analysis of the susceptibility of A. fumigatus and A. nidulans cell wall and signalling pathway mutants to CW-11 suggests that its mode of action differs from that of the antifungals caspofungin and voriconazole.

This work demonstrates the feasibility of using a conditional Aspergillus mutant to conduct a small-molecule library screen to identify novel ‘hit’ compounds affecting cell wall integrity.

The inhibitors cyanide and salicylhydroxamic acid (SHAM) were each combined with azoles to examine the effect of the combinations on C. albicans. C. albicans strains deleted for the alternative oxidase (Aox) were also examined for susceptibility to azoles and for the generation of intracellular reactive oxygen species (ROS). A chequerboard microdilution assay was performed on several C. albicans clinical strains including azole-resistant isolates to explore the combined effect of fluconazole and inhibitors of Aox.

The induction of the alternative respiratory pathway by cyanide decreased susceptibility to azoles, while the inhibition of alternative respiration by SHAM increased azole susceptibility. It was found that ROS production was increased in the absence of Aox in C. albicans upon treatment by antifungals such as miconazole and benomyl. The combination of fluconazole with SHAM resulted in a synergistic effect on the killing of C. albicans clinical isolates.

These results demonstrate that the induction of the alternative respiratory pathway confers reduced susceptibility to antifungal azoles, potentially through a mechanism that involves decreased intracellular ROS production during exposure to antifungal agents.

Spontaneous mutants were generated by isoniazid overdose and limited broth dilution, while for genetically modified mutants sense–antisense DNA technology was used. Southern hybridization and immunoprecipitation were both used to identify the InhA homologue in M. aurum. The latter method was further used to compare the level of InhA expression in M. aurum with that in corresponding mutants. Isoniazid/ethionamide susceptibility modulation was examined in vitro and ex vivo using a resazurin assay as well as by cfu counting. In addition, circular dichroism and the bacterial two-hybrid system were exploited to investigate the interaction of InhA with other enzymes of the FAS-II pathway.

A Mycobacterium tuberculosis InhA homologue was detected in M. aurum. Susceptibility to isoniazid/ethionamide was significantly altered in genetically modified mutants and simultaneously InhA was overexpressed in both spontaneous and genetically modified mutants. InhA interacts with other FAS-II enzymes of M. aurum in vivo.

Close resemblance of isoniazid/ethionamide action on InhA between M. tuberculosis and M. aurum further supports the use of fast-growing and intracellularly surviving drug-resistant M. aurum to substitute for highly virulent, extremely slow-growing M. tuberculosis strains in the early stage of antituberculosis inhibitor screening.

In vitro PDI studies employing Mycobacterium smegmatis as a surrogate for Mycobacterium tuberculosis were performed examining photosensitizer (PS) type, concentration and light dose. M. smegmatis was grown to a concentration of 10⁸ colony forming units (cfu) per mL, resuspended in PBS–0.5% Tween-80-containing buffer, incubated with the PS for 5 min and subsequently illuminated with white light (400–700 nm) at a fluence rate of 60 mW/cm² for 1, 5, 15 or 30 min (equivalent to 3.4, 18, 54 or 108 J/cm²). The percentage survival was determined by the ratio of the colony count from illuminated and non-illuminated control cell suspensions. The PSs examined were 5,10,15,20-tetrakis(1-methyl-4-pyridinyl)porphyrin tetratosylate (TMPyP), 5,10,15,20-tetrakis(4-N,N,N-trimethylanilinium)porphyrin tetrachloride (TNMAP), methylene blue (MB), 5,10,15,20-tetrakis(4-sulphonatophenyl)porphyrin (TSPP), 5,10,15,20-tetrakis(4-carboxyphenyl)porphyrin-Pd(II) (TCPP-Pd) and phthalocyanine tetrasulphonic acid (PhCS).

Our best results demonstrate that PDI of M. smegmatis can achieve a noteworthy 5–6 log unit reduction in cfu (99.999% + viable cell eradication) when cationic PSs are employed in the nanomolar concentration range. Anionic PSs did not effectively mediate PDI of mycobacteria due to their inability to associate with the negatively charged mycobacterial cell membrane.

PDI of M. smegmatis was found to be highly efficient in reducing the number of viable cells in vitro when cationic PSs were employed.

We determined the MICs of rifampicin, isoniazid and ethambutol for M. tuberculosis using a Middlebrook 7H10 dilution method for 90 consecutive clinical isolates, 8 resistant strains and 16 isolates from the WHO proficiency test panel. M. tuberculosis H37Rv was used for quality control and susceptibility results using 7H10 were compared with the results obtained with BACTEC460.

The agreement with BACTEC460 was very high for isoniazid (99.1%) and rifampicin (99.1%) but lower for ethambutol (94.7%). Intra- and inter-assay variation was below one MIC dilution. The MIC distributions for isoniazid and rifampicin provided a clear separation between susceptible and resistant strains. Regarding ethambutol, the current breakpoint for 7H10 (5 mg/L) is close to the wild-type and all strains (n = 6) showing a disagreement between BACTEC460 and 7H10 were distributed very close to the breakpoint (MIC 4–8 mg/L). This problematic relation was confirmed by investigating isolates from the WHO panel with an agreement <95% (64%–88% among 26 laboratories, n = 4) for which the MICs were 4–8 mg/L.

Utilizing the wild-type MIC distribution was found to be as useful in M. tuberculosis as in other bacteria when setting clinical breakpoints. We suggest that the present clinical breakpoints should be re-evaluated, taking into account wild-type MIC distributions and available pharmacokinetic data.

Results were generated by three laboratories: the Instituto Adolfo Lutz (IAL) Mycobacteria Reference Laboratory and two IAL Regional Laboratories in Santo André and Sorocaba, São Paulo State, Brazil. One hundred and twenty M. tuberculosis strains were simultaneously tested using NRA and the proportion method (PM), while 117 strains were tested using both NRA and BACTEC MGIT 960 (M960).

Repeatability analysis of NRA results showed rates of 100% for isoniazid and ethambutol and 97% for streptomycin and rifampicin susceptibility detection, representing substantial agreement. McNemar testing of the data also indicates that NRA and PM, as well as NRA and M960, do not differ significantly. On average, NRA results were available after 10 days.

The data demonstrate that NRA is reliable for susceptibility testing of isoniazid and rifampicin, the two most important drugs for the treatment of tuberculosis. In addition, the reduction in the time necessary to obtain susceptibility results is of fundamental importance.

MIC and MBC determinations were performed according to CLSI guidelines. Linezolid-resistant bacterial strains were generated in-house; inoculum effect, pH effect and kill kinetics experiments were performed as per standard protocols.

OCID0050 demonstrated better inhibitory potency against many of the tested clinically significant strains by generally showing 2–4-fold lower MICs than linezolid. In addition, it has higher inhibitory activity against linezolid-resistant strains.

The bacteriostatic and bactericidal activities of ceftazidime, ciprofloxacin, meropenem, minocycline, tobramycin and trimethoprim/sulfamethoxazole were determined against 38 B. cepacia complex strains. MICs and minimal biofilm inhibitory concentrations (MBICs) were determined using a traditional broth microdilution method and a novel resazurin-based viability staining, respectively. The bactericidal effects of the investigated antibiotics (using antibiotic concentrations corresponding to 10 x MIC; except for tobramycin, for which a final concentration of 4 x MIC was tested) on stationary phase planktonic cultures and on 24-h-old biofilms were evaluated using conventional plate count methods.

Our results confirm the innate resistance of B. cepacia complex organisms to six first-line antibiotics used to treat infected cystic fibrosis patients. All antibiotics showed similar bacteriostatic activities against exponentially growing B. cepacia complex planktonic cells and freshly adhered sessile cells (4 h). In addition, most of the antibiotics showed similar bactericidal effects on stationary phase planktonic cultures and on young and older biofilms.

Despite the general assumption that sessile cells show a decreased susceptibility to antibiotics, our data indicate similar bacteriostatic and bactericidal activity of six selected antibiotics against planktonic and sessile B. cepacia complex bacteria.

Antibacterial activity of squalamine and two ASDs (1 and 2) was evaluated against 135 MDR Gram-negative and Gram-positive bacteria using the broth microdilution method for MIC determination.

For Gram-negative bacteria, MICs ranged from 2 to 128 mg/L. Resistance to colistin and mucoidity were significantly associated with higher MICs of squalamine and ASDs 1 and 2. Tested compounds were active against various Gram-positive bacteria with MIC values varying from 0.5 to 8 mg/L, with the exception of two capsulated isolates of Streptococcus pneumoniae demonstrating MICs of 32 mg/L.

In this study, we present new findings concerning the antibacterial potential of ASDs against MDR bacteria. Colistin-resistant, mucoid and capsulated bacteria were found to exhibit decreased susceptibility to ASDs indicating that these compounds might share some mechanistic aspects with polymyxins towards Gram-negative bacteria. However, ASDs were remarkably active against Gram-positive species suggesting different mechanisms of action towards Gram-positive and Gram-negative bacteria. As tested ASDs exhibited elevated MICs in some cases, we believe that these compounds may be developed to be locally administrated as aerosols rather than via systemic administration routes. Further work is warranted to evaluate their in vivo efficacy in aerosol formulations using a lung-infected animal model.

S. aureus ATCC 43300 was exposed to once-daily dosing of daptomycin at subtherapeutic ratios of 24 h area under the curve (AUC₂₄) to the MIC (32 and 64 h). To provide an integral presentation of the time course of mutants grown on agar plates containing 2x and 4x the MIC of daptomycin, areas under the bacterial mutant kinetic curves (AUBC_Ms) were calculated.

Daptomycin-resistant S. aureus mutants were enriched gradually over the entire treatment duration, with systematic increases in AUBC_M and concomitant decreases in susceptibility. AUBC_M analyses were also applied to resistance data reported from other studies with S. aureus exposed to daptomycin and garenoxacin over a wide range of AUC₂₄/MIC ratios. Although the maximal AUBC_Ms were greater with longer than with shorter exposures, the treatment or observation durations did not influence the predicted anti-mutant AUC₂₄/MIC ratios.

These findings suggest that the duration of in vitro simulated antibiotic exposure is important for estimates of the maximal enrichment of resistant mutants but not for the prediction of the anti-mutant AUC₂₄/MIC ratio.

Time–kill curves were performed in Mueller–Hinton broth (MHB) and in pooled human CSF using fosfomycin concentrations ranging from 0.25x to 8x MIC for a clinical Staphylococcus aureus isolate. To estimate the activity of fosfomycin at the target site, the concentration–time curve measured in CSF of a patient at steady state was simulated in vitro in human CSF using two S. aureus isolates.

In CSF a higher fosfomycin concentration (8x MIC) was required to achieve sustained bacterial killing than in MHB (1x MIC). In vitro simulation of the pharmacokinetic profile measured in CSF of the selected patient showed initial killing, but terminal re-growth of both test strains.

The antibacterial activity of fosfomycin is lower in CSF than in MHB, and drug concentrations slightly exceeding the MIC may not be sufficient to achieve bactericidal effects in the CNS.

Nine E. coli isolates were studied, including three ESBL-producing isolates, three AmpC-producing isolates and three isolates demonstrating CRS (ertapenem MIC ≥ 0.12 mg/L). The pharmacodynamic model was inoculated with organisms at 1 x 10⁶ cfu/mL and tigecycline dosed once every 24 h to simulate the fC_max (free peak serum concentration) and t_1/2 (serum half-life) obtained after standard dosing of 100 mg intravenously every 24 h (fC_max, 0.15 mg/L; t_1/2, 42 h). Samples were collected over 48 h.

For isolates with a tigecycline fAUC₂₄/MIC of 2.0 (tigecycline MIC = 0.5 mg/L), tigecycline demonstrated bacteriostatic activity with <1 log₁₀ reduction in bacterial growth compared with the initial inoculum at 12, 24 and 48 h. Against the two isolates for which the tigecycline fAUC₂₄/MIC was 4.0 (tigecycline MIC = 0.25 mg/L), tigecycline demonstrated bacteriostatic activity with ~1.5 log₁₀ reduction in bacterial growth compared with the initial inoculum at 12, 24 and 48 h. Against the two isolates for which the tigecycline fAUC₂₄/MIC was 8.0 (tigecycline MIC = 0.12 mg/L), tigecycline demonstrated bacteriostatic activity with ~2.0 log₁₀ reduction in bacterial growth compared with the initial inoculum at 12, 24 and 48 h.

Tigecycline demonstrated ~1–2 log₁₀ killing against E. coli harbouring ESBLs, AmpC β-lactamases and CRS when simulating clinically achievable serum concentrations, and represents a potential therapy for infections caused by these isolates.

Clinical isolates of CF Pseudomonas aeruginosa, Staphylococcus aureus, Haemophilus influenzae, Stenotrophomonas maltophilia, Burkholderia cepacia complex, Escherichia coli and Klebsiellia spp. were evaluated by MIC, MBC, post-antibiotic effect (PAE), synergy, time–kill, a rat pneumonia model and spontaneous mutation frequency (SMF).

FTI showed high activity against E. coli, H. influenzae, S. aureus and Klebsiella spp. For the S. aureus strains, 75% of which were methicillin resistant (MRSA), FTI had a lower MIC₉₀ than tobramycin. For P. aeruginosa, FTI had a lower MIC₉₀ than fosfomycin, but tobramycin was more active than either. Synergy studies showed no antagonism between fosfomycin and tobramycin, and 93% of the isolates demonstrated no interaction. FTI was rapidly bactericidal and exhibited concentration-dependent killing in time–kill studies. In the rat pneumonia model, FTI and tobramycin demonstrated bactericidal killing of P. aeruginosa; both were more active than fosfomycin alone. The SMF for S. aureus resistance to FTI was 2–4 log₁₀ lower than that for tobramycin and 2–7 log₁₀ lower than that for fosfomycin. For P. aeruginosa, the FTI SMF was 2–3 log₁₀ lower than that for fosfomycin and 1–2 log₁₀ lower than that for tobramycin.

FTI is a broad-spectrum antibiotic combination with high activity in vitro and in vivo. These data suggest FTI could be a potential treatment for respiratory infections caused by Gram-positive and Gram-negative aerobic bacteria.

Single doses of tigecycline 50 and 25 mg/kg were administered to neutropenic ICR mice with or without the presence of an Acinetobacter baumannii lung infection. Serum samples were gathered at 0.5–24 h after tigecycline administration; bronchoalveolar lavage was conducted at 1, 1.5, 4 and 8 h. Tigecycline concentrations were determined by HPLC. Comparisons of ELF penetration in infected and uninfected lungs were based on the ratios of the AUC_0–8 in ELF and the free AUC_0–8 in serum. AUCs were calculated by the trapezoidal rule.

The group without pulmonary infection displayed an ELF penetration ratio of 8.1 and 6.2 for the 50 and 25 mg/kg doses, respectively. The respective penetration ratios in the infected lungs were 23.3 and 12.9.

While tigecycline exhibits excellent ELF penetration in healthy and infected murine lungs, the presence of infection greatly enhances penetration. Moreover, increased systemic exposures of tigecycline result in greater ELF penetration, regardless of infection status. When future tigecycline clinical trials for the treatment of pneumonia are considered, escalated doses may reap greater than expected benefits towards achieving adequate pharmacodynamic indexes within the lungs.

Plasma drug concentrations (48 samples from 10 adult patients with haematological malignancies) were determined chromatographically, and used for population PK modelling and Monte Carlo simulation to evaluate the ability of regimens (1 h infusions) to attain genus-dependent PK–PD targets, namely fungistatic and fungicidal targets against Candida spp. [area under the plasma unbound (1%) drug concentration–time curve over 24 h/MIC (fAUC/MIC) = 10 and 20] and an effective concentration target against Aspergillus spp. (plasma unbound drug concentration = 0.05 mg/L).

Mean (variance) values for two-compartment PK model parameters were: clearance, 0.762 L/h (15.4%); volume of central compartment, 9.25 L (24.6%); intercompartmental clearance, 7.02 L/h (fixed); and volume of peripheral compartment, 8.86 L (71.8%). The Monte Carlo simulation demonstrated that 50 mg once daily and 100 mg once daily for the fungistatic and fungicidal targets achieved a >95% probability of target attainment against Candida spp. To achieve such probability against Aspergillus spp., 250 mg once daily or 100 mg twice daily was required.

These results rationalize the approved micafungin dosages for Candida infections (50 mg once daily for prophylaxis and 100–150 mg once daily for treatment), and on the basis of these results we propose a PK–PD-based dosing strategy for Aspergillus infections. A regimen of 200–250 mg/day should be initiated to ensure the likelihood of a favourable outcome. The regimen can be optimized by decreasing the dosing interval.

From 1 July 2008 to 1 March 2009, 34 consecutive HIV-infected patients with detectable viral load during the last 6 months began an 8 day exposure to maraviroc (MCT group); six HIV-infected patients without antiretroviral therapy received no treatment (control group). Plasma viral load was evaluated on days 0, 2, 5 and 8. Baseline Trofile^® was performed in MCT group patients. The maraviroc clinical test (MCT) was considered positive if viral load was undetectable (<40 HIV-RNA copies/mL) or a reduction ≥1 log₁₀ HIV-RNA copies/mL was achieved after 8 days of maraviroc exposure.

Global concordance between MCT and Trofile^® was 93.5%. In patients with R5 virus according to Trofile^®, MCT was positive in 19/20 (concordance 95%); in patients with dual/mixed virus, MCT was negative in 10/11 (concordance 90.9%). An additional phenotypic tropism assay was performed in patients with discordance between MCT and Trofile^®, being concordant with MCT in both cases. Three patients showed a non-reportable Trofile^® result, and all of them achieved undetectability after MCT.

A clinical approach like short-term maraviroc exposure could be an additional resource to genetic and phenotypic HIV tropism assays. This clinical approach shows high concordance with Trofile^®, and could allow patients with non-reportable results by Trofile^® to benefit from maraviroc therapy.

Thirty patients qualified for inclusion and were reviewed. Patients were divided into two groups: one group received trimethoprim–sulfamethoxazole plus prednisolone (18 patients); and the other group received trimethoprim–sulfamethoxazole alone (12 patients).

The two groups were comparable at baseline, except for the severity of the P. jiroveci pneumonia. Hyperkalaemia developed in seven patients: all in the prednisolone and trimethoprim–sulfamethoxazole group. The greater incidence of hyperkalaemia in this group is surprising and was counter to our expectation.

Although it is possible that there is an unexplained interaction between trimethoprim and prednisolone, we postulate that our observation is a result of the catabolic effect of prednisolone. The patients treated with trimethoprim–sulfamethoxazole plus prednisolone appear to be more likely to develop hyperkalaemia than patients treated with trimethoprim–sulfamethoxazole alone.

A questionnaire survey on antibiotic stewardship factors was completed by 170 hospitals from 32 European countries. Data on committees, antibiotic formularies and policies addressing empirical therapy and prophylaxis were collated. Data on antibiotic use, expressed as defined daily doses per 100 occupied bed-days (DDD/100 BD), were provided by 139 hospitals from 30 countries, and 124 hospitals provided both data sets. Six key indicator stewardship variables were analysed by European region, case mix and antibiotic consumption.

Hospitals from Northern and Western Europe were more likely to convene antibiotic committees or drugs and therapeutic committees compared with those from Southern and South-Eastern Europe (P < 0.001). One-fifth of hospitals had neither an antibiotic committee nor a policy. Hospital antibiotic policies commonly included recommendations on individual drugs, drug choices, dosage, duration and route but were less likely to contain information on side effects and cost. There were no significant differences by median total (J01) antibiotic consumption, although other antibiotic subgroups differed by stewardship indicators.

Policies and practices relating to antibiotic stewardship varied considerably across European hospitals. These data provide a benchmark for newer European strategies tackling antibiotic resistance. More work is required to achieve harmonization of recommended practice, particularly in hospitals from Southern Europe.