This article appears in the following Journal of Antimicrobial Chemotherapy issue: The British Society for Antimicrobial Chemotherapy Resistance Surveillance Project 1999/2000-2006/7 [View the issue table of contents]
Articles |
Non-susceptibility trends among Enterobacteriaceae from bacteraemias in the UK and Ireland, 2001–06
1 Health Protection Agency Centre for Infections, 61 Colindale Avenue, London NW9 5EQ, UK 2 Department of Medical Microbiology, Southmead Hospital, Bristol BS10 5NB, UK
* Correspondence address. Antibiotic Resistance Monitoring and Reference Laboratory, HPA Centre for Infections, 61 Colindale Avenue, London NW9 5EQ, UK. Tel: +44-20-8327-7223; Fax: +44-20-8327-6264; E-mail: david.livermore{at}hpa.org.uk
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Background: Enterobacteriaceae are common agents of bacteraemia, with Escherichia coli accounting for 20% of the cases. Reflecting this importance, members of the family constitute 4 of the 12 collection groups in the British Society for Antimicrobial Chemotherapy (BSAC) Bacteraemia Surveillance Programme.
Methods: MICs from the BSAC surveillance programme were reviewed, along with bacteraemia reports received by the Health Protection Agency (HPA) via its CoSurv/LabBase system. Isolates with unusual resistances were subjected to molecular analysis.
Results: The BSAC and HPA systems both revealed dramatically increasing resistance to cephalosporins, ciprofloxacin and gentamicin among E. coli and Klebsiella spp., with cephalosporin resistance largely contingent on the spread of CTX-M extended-spectrum β-lactamases (ESBLs); fluoroquinolone resistance also increased among Proteus mirabilis and ESBL-negative E. coli. Carbapenem resistance remained extremely rare, but two Enterobacter spp., from the same patient in different years, had KPC carbapenemases, while a few isolates had carbapenem resistance contingent upon combinations of β-lactamase and impermeability, and ertapenem MICs for AmpC-derepressed Enterobacter spp. rose over time. Three new agents—ceftobiprole, doripenem and tigecycline—were tested. Ceftobiprole was broadly active, except against ESBL producers and Klebsiella oxytoca hyperproducing K1 enzyme, and was variable against AmpC-derepressed Enterobacter spp. and against Proteus vulgaris. Doripenem was more potent than imipenem. Tigecycline was almost universally active against E. coli, but low-level non-susceptibility (MIC 2 mg/L) was frequent among Klebsiella spp.
Conclusions: E. coli and Klebsiella spp. showed dramatic shifts, with sharply rising non-susceptibility to cephalosporins, ciprofloxacin and gentamicin. The rise in cephalosporin resistance reflected dissemination of CTX-M ESBLs. Carbapenems remain broadly active and new agents offer potential.
Keywords: BSAC , HPA , surveillance
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Introduction |
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Escherichia coli accounts for around one-fifth of all bacteraemias in the UK, a proportion only rivalled by Staphylococcus aureus and at least double that for any other pathogen. Other important Enterobacteriaceae in bloodstream infections include Klebsiella spp., causing 4.7% to 6.0% of bacteraemias; Proteeae, causing 2.6% to 4.1% and Enterobacter spp. causing 2.6% to 3.3%.1,2 These proportions have been stable over the past 15 years but there is evidence, from reports to the Health Protection Agency (HPA) and the European Antimicrobial Resistance Surveillance System (http://www.earss.rivm.nl), of rising resistance, particularly in E. coli and to fluoroquinolones and cephalosporins.3 The rise in cephalosporin resistance may be attributable to the spread of extended-spectrum β-lactamases (ESBLs), particularly CTX-M types.4
The British Society for Antimicrobial Chemotherapy (BSAC) Bacteraemia Resistance Surveillance Programme is structured to reflect the importance of Enterobacteriaceae, which comprise 4 of the 12 collection groups (E. coli, Klebsiella spp., Enterobacter spp. and Proteeae), while Serratia and Citrobacter spp. are well represented in the ‘other Gram-negative’ group. This analysis reviews the BSAC results from 2001 to 2006 along with Hospital Trusts’ bacteraemia data, as reported to the HPA under its LabBase/CoSurv system which covers England, Wales and Northern Ireland but not Scotland nor the Irish Republic.5 Over 90% of Hospital Trusts report to the HPA, and the system captures data for around 70% of all bacteraemias in its geographic ambit. These reports are complementary to the BSAC surveillance, with a much larger sample, but with less standardization of methods or antimicrobial panels, and without the facility for molecular investigation of unusual isolates.
The present paper also reviews the activity of the newer broad-spectrum antibiotics included in the BSAC surveillance, specifically ceftobiprole, doripenem and tigecycline.
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Materials and methods |
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The collection, identification and susceptibility testing protocols of the BSAC surveillance are described elsewhere in this supplement.6 Isolates inferred to have ESBLs were investigated for blaCTX-M by multiplex PCR;7 those non-susceptible to carbapenems were screened for blaKPC genes by specific PCR8 and for metallo-β- lactamase genes either by specific PCR9 or, from 2005, by multiplex PCR.10 The HPA LabBase/CoSurv system for the collection of laboratories’ voluntarily submitted data on bacteraemia isolates has been described previously.5
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Results |
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Pathogens, patients and sources
From 2001 to 2006, the BSAC Bacteraemia Resistance Surveillance Programme collected 1480 E. coli, 1419 Klebsiella spp., 1220 Enterobacter spp., 1291 Proteeae and, among the ‘other Gram-negative bacteria’, 437 Serratia spp., 170 Citrobacter spp., 23 Raoultella terrigena, 19 Pantoea and 9 isolates of enterobacterial genera with fewer than five representatives each. We undertook analysis wherever there were more than 100 isolates of a species or genus, but excluded Citrobacter spp. on the ground that the collection was evenly split between Citrobacter freundii (n = 88) and Citrobacter koseri (diversus) (n = 61)—species that differ in their β-lactamase and resistance profiles.11
There were differences in sources between the E. coli and Proteeae isolates, on the one hand, and the Klebsiella, Enterobacter and Serratia groups on the other hand (Table 1). Specifically: (i) E. coli and Proteeae were more often from the oldest patients, with a mode age 
80 years, compared with 70–79 years for Klebsiella, Enterobacter and Serratia; (ii) outpatients and those hospitalized for <48 h accounted for just over half of the E. coli and Proteeae bacteraemias but <40% of the Enterobacter, Klebsiella and Serratia groups; (iii) around 17% of the E. coli and Proteeae were from patients presenting at the accident and emergency department, compared with <10% for the other species, where intensive care, haematology and oncology were frequent sources; and (iv) lastly, >40% of E. coli and Proteeae bacteraemias had a genitourinary origin, compared with 13% to 23% for Klebsiella, Enterobacter and Serratia spp., where ‘unknown’ was the most-cited source, >34% for each genus.
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Resistance trends
Many prevalence rates for non-susceptibility in the BSAC data set showed sizeable year-to-year fluctuation, probably reflecting the small sample sizes (
250 isolates per group per year) and the fact that several breakpoints cut the MIC distributions for widespread resistance types [European Committee on Antimicrobial Susceptibility Testing (EUCAST) breakpoints aim not to split MIC distributions for wild-type populations, but may do so for those with common modes of acquired resistance]. Consequently, we only accepted trends as convincing when: (i) they were apparent for both the proportion non-susceptible and the proportion resistant, and (ii) when they were supported by trends in the HPA CoSurv/LabBase data, with its much larger sample size.5 We believe that this strategy is robust, but note two limitations: (i) reporting to the HPA system is as ‘susceptible’ and ‘non-susceptible’, with no distinction of intermediate and resistant; and (ii) reported categorizations are based on the definitions of non-susceptibility used by the source laboratories at the time of reporting. In most cases, these were the then-current BSAC values, not the EUCAST-harmonized breakpoint values applied here to the BSAC surveillance data; however, a sizeable, if diminishing, minority of laboratories used Stokes’ method in the earlier years, while a small but growing minority use automated systems, some of them calibrated, by default or intention, to CLSI criteria.
E. coli is the commonest agent of bacteraemia and, among all those considered in the BSAC surveillance, is the one showing the most striking changes in resistance. The BSAC data indicated significant up-trends in non-susceptibility and resistance to ceftazidime, cefotaxime, ciprofloxacin and gentamicin (P < 0.0001, except gentamicin, P = 0.0035), with corresponding shifts also evident in the LabBase/CoSurv reports to the HPA (Table 2). Significant up-trends in non-susceptibility, in both the BSAC and HPA datasets, to cefuroxime and piperacillin/tazobactam, which have no intermediate category, also were seen. Increasing non-susceptibility to amoxicillin/clavulanate too was apparent in both datasets, and although this latter trend was not confirmed by rising resistance in the BSAC data, we accept it as significant because (i) it parallels the trends for piperacillin/tazobactam, and (ii) frank resistance to amoxicillin/clavulanate (MIC > 16 + 8 mg/L) is difficult to achieve when many E. coli isolates are inhibited by clavulanate alone at 8 mg/L. The BSAC data indicated rising non-susceptibility to cefoxitin (P < 0.001) but results for this drug are rarely reported under LabBase surveillance, precluding confirmation, and we remain sceptical, since the single cefoxitin breakpoint lies at the upper edge of the normal MIC distribution, exacerbating the risk of categorization errors. Both surveillance systems revealed a high proportion of E. coli non-susceptible to amoxicillin: the HPA LabBase data suggested that this fraction was still rising (P < 0.0001) but this was not supported by the BSAC data.
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Non-susceptibility to carbapenems and tigecycline was extremely rare in E. coli. An apparently falling non-susceptibility trend for imipenem in the LabBase data set is certainly spurious and perhaps reflects an improvement in the quality control of laboratories' susceptibility testing. Nevertheless, two imipenem non-susceptible (MIC 8 and 16 mg/L) E. coli were collected by the BSAC surveillance in 2006 (Table 3), with none found previously. They were from separate centres. One, EO1441, had unstable imipenem resistance, lost on subculture, contingent on a combination of a CTX-M ESBL plus impermeability; the other, EO1452, had narrow-spectrum carbapenem resistance and remains under study. Four tigecycline non-susceptible E. coli were collected, scattered through the surveillance, all with ‘intermediate’ MICs of 2 mg/L.
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From 2002, the nature of cephalosporin resistance was investigated for the BSAC collection. The number of isolates with non-CTX-M ESBLs fluctuated from two to six per annum, without trend, while the number with CTX-M ESBLs rose from 2 in 2002 to 27 in 2006, exceeding 10% of all E. coli collected in the latter year (Table 4). Resistances to amoxicillin/clavulanate, ceftazidime, cefotaxime, ciprofloxacin, gentamicin, cefoxitin, cefuroxime and piperacillin/tazobactam were strongly associated with ESBL production (P < 0.0001) and so the rises in non-susceptibility to these drugs were strongly associated with the proliferation of ESBL producers. In addition, there was also an independent trend of increasing ciprofloxacin non-susceptibility and resistance (P < 0.001) among isolates lacking ESBLs.
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Logistic regression analysis of the BSAC data set, adjusted for inter-centre variation,12 implicated hospitalization for >48 h before acquisition of infection and male sex as independent predictors of increased resistance or non-susceptibility in E. coli; hospital-acquired isolates were more often ESBL producers, but an association of male sex and resistance was only partly ESBL-related.
Of the two new antibiotics tested, doripenem had a unimodal MIC distribution, centred at 0.015 mg/L, and all MIC values lay between 0.008 and 0.06 mg/L, except for the two imipenem-resistant isolates detailed in Table 3. Ceftobiprole had MICs of 0.06–0.5 mg/L for isolates without ESBLs but values for ESBL producers were 0.5 to 128 mg/L, with 94% above 4 mg/L.
Among the 1419 Klebsiella spp. collected in the BSAC surveillance, 1059 were Klebsiella pneumoniae and 325 Klebsiella oxytoca; the remaining 35 were not identified to species level. A further 23 isolates collected in 2004–06 initially were identified as Klebsiella terrigena but were re-assigned to the ‘other Gram-negative group’ following reclassification of this species into the genus Raoultella; prior to 2004, any R. terrigena isolates were not identified to species level and so remain included as Klebsiella spp. Susceptibility results for the pooled species are shown in Table 2 but K. pneumoniae more often produced ESBLs than all other Klebsiella species combined (P = 0.0004).
ESBL production, gentamicin non-susceptibility, gentamicin resistance and ceftazidime resistance all showed highly significant rises over time among Klebsiella spp. (P < 0.0001) in the BSAC collection. There was weaker evidence (P < 0.01) for rises in ceftazidime and cefotaxime non-susceptibility and ciprofloxacin resistance, and also for a rise in non-susceptibility to amoxicillin/clavulanate. All these observations were supported by the trends in reports to the HPA (P < 0.005 in all cases), which additionally indicated a rise in non-susceptibility to cefuroxime. Neither the BSAC nor the HPA surveillance programmes suggested any significant change in the prevalence of non-susceptibility to piperacillin/tazobactam and both confirmed imipenem as retaining near universal activity throughout. An apparent fall in tigecycline non-susceptibility in the BSAC collection was probably an artefact, reflecting a high rate in 2002, when the risk of the compound being oxidatively deactivated during susceptibility testing was not fully appreciated.13
Multiplex PCR revealed that the proportion of ESBL-producing Klebsiella spp. with CTX-M enzymes increased from 1/11 in 2002 to 24/31 in 2006 (Table 2) (P < 0.0001). Based on the phenotype (high-level resistance to piperacillin/tazobactam and cefuroxime coupled with susceptibility to ceftazidime and a borderline result for cefotaxime),11 16/325 K. oxytoca isolates hyperproduced K1 enzyme. These numbers were too small for trend analysis but, clearly, there was no major increase.
A single imipenem non-susceptible K. pneumoniae (MIC 4 mg/L) was collected in 2001 but was not investigated, since the MIC was below the then breakpoint, which was later reduced from 4 to 2 mg/L. Its phenotype was otherwise unexceptional (Table 3), and we view the result as of doubtful significance. The collection included eight Klebsiella isolates that were non-susceptible to ertapenem, five with MICs of 1 mg/L and three fully resistant with MICs of 2–8 mg/L. All were collected in 2004 or later. Four had ESBLs, three of them Group 1 CTX-M types; the fourth (with an ertapenem MIC of 8 mg/L) had a non-CTX-M ESBL. Another was a K. oxytoca isolate hyperproducing K1 enzyme while the remaining three (all with ertapenem MICs of 1 mg/L) were susceptible to cefotaxime and ceftazidime. In no case was the imipenem MIC > 1 mg/L, and all those tested were susceptible to meropenem and doripenem at 0.25 mg/L. Such phenotypes suggest impermeability, particularly when an ESBL was also present,14 but details were not investigated. Tigecycline was less active against Klebsiella spp. than against E. coli, with 6% to 11% of the isolates non-susceptible (MIC > 1 mg/L) in each of the years 2003–2006, and 0–4% resistant (MIC > 2 mg/L).
Ceftobiprole had MICs of 0.06–0.5 mg/L for over 90% of cefotaxime-susceptible Klebsiella spp., whereas MICs were >4 mg/L for 96/99 ESBL producers. A distinction from other oxyimino-cephalosporins except cefuroxime and ceftriaxone11 was that ceftobiprole also lost activity against most (15/16) K. oxytoca isolates inferred to hyperproduce K1 enzyme, with MICs of 32–128 mg/L.
Logistic regression analysis indicated hospital acquisition and species (K. pneumoniae versus K. oxytoca versus other) as independent predictors of non-susceptibility or resistance to cephalosporins and gentamicin; the effect of hospital acquisition arose largely from an increased proportion of ESBL producers.
The 1220 Enterobacter spp. isolates comprised 957 Enterobacter cloacae, 122 Enterobacter aerogenes and 141 not identified to species level: 35 of the latter were identified as Enterobacter sakazakii by API20E (bioMerieux, Marcy l'Etoile, France) but we are sceptical of this result, as many were inducible or derepressed for AmpC, whereas true E. sakazakii have only basal AmpC expression.15 There was a variation in susceptibility by species, with E. cloacae generally the least susceptible to ciprofloxacin (P = 0.00016), ertapenem (P = 0.0019) and tigecycline (P = 0.0044).
Trend analysis for the BSAC data, with all Enterobacter species pooled, indicated a rising non-susceptibility (P = 0.0012) and frank resistance (P = <0.00001) for ertapenem, as discussed below, and also a falling trend, almost certainly spurious, in non-susceptibility to tigecycline, which became insignificant if the 2002 data were discounted. Non-susceptibility rates for many other antimicrobials were high but lacked a temporal trend in the BSAC data set. Reports to the HPA broadly supported this lack of trends, but did indicate progressive increases in the prevalence of cefotaxime and ceftazidime non-susceptibility, from around 33% in 2001 to 41% to 42% in 2006 (P < 0.0001).
According to the year of isolation, between one-seventh and one-quarter of the oxyimino-cephalosporin-resistant Enterobacter spp. proved to have ESBLs rather than derepressed AmpC (which is widely perceived as the ‘classical’ route to resistance in Enterobacter spp.). Unlike among E. coli and Klebsiella spp., non-CTX-M ESBLs remained predominant. Only one ESBL producer was an E. aerogenes, whereas the remainder were E. cloacae (n = 90) or were not identified to species level (n = 20).
The MIC distribution of ertapenem, unlike that of imipenem, had a bimodal character for Enterobacter spp. (compare Figure 1a and b). Based on the 2004–06 results, when mechanisms were investigated, the high-MIC peak largely comprised AmpC-derepressed organisms and its value tended to increase over time, from 0.25 mg/L in 2002 to 1–2 mg/L in 2006 and thus from susceptible, through intermediate, to resistant (Table 5). There was no parallel shift in the mode MIC for the susceptible population, which oscillated from 0.015 to 0.03 mg/L, without trend.
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Single imipenem non-susceptible E. cloacae isolates were collected in 2003 and 2004, then four in 2006 (Table 3). The 2003 isolate, E624, which was the first producer of a KPC carbapenemase to be recorded in the UK, had a novel KPC variant, KPC-4, with Pro-103
Arg, Ser-174Gly and Val-239Gly substitutions compared with KPC-1.16 The gene sequence for the enzyme was deposited with GenBank as AY700571. The second producer, E877, was from the same hospital in the following year and had a PFGE profile identical to E624. It was believed to have been from the same patient, although this proved impossible to verify retrospectively, since the BSAC data collection is anonymized. Among the four imipenem-non- susceptible Enterobacter isolates in 2006, three were most resistant to ertapenem and least so to meropenem and doripenem; all lacked blaKPC or metallo-carbapenemase genes (Table 3). Such patterns suggest a combination of AmpC derepression coupled with impermeability.14 The imipenem MIC for the remaining isolate was only 4 mg/L, and since this organism was otherwise susceptible, it was not investigated further. Among the new agents tested, doripenem MICs were unimodally distributed, like those of imipenem and unlike those of ertapenem (Figure 1); the modal value was 0.03 mg/L, compared with 0.5 mg/L for imipenem. Ceftobiprole had a modal MIC of 0.06 mg/L for cephalosporin-susceptible Enterobacter spp. compared with 0.25 mg/L for cefotaxime or ceftazidime. Unlike these earlier cephalosporins, however, ceftobiprole retained activity against many AmpC-derepressed strains, with a mode MIC of 4 mg/L, albeit with much scatter. ESBL producers again had raised MICs, mostly >8 mg/L (Figure 1c).
Hospital acquisition and ICU location were predictors of resistance for Enterobacter spp., with the effect of hospital acquisition partly, but not entirely, linked to the increasing proportions of multiresistant ESBLs producers. Non-susceptibility varied significantly among species.
The 1291 Proteeae comprised 1017 Proteus mirabilis, 166 Morganella morganii, 66 Proteus vulgaris, 24 Providencia stuartii, 9 P. penneri and 9 P. rettgeri. Because of the small numbers of other species, analysis was largely restricted to P. mirabilis, which showed increasing non-susceptibility and resistance only for ciprofloxacin, as confirmed by both BSAC and HPA data (P < 0.0026). Trends of declining non-susceptibility to imipenem in the BSAC data were unconvincing, as P. mirabilis has inherently borderline susceptibility to this drug. Moreover, while the proportion of isolates non-susceptible ranged widely between years, only two BSAC isolates had frank imipenem resistance (MIC > 8 mg/L). Less fluctuation in the non-susceptible proportion was seen in reports to the HPA, probably (i) because of the larger numbers tested, (ii) because the laboratories mostly used susceptibility definitions corresponding to the former BSAC breakpoint of 4 mg/L, not the EUCAST harmonized value of 2 mg/L, and (iii) because testing was continuous, with less potential for run-to-run experimental variation.
Only one ESBL-producing P. mirabilis isolate, with a non-CTX-M enzyme, was identified during the 6 years of BSAC surveillance (Table 4) and even resistance to amoxicillin was only a little more than half as prevalent as among E. coli isolates (Table 2). Among newer agents, doripenem had a modal MIC of 0.12 mg/L, with no values above 1 mg/L, whereas ceftobiprole had a modal MIC of 0.06 mg/L, with values >1 mg/L for just two P. mirabilis isolates, one of them the ESBL producer.
None of the factors investigated by logistic regression (hospital acquisition, specialty, focus of infection, age or sex) was a significant predictor of non-susceptibility in P. mirabilis, though analysis was weak owing to the small proportion of non-susceptible isolates.
Since few M. morganii isolates were collected, comment must be cautious. Most cephalosporin resistance was associated with derepression of AmpC enzymes, but one isolate, collected in 2006, had a Group 1 CTX-M enzyme. Based on the larger numbers of isolates represented in the HPA data set, there did appear to be rising resistance rates to cefotaxime, ceftazidime and ciprofloxacin. Ceftobiprole retained good activity against all six AmpC-derepressed Morganella spp., with MICs of 0.03–0.12 mg/L, but had variable activity against some cefotaxime- and ceftazidime-susceptible P. vulgaris isolates, with MICs up to 16 mg/L.
The 437 Serratia spp. isolates collected in the BSAC surveillance comprised 376 S. marcescens, 48 S. liquefaciens and 13 of other or unidentified species. Analysis is complicated by these small numbers and the fact that the isolates, as members of the other ‘Gram-negatives group’, were unevenly sourced from the participating hospitals. There was no evidence of rising resistance, though it is striking that resistance to ciprofloxacin was more prevalent than in any other enterobacterial genus—a result supported by bacteraemia reports to the HPA (Table 2). The BSAC data suggested falling non-susceptibility to gentamicin, but this was not confirmed by reports to the HPA and was contingent on a sharp fall from an unusually high proportion in 2001, which may have reflected sampling variation. Reports to the HPA indicated a rising trend of ceftazidime resistance, not reflected in the BSAC collection, nor caused by any mechanism of which we are presently aware.
Phenotypic assessment of the proportion of Serratia isolates derepressed for AmpC was undertaken from 2004 onwards for the BSAC collections, with high rates found (Table 4). This mechanism is associated with resistance to cefotaxime but not ceftazidime in this genus.11 A single isolate, collected in 2005, produced an ESBL, and 11/387 had intermediate or full resistance to ertapenem, whereas none was non-susceptible to imipenem.
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Discussion |
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Of all the bacteria considered in the BSAC programmes, E. coli shows the most striking and disturbing changes, with sharp rises in resistance to cephalosporins, ciprofloxacin and gentamicin. These shifts are important because E. coli is the commonest agent of bacteraemia1,2 and, as recently as 2000, remained one of the most susceptible members of the Enterobacteriaceae. Although ciprofloxacin resistance rates were rising slowly in the species by then, they remained half those for Klebsiella and Enterobacter spp.17 Moreover, E. coli had minimal production of ESBLs.4 All this has changed in the opening years of the present century: among E. coli from bacteraemias, the prevalence rates of resistance to ciprofloxacin and oxyimino-cephalosporins have risen to match or exceed those in Klebsiella spp. (Table 2). Similar trends are becoming apparent for E. coli elsewhere in Europe, Asia and Canada though not, as yet, in the USA.4 Nearly, all the rise in cephalosporin resistance reflects the proliferation of Group 1 CTX-M enzymes, principally CTX-M-15, and is contingent on both the dissemination of epidemic strains and the spread of plasmids among strains.18 The blaCTX-M-15 gene generally is linked to aac6'-1b-cr encoding an amikacin acetyltransferase that can also deactivate ciprofloxacin and to blaOXA-1 encoding an inhibitor-resistant penicillinase.19 These linkages explain the associated rises in non-susceptibility to cephalosporins, ciprofloxacin and β-lactamase inhibitor combinations found here, though it should be added that fluoroquinolone resistance is also rising in frequency among the ESBL-negative strains and that its prevalence is around double that of ESBL production.
K. pneumoniae recapitulates many of the patterns described for E. coli, albeit less dramatically, with both the BSAC and HPA surveillance programmes indicating significantly rising non-susceptibility to cephalosporins, quinolones and gentamicin and with the production of CTX-M enzymes becoming a major component of cephalosporin resistance. However, CTX-M ESBLs have not become so completely dominant over non-CTX-M types as in E. coli, and the rises in both cephalosporin and quinolone resistance show some sign of levelling off.
None of the other species showed changes of the magnitude or significance of those for E. coli and Klebsiella spp., though the high or rising rates of ciprofloxacin resistance in Serratia spp. and P. mirabilis are notable, as is the observation, based on the BSAC surveillance, that a sizeable fraction of the oxyimino-cephalosporin resistance seen in Enterobacter spp. involves ESBLs—most of them non-CTX-M types—rather than derepressed AmpC enzymes. This is almost certainly an underestimate, as ESBLs are difficult to detect when co-produced with AmpC enzymes.20 It is unclear why plasmids encoding CTX-M enzymes have not yet proliferated in Enterobacter. A curious observation, apparent in reports to the HPA but not the BSAC surveillance, was the rising ceftazidime non-susceptibility in Serratia spp. (Table 2). Without confirmation among collected isolates, we cannot propose a mechanism, but would note: (i) derepression of AmpC typically does not confer ceftazidime resistance in this genus;11 and (ii) we are unaware of any evidence for significant ESBL spread in Serratia spp.
The spread of quinolone resistance and of ESBLs is driving the use of carbapenems, with few alternatives in reserve. A serious problem will arise if carbapenem resistance accumulates, as it is now doing in the USA with the spread of KPC β-lactamases.21 In the UK, however, carbapenem resistance remains extremely rare in Enterobacteriaceae, except for: (i) intermediate imipenem resistance in Proteeae; and (ii) ertapenem non-susceptibility in Enterobacter spp.—both traits that have become more prominent owing to the reductions in imipenem and ertapenem breakpoints adopted during the EUCAST harmonization process (from 4 to 2 and 2 to 0.5 mg/L, respectively).
Proteeae are less susceptible than other Enterobacteriaceae to imipenem and EUCAST cites an epidemiological cut-off of 4 mg/L (http://www.eucast.org), thus exceeding the susceptible breakpoint; likewise three of eight studies cited by Wiedemann and Grimm22 in Lorian's Antibiotics in Laboratory Medicine report an MIC50 of 4 mg/L and six quote an MIC90 of 4–8 mg/L. This ‘non-susceptibility’ is therefore nothing new and there is no evidence of clinical significance. The issue of ertapenem versus Enterobacter spp. is more interesting. Here, the change from BSAC breakpoints of S 2 mg/L, R > 2 mg/L to the EUCAST's values of S 0.5, R > 1 mg/L has resulted in many AmpC-derepressed isolates being re-categorized as non-susceptible (Figure 1) but, beneath this, there is a further trend (Table 5) whereby MICs for AmpC-derepressed isolates have crept up by two dilutions over the surveillance period. The reasons for this are not clear, but plausibly might relate to increased levels of AmpC enzyme or to decreased permeability.14 A trivial explanation, based on experimental error, seems unlikely in the absence of any parallel shift in the modal MICs for cephalosporin-susceptible Enterobacter.
Aside from these species-related effects, carbapenem resistance was extremely rare, with only nine imipenem-non- susceptible isolates among the 6068 reviewed here (Table 3). Two of these (K. pneumoniae K77 and Enterobacter spp. E1359) were marginal cases, with imipenem MICs of 4 mg/L, indicating intermediate status, and with little or no cross-resistance. The other seven were more convincing and comprised: (i) two E. coli isolates, both collected in 2006, one with a combination of ESBL and impermeability and the other with a still undefined mechanism; and (ii) two Enterobacter spp., from different years but probably from the same patient, with KPC carbapenemases and three Enterobacter spp. with combinations of AmpC and impermeability.
Detection of KPC carbapenemases is a major concern: Klebsiella clones with these enzymes have spread extensively in the USA, first in New York and the Atlantic coast but now more widely.21 They have also been reported in multiple species in Colombia and Israel.23,24 Most producers are resistant to β-lactams and quinolones, but susceptible to tigecycline and polymyxins and, as here, variably susceptible to aminoglycosides. So far the only UK reports are the present Enterobacter isolates, a recent K. pneumoniae from a urinary infection in Scotland25 and a further K. pneumoniae epidemiologically linked to Israel (Antibiotic Resistance Monitoring and Reference Laboratory, data on file). Carbapenem resistance contingent on combinations of impermeability and AmpC, as found in Enterobacter E1285, E1306 and E1415 (Table 3), is a scattered phenotype in the UK;14 and the continued relative susceptibility of these organisms to doripenem (MIC 0.25–2 mg/L) deserves further investigation. Ongoing research suggests that a variety of porin gene mutations and inactivations may generate the phenotype, which is occasionally selected during therapy.26 A mitigating factor is that this type of resistance is commonly unstable and we are unaware of such strains ever having caused sizeable outbreaks. The same broad type of resistance can also arise in Klebsiella spp.,14 but only eight marginal examples were seen in the BSAC collection, with ertapenem MICs of 1–8 mg/L and with retained susceptibility to all other carbapenems.
Two yet-to-be-licensed agents, ceftobiprole and doripenem, were tested from 2004 and 2005, respectively. Ceftobiprole is notable as the first anti-MRSA cephalosporin to approach market, but also has microbiologically similar anti-enterobacterial activity to cefepime. We found it active against Enterobacteriaceae except for: (i) ESBL producers; (ii) K. oxytoca hyperproducing K1 β-lactamase; and (iii) some P. vulgaris. All six AmpC- derepressed M. morganii and many derepressed Enterobacter and Serratia spp. isolates were susceptible to ceftobiprole at 1 mg/L; nevertheless, the MIC scatter was wide for the latter groups, with values from 0.06 to 128 and from 0.06 to 8 mg/L, respectively. This scatter remains to be explained but might relate to the amount of enzyme and/or strain permeability. In this context, we noted a relationship between the MICs of ertapenem and ceftobiprole for AmpC-derepressed enterobacters, such that geometric mean ceftobiprole MICs for ertapenem-susceptible (MIC 0.5 mg/L), -intermediate (1 mg/L) and -resistant (>1 mg/L) isolates were 0.42, 2.5 and 5.4 mg/L, respectively. In the case of AmpC-derepressed Serratia spp., there was a relationship between the MICs of cefotaxime and ceftobiprole, suggesting that both depended on the same factors. Doripenem was highly active, with results generally supporting those of others, at about three doubling dilutions below those of imipenem.27
In summary, if the 1990s was the decade when MRSA became prevalent in bacteraemia,28 then the ‘Noughties’ are when E. coli turned nasty, with dramatic rises in resistance to quinolones and, cephalosporins. These shifts are apparent in K. pneumoniae too, albeit less strongly, but not in Enterobacter, Proteus or Serratia. Fortunately, imipenem remains active against the huge majority of multiresistant Enterobacteriaceae, though the occurrence of two isolates with KPC carbapenemases is disturbing, while the adoption of EUCAST breakpoints has moved many AmpC-derepressed Enterobacter spp. into the ertapenem-intermediate and -resistant groups, with a trend to greater resistance. Doripenem and ceftobiprole have some advantages over earlier analogues against Enterobacteriaceae, but do not represent a generational leap. Tigecycline is a generational leap compared with earlier tetracyclines, overcoming practically all acquired resistance;29 nevertheless, its MICs are high for many Enterobacteriaceae other than E. coli when compared with the low serum levels achieved.30
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The BSAC Bacteraemia Resistance Surveillance Programme 2001–06 has received financial support from AstraZeneca, Basilea, Cubist, Johnson & Johnson, Merck Sharp & Dohme, Novartis, Pfizer, Theravance and Wyeth or their predecessors. The BSAC funds the work of the Resistance Surveillance Coordinator (R. R.) and Resistance Surveillance Working Party.
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This article is part of a Supplement sponsored by the British Society for Antimicrobial Chemotherapy.
D. M. L. has shareholdings in AstraZeneca, Pfizer, Schering Plough and GlaxoSmithKline, held within a diversified portfolio, as Enduring Attorney, and also manages family holdings, including in GlaxoSmithKine, Dechra and Eco-Animal Health, also within a diversified portfolio. He has accepted grants, advisory invitations, speaking invitations and conference invitations from most major pharmaceutical companies and many biotechnology companies. He is also employed within the UK public sector and is subject to influence by the HPA's views of antibiotic prescribing, policy and usage. All other authors have none to declare.
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We are grateful to all who have contributed to the success of the BSAC Resistance Surveillance Project, in particular to the many laboratories that have collected isolates and all who have played a part in testing them [see page ii10 (Acknowledgements)]. Additional information on the isolates collected in the Project is available on the BSAC surveillance website (www.bsacsurv.org, or through a link on the BSAC homepage www.bsac.org.uk). See page ii12 (Publications) for a full list of previous publications from the Project, some of which may include parts of the information presented here. We are grateful also to all the laboratories that have contributed to the HPA's LabBase/CoSurv system.
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