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JAC Advance Access originally published online on September 29, 2007
Journal of Antimicrobial Chemotherapy 2007 60(5):1010-1017; doi:10.1093/jac/dkm348
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© The Author 2007. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org

Characterization of Pseudomonas aeruginosa isolated from clinical and environmental samples in Minia, Egypt: prevalence, antibiogram and resistance mechanisms

Gamal F. Gad1, Ramadan A. El-Domany2, Sahar Zaki3 and Hossam M. Ashour4,*

1 Department of Microbiology and Immunology, Faculty of Pharmacy, Minia University, Egypt; 2 Department of Microbiology and Immunology, Faculty of Pharmacy, Helwan University, Egypt; 3 Department of Environmental Biotechnology, Genetic Engineering and Biotechnology Research Institute, Mubarak City for Scientific Research and Technology Applications, Alexandria, Egypt; 4 Department of Microbiology and Immunology, Faculty of Pharmacy, Cairo University, Egypt

* Corresponding author. Tel: +20-106522867; Fax: +20-238371549; E-mail: hossamking{at}mailcity.com

Received 4 July 2007; returned 2 August 2007; revised 11 August 2007; accepted 13 August 2007


   Abstract
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Objectives: To assess the prevalence, levels of antimicrobial susceptibility and resistance mechanisms of Pseudomonas.

Methods: A total of 445 clinical isolates and 200 environmental isolates were collected from three hospitals in Minia, Egypt. The MICs of different antibiotics were determined using the agar dilution method. The isolates were tested for ß-lactamase production and for the presence of efflux pumps.

Results: Out of the 445 clinical specimens, 107 Pseudomonas strains (24%) and 81 Pseudomonas aeruginosa strains were isolated (18.2%). Out of the 200 environmental specimens, 57 Pseudomonas strains (28.5%) and 39 P. aeruginosa strains were isolated (19.5%). Amikacin was the most active drug against P. aeruginosa followed by meropenem, cefepime and fluoroquinolones. P. aeruginosa was highly resistant to all other antibiotics tested. The environmental isolates of P. aeruginosa exhibited higher antibiotic resistance than clinical isolates. Mechanisms of resistance used by P. aeruginosa included ß-lactamase production and multiple drug resistance efflux pumps. Our results showed that 29 (36%) of the clinical P. aeruginosa isolates and 37 (95%) of the environmental P. aeruginosa isolates were ß-lactamase producers. In addition, P. aeruginosa isolates effectively used an efflux-mediated mechanism of resistance against ciprofloxacin and meropenem, but not gentamicin or cefotaxime.

Conclusions: This study examined the prevalence of P. aeruginosa, and its susceptibility patterns to different antibiotics. The presence of antibiotic-resistant P. aeruginosa isolates could be attributed to ß-lactamase production and the use of multiple drug resistance efflux pumps.

Keywords: nosocomial infections , antibiotics , ß-lactamases , efflux pumps


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The Gram-negative bacterium Pseudomonas aeruginosa is a ubiquitous aerobe that is present in water, in soil and on plants. Moreover, P. aeruginosa can be frequently isolated from tap water in patient rooms.1 However, clinical isolates of P. aeruginosa appear to be more resistant to amoebal ingestion than environmental isolates.2

P. aeruginosa accounts for a significant proportion of nosocomial infections.3 A general problem with nosocomial infections is the tendency of nosocomial pathogens to acquire new antibiotic resistance.4 Multidrug-resistant (MDR) strains of P. aeruginosa are often isolated among patients suffering from nosocomial infections, particularly those in the intensive care unit (ICU).5 Thus, infections caused by P. aeruginosa are particularly problematic because the organism is inherently resistant to many drug classes and is able to acquire resistance to all effective antimicrobial drugs.6

As an opportunistic infectious pathogen, P. aeruginosa can often lead to life-threatening diseases. For example, P. aeruginosa is the main cause of mortality in cases of polymicrobial bacteraemia,7 and the second most common bacterium causing sepsis in the ICU.8 In addition, P. aeruginosa has been implicated in urinary tract infections, burn wounds, ventilator-associated pneumonia and multi-organ system failure.9–12

Therefore, it was important to study the susceptibility patterns of P. aeruginosa isolates to some commonly used antibiotics in Egypt. Use of the antibiogram as an epidemiological indicator for our isolates can help us make the best use of these antimicrobial agents in the management of P. aeruginosa infections. We also studied the prevalence of Pseudomonas species infections, especially P. aeruginosa, in hospitalized patients and in the hospital environment in Egypt. The high intrinsic antibiotic resistance of this organism is attributed to factors such as active drug efflux and ß-lactamase production.13 Thus, we concluded the study by testing these two possible mechanisms of resistance of the isolated P. aeruginosa to different antibiotics.


   Materials and methods
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Study design

A total of 445 clinical specimens were examined; 100 urine specimens (from patients with urinary tract infection), 45 stool specimens (from patients with gastrointestinal track infections), 50 sputum specimens (from patients with respiratory tract infections), 8 ear swab specimens (from patients with ear infections), 100 wound swab specimens, 45 abscess swab specimens and 25 burn swab specimens (from patients with wounds, abscesses, or burns). All specimens were collected from El-Minia University Hospital, El-Minia General Hospital and El-Minia Chest Hospital in Egypt. In addition, 200 environmental specimens were randomly collected from furniture, medical appliances and the surroundings of the same hospitals such as patients' beds, tables, ward sinks and surgical equipment. All specimens were examined for the existence of Pseudomonas species, and P. aeruginosa by standard procedures.14,15 Ethical approval to perform the study was obtained from the management boards of these hospitals and the Egyptian Ministry of Health and Population.

PCR detection of P. aeruginosa

This method was used before by Abd-El-Haleem et al.16 for the detection of P. aeruginosa. Total bacterial DNA was prepared using the boiling approach. Bacterial cells were pelleted by centrifugation, resuspended in 50 µL of TE buffer and then lysed by boiling for 10 min. The lysate was centrifuged and the supernatant was transferred to a new tube. The crude cell lysate was used directly for PCR.

The primer pair PaLif (5'-ATGGAAATGCTGAAATTCGGC-3') and PaLir (5'-CTTCTTCAGCTCGACGCGACG-3') was selected in order to amplify conserved regions of a target gene in P. aeruginosa and thus generate a PCR amplicon with a certain molecular weight (504 bp) that can be identified by gel electrophoresis.

PCR assays were performed in a 50 µL volume with 2 U of DNA Taq polymerase (GIBCO PRL) in a thermal cycler (PTC-100 MJ Research, Watertown MA, USA). After initial denaturation for 2 min at 94°C, 30 cycles were performed (the conditions for each cycle were: 30 s at 94°C, 30 s at 51°C and 1 min at 72°C). The final cycle was followed by 72°C incubation for 7 min. A reaction mixture containing sterile water was included as a negative control and a purified DNA mixture of the targeted bacteria was included as a positive control. The amplified PCR products were analysed by gel electrophoresis in 2% agarose gels stained with ethidium bromide, and then visualized and photographed in a Multi-Image light cabinet (Alpha Innotech Corporation, USA).

Antibiotics

The following antibiotics were obtained from the Egyptian market: ampicillin (Nile Pharmaceutical Company, Cairo, Egypt), ampicillin/sulbactam (Medical Union Pharmaceutical Company, Cairo, Egypt), amoxicillin (Egyptian International Pharmaceutical Industries Company; EIPICO, Cairo, Egypt), amoxicillin/clavulanate (Medical Union Pharmaceutical Company), cefalexin (Glaxo Wellcome, Cairo, Egypt), cefuroxime (Glaxo Wellcome), cefotaxime (Aventis Pharma, Cairo, Egypt), cefoperazone (Pharco Pharmaceuticals, Cairo, Egypt), ceftriaxone (EIPICO), cefepime (Bristol Myers Squibb; BMS, Cairo, Egypt), meropenem (Astra-Zeneca, Cairo, Egypt), gentamicin (Memphis for Pharmaceutical Chemical Industries Co., Cairo, Egypt), amikacin (Bristol Myers Squibb) and chloramphenicol (Chemical Industries Development; CID, Cairo, Egypt). Tetracycline, ciprofloxacin, levofloxacin, ofloxacin, norfloxacin and azithromycin were obtained from Sigma-Aldrich (St Louis, MO, USA).

Determination of MICs

The MICs of different antibiotics were determined by the agar dilution method, according to the CLSI (formerly known as the NCCLS) (1997), on Mueller–Hinton agar (MHA). Overnight cultures of tested organisms on Mueller–Hinton broth (MHB) were diluted to the initial cell density of ~107 cfu/mL with fresh MHB. Inocula of ~105 cfu per spot were applied to the surface of dry MHA plates containing graded concentrations (from 1–1024 mg/L) of the respective antibiotics. Plates were incubated at 37°C for 18–24 h and MICs were calculated. Spots with the lowest concentrations of antibiotic showing no growth were defined as the MIC.

ß-Lactamase detection

The tested isolates were tooth-picked onto the surface of nutrient agar plates. After overnight incubation at 37°C, the plates were overlaid with 1% molten agarose containing 0.2% soluble starch and 1% penicillin. The plates were incubated for 15 min at room temperature to solidify, and iodine solution was poured onto the agar plates. After 10 s, the residual iodine solution was damped out and the plates were incubated at room temperature until a discolouration zone appeared around ß-lactamase-producing colonies.17 Thus, the presence of a clear zone around bacterial growth is indicative of ß-lactamase production.

Study of efflux system

We employed the method of Lomovskaya et al.18 to study the efflux system of P. aeruginosa isolates. The MICs of four antibiotics of different groups (ciprofloxacin, meropenem, cefotaxime and gentamicin) for 25 MDR P. aeruginosa isolates were examined in the presence and absence of 10 µM of the efflux inhibitor carbonyl cyanide m-chlorophenylhydrazone (CCCP) (Sigma). The reduction in MIC of a certain antibiotic with CCCP is an indication of resistance to this antibiotic mediated by an efflux system.


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Prevalence of Pseudomonas species in clinical and environmental specimens

Out of the 445 clinical specimens, 107 Pseudomonas strains (24%) and 81 P. aeruginosa strains were isolated (18.2%). Out of the 200 environmental specimens, 57 Pseudomonas strains (28.5%) and 39 P. aeruginosa strains were isolated (19.5%), as shown in Tables 1 and 2. From Table 1, the percentage of P. aeruginosa isolates with respect to the total number of clinical Pseudomonas isolates can be calculated as 81/107 x 100 = 75.7%. From Table 2, the percentage of P. aeruginosa isolates with respect to the total number of environmental Pseudomonas isolates can be calculated as 39/57 x 100 = 68.4%.


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  		Table 1. Prevalence of Pseudomonas species in clinical specimens

 


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  		Table 2. Prevalence of Pseudomonas species in environmental specimens

 
To further examine the percentage of P. aeruginosa isolates with respect to the total number of Pseudomonas isolates, 40 random Pseudomonas strains (20 clinical and 20 environmental) were examined biochemically and by PCR followed by gel electrophoresis and results were comparable (Figure 1). The average percentage of P. aeruginosa isolates with respect to the total number of clinical Pseudomonas isolates was 80%, whereas the average percentage of P. aeruginosa isolates with respect to the total number of environmental Pseudomonas isolates was 57.5%. Thus, the biochemical and molecular methods for identification of P. aeruginosa did not show a significant difference from our standard procedures for detecting the bacterium.14,15


Figure 1
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  		Figure 1. Gel electrophoresis of the PCR-amplified products for detection of clinical (lanes 1–20) and environmental (lanes 21–40) Pseudomonas isolates.

 
Antibiotic susceptibility and determination of MICs

Tables 3 and 4 show the respective MIC distributions of different antibiotics for clinical isolates (81 isolates) and environmental isolates (39 isolates) of P. aeruginosa. Tables 5 and 6 show the MIC90s (MIC required to inhibit the growth of 90% of organisms) of each antibiotic, and whether the bacteria were susceptible, intermediately susceptible or resistant to each antibiotic.


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  		Table 3. MICs of different antibiotics for clinical P. aeruginosa isolates (81 isolates)

 


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  		Table 4. MICs of different antibiotics for environmental P. aeruginosa isolates (39 isolates)

 


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  		Table 5. Antibiotic susceptibility of clinical P. aeruginosa isolates

 


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  		Table 6. Antibiotic susceptibility of environmental P. aeruginosa isolates

 
The breakpoint MIC of a drug is the highest concentration that can be safely attained in blood using the recommended dosing regimen. Organisms are considered susceptible to a drug if the MIC is below the breakpoint MIC. Organisms characterized by intermediate susceptibility are inhibited at concentrations that approach breakpoint. The MIC for a resistant organism surpasses the breakpoint MIC of the drug, and for that drug the risk of toxicity outweighs the potential benefits of therapy.

Among the antibiotics tested, amikacin was the most active drug against P. aeruginosa followed by meropenem, cefepime and fluoroquinolones. P. aeruginosa was highly resistant to all other antibiotics tested. In addition, the environmental isolates of P. aeruginosa exhibited higher antibiotic resistance than clinical isolates.

Resistance through ß-lactamase production

All P. aeruginosa isolates were subjected to ß-lactamase detection. The presence of a clear zone around bacterial growth was indicative of ß-lactamase production as described in the Materials and methods section. Our results showed that 29 (36%) of the clinical P. aeruginosa isolates and 37 (95%) of the environmental P. aeruginosa isolates were ß-lactamase producers.

Resistance through the efflux system

MDR nosocomial infections by P. aeruginosa are increasing worldwide.19 The evolution of MDR bacteria can be attributed to the uncontrolled extensive use of antibiotics in hospitals and the community.20 The evolution of MDR strains can be caused by an active efflux system that expels antibiotics from the cell.21 Table 7 shows the MICs of four antibiotics (ciprofloxacin, gentamicin, cefotaxime and meropenem) for 25 different MDR P. aeruginosa isolates in the presence and absence of the efflux inhibitor (CCCP). As can be seen from the table, the addition of CCCP enhanced the activities of ciprofloxacin and meropenem, but not gentamicin or cefotaxime. These results emphasized the presence of an efflux-mediated resistance in the tested strains to ciprofloxacin and meropenem, but not to gentamicin or cefotaxime.


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  		Table 7. Effect of adding CCCP on antibiotic resistance pattern of P. aeruginosa isolates

 

   Discussion
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In this study, P. aeruginosa was the most prevalent species in the isolated strains. P. aeruginosa represented 75.7% of clinical Pseudomonas isolates and 18.2% of all clinical specimens. It also represented 68.4% of environmental Pseudomonas isolates and 19.5% of all environmental specimens.

The effectiveness of amikacin against P. aeruginosa is corroborated by data from other groups.22,23 One earlier study reported that meropenem was the most effective antibiotic against P. aeruginosa.24 However, more recent studies demonstrated the evolution of meropenem-resistant strains of P. aeruginosa.25,26 Our study revealed moderate activity of quinolones towards P. aeruginosa. Whereas others reported similar rates of resistance to quinolones,22,27 Corona-Nakamura et al.28 showed that P. aeruginosa was absolutely susceptible to ciprofloxacin. This discrepancy can be attributed to the continuous development of MDR strains of P. aeruginosa in different parts of the world.

The resistance pattern of P. aeruginosa to cephalosporins was consistent with the one reported by Yetkin et al.23 who showed that the percentage of resistance to cephalosporins was in the range of 27% to 88%. Results in Tables 5 and 6 demonstrated that cefepime was the most active cephalosporin against P. aeruginosa. This is consistent with reports from several groups.24,28,29 Additionally, the P. aeruginosa resistance pattern to gentamicin was close to the one reported by Muller-Premru and Gubina.30

In order to define the main mechanisms used by P. aeruginosa to resist antibiotics, we tested for ß-lactamase production and for the possess of efflux-mediated resistance. P. aeruginosa were previously shown to use ß-lactamase-mediated resistance to antibiotics.31–33 We observed high levels of ß-lactamase production in P. aeruginosa isolates (36% in clinical isolates and 95% in environmental isolates) in Minia similar to what has been reported elsewhere.29 P. aeruginosa isolates in Minia also used efflux-mediated resistance to ciprofloxacin and meropenem, but not gentamicin or cefotaxime. Efflux-mediated fluoroquinolone resistance of P. aeruginosa was reported in several studies.34–36 Moreover, Pai et al.37 reported that overproduction of the MexAB-OprM efflux system was associated with clinical episodes of carbapenem resistance in P. aeruginosa. Therefore, mechanisms of resistance used by P. aeruginosa isolates from Minia included ß-lactamase production and the use of multiple drug resistance efflux pumps.


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No specific funding has been received to conduct this study.


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None to declare.


   Acknowledgements
 
We are grateful to the management and medical stuff of the El-Minia University hospital, El-Minia General hospital and El-Minia Chest hospital for assistance in sample collection. We also remain indebted to Minia University, Helwan University, Cairo University and Mubarak City for the equipment and facilities put at our disposal.


   References
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1 Valles J, Mariscal D, Cortes P, et al. Patterns of colonization by Pseudomonas aeruginosa in intubated patients: a 3-year prospective study of 1,607 isolates using pulsed-field gel electrophoresis with implications for prevention of ventilator-associated pneumonia. Intensive Care Med (2004) 30:1768–75.[Web of Science][Medline]

2 Fenner L, Richet H, Raoult D, et al. Are clinical isolates of Pseudomonas aeruginosa more virulent than hospital environmental isolates in amebal co-culture test? Crit Care Med (2006) 34:823–8.[CrossRef][Web of Science][Medline]

3 Blanc DS, Petignat C, Janin B, et al. Frequency and molecular diversity of Pseudomonas aeruginosa upon admission and during hospitalization: a prospective epidemiologic study. Clin Microbiol Infect (1998) 4:242–7.[Medline]

4 Livrelli V, De Champs C, Di Martino P, et al. Adhesive properties and antibiotic resistance of Klebsiella, Enterobacter, and Serratia clinical isolates involved in nosocomial infections. J Clin Microbiol (1996) 34:1963–9.[Abstract]

5 Tassios PT, Gennimata V, Spaliara-Kalogeropoulou L, et al. Multiresistant Pseudomonas aeruginosa serogroup O:11 outbreak in an intensive care unit. Clin Microbiol Infect (1997) 3:621–8.[Medline]

6 Gales AC, Jones RN, Turnidge J, et al. Characterization of Pseudomonas aeruginosa isolates: occurrence rates, antimicrobial susceptibility patterns, and molecular typing in the global SENTRY Antimicrobial Surveillance Program, 1997–1999. Clin Infect Dis (2001) 32(Suppl 2):S146–55.[CrossRef][Web of Science][Medline]

7 Marra AR, Bar K, Bearman GM, et al. Systemic inflammatory response syndrome in nosocomial bloodstream infections with Pseudomonas aeruginosa and Enterococcus species: comparison of elderly and nonelderly patients. J Am Geriatr Soc (2006) 54:804–8.[CrossRef][Web of Science][Medline]

8 Vincent JL, Sakr Y, Sprung CL, et al. Sepsis in European intensive care units: results of the SOAP study. Crit Care Med (2006) 34:344–53.[CrossRef][Web of Science][Medline]

9 Harjai K, Mittal R, Chhibber S, et al. Contribution of Tamm-Horsfall protein to virulence of Pseudomonas aeruginosa in urinary tract infection. Microbes Infect (2005) 7:132–7.[CrossRef][Web of Science][Medline]

10 Rello J, Allegri C, Rodriguez A, et al. Risk factors for ventilator-associated pneumonia by Pseudomonas aeruginosa in presence of recent antibiotic exposure. Anesthesiology (2006) 105:709–14.[CrossRef][Web of Science][Medline]

11 Altoparlak U, Aktas F, Celebi D, et al. Prevalence of metallo-ß-lactamase among Pseudomonas aeruginosa and Acinetobacter baumannii isolated from burn wounds and in vitro activities of antibiotic combinations against these isolates. Burns (2005) 31:707–10.[CrossRef][Web of Science][Medline]

12 Guidet B, Aegerter P, Gauzit R, et al. Incidence and impact of organ dysfunctions associated with sepsis. Chest (2005) 127:942–51.[CrossRef][Web of Science][Medline]

13 Hancock RE, Speert DP. Antibiotic resistance in Pseudomonas aeruginosa: mechanisms and impact on treatment. Drug Resist Updat (2000) 3:247–55.[CrossRef][Web of Science][Medline]

14 Collee JG, Fraser AG, Marmion BP, et al. Mackie & McCartney Practical Medical Microbiology (1996) 14th Edition. New York: Churchill Livingstone.

15 Barrow GI, Feltham RKA. Cowan and Steel's Manual for the Identification of Medical Bacteria (1993) Third Edition. Cambridge, UK: Cambridge University Press.

16 Abd-El-Haleem D, Kheiralla ZH, Zaki S, et al. Multiplex-PCR and PCR-RFLP assays to monitor water quality against pathogenic bacteria. J Environ Monit (2003) 5:865–70.[CrossRef][Web of Science][Medline]

17 Sykes RB. Methods for detecting ß-lactamase. In: Laboratory Methods in Antimicrobial Chemotherapy—Reeves DS, Phillips I, Williams JD, et al, eds. (1978) Edinburgh, UK: Churchill Livingstone. 64–9.

18 Lomovskaya O, Warren MS, Lee A, et al. Identification and characterization of inhibitors of multidrug resistance efflux pumps in Pseudomonas aeruginosa: novel agents for combination therapy. Antimicrob Agents Chemother (2001) 45:105–16.[Abstract/Free Full Text]

19 Hsueh PR, Teng LJ, Yang PC, et al. Persistence of a multidrug-resistant Pseudomonas aeruginosa clone in an intensive care burn unit. J Clin Microbiol (1998) 36:1347–51.[Abstract/Free Full Text]

20 Pellegrino FL, Teixeira LM, Carvalho Md Mda G, et al. Occurrence of a multidrug-resistant Pseudomonas aeruginosa clone in different hospitals in Rio de Janeiro, Brazil. J Clin Microbiol (2002) 40:2420–4.[Abstract/Free Full Text]

21 Kohler T, van Delden C, Curty LK, et al. Overexpression of the MexEF-OprN multidrug efflux system affects cell-to-cell signaling in Pseudomonas aeruginosa. J Bacteriol (2001) 183:5213–22.[Abstract/Free Full Text]

22 Bratu S, Quale J, Cebular S, et al. Multidrug-resistant Pseudomonas aeruginosa in Brooklyn, New York: molecular epidemiology and in vitro activity of polymyxin B. Eur J Clin Microbiol Infect Dis (2005) 24:196–201.[CrossRef][Web of Science][Medline]

23 Yetkin G, Otlu B, Cicek A, et al. Clinical, microbiologic, and epidemiologic characteristics of Pseudomonas aeruginosa infections in a University Hospital, Malatya, Turkey. Am J Infect Control (2006) 34:188–92.[CrossRef][Web of Science][Medline]

24 Gales AC, Sader HS, Jones RN. Urinary tract infection trends in Latin American hospitals: report from the SENTRY antimicrobial surveillance program (1997–2000). Diagn Microbiol Infect Dis (2002) 44:289–99.[CrossRef][Web of Science][Medline]

25 Maniati M, Ikonomidis A, Mantzana P, et al. A highly carbapenem-resistant Pseudomonas aeruginosa isolate with a novel blaVIM-4/blaP1b integron overexpresses two efflux pumps and lacks OprD. J Antimicrob Chemother (2007) 60:132–5.[Abstract/Free Full Text]

26 Pournaras S, Maniati M, Spanakis N, et al. Spread of efflux pump-overexpressing, non-metallo-ß-lactamase-producing, meropenem-resistant but ceftazidime-susceptible Pseudomonas aeruginosa in a region with blaVIM endemicity. J Antimicrob Chemother (2005) 56:761–4.[Abstract/Free Full Text]

27 Sahm DF, Critchley IA, Kelly LJ, et al. Evaluation of current activities of fluoroquinolones against gram-negative bacilli using centralized in vitro testing and electronic surveillance. Antimicrob Agents Chemother (2001) 45:267–74.[Abstract/Free Full Text]

28 Corona-Nakamura AL, Miranda-Novales MG, Leanos-Miranda B, et al. Epidemiologic study of Pseudomonas aeruginosa in critical patients and reservoirs. Arch Med Res (2001) 32:238–42.[CrossRef][Web of Science][Medline]

29 Gencer S, Ak O, Benzonana N, et al. Susceptibility patterns and cross resistances of antibiotics against Pseudomonas aeruginosa in a teaching hospital of Turkey. Ann Clin Microbiol Antimicrob (2002) 1:2.[CrossRef][Medline]

30 Muller-Premru M, Gubina M. Serotype, antimicrobial susceptibility and clone distribution of Pseudomonas aeruginosa in a university hospital. Zentralbl Bakteriol (2000) 289:857–67.[Web of Science][Medline]

31 al Naiemi N, Duim B, Bart A. A CTX-M extended-spectrum ß-lactamase in Pseudomonas aeruginosa and Stenotrophomonas maltophilia. J Med Microbiol (2006) 55:1607–8.[Free Full Text]

32 Wang C, Cai P, Chang D, et al. A Pseudomonas aeruginosa isolate producing the GES-5 extended-spectrum ß-lactamase. J Antimicrob Chemother (2006) 57:1261–2.[Free Full Text]

33 Bell SM, Pham JN, Gatus BJ, et al. Isolation of an extended spectrum ß-lactamase producing Pseudomonas aeruginosa from a patient in a Sydney hospital. Pathology (2007) 39:189–90.[CrossRef][Medline]

34 Llanes C, Hocquet D, Vogne C, et al. Clinical strains of Pseudomonas aeruginosa overproducing MexAB-OprM and MexXY efflux pumps simultaneously. Antimicrob Agents Chemother (2004) 48:1797–802.[Abstract/Free Full Text]

35 Poole K. Efflux-mediated multiresistance in Gram-negative bacteria. Clin Microbiol Infect (2004) 10:12–26.[Web of Science][Medline]

36 Oh H, Stenhoff J, Jalal S, et al. Role of efflux pumps and mutations in genes for topoisomerases II and IV in fluoroquinolone-resistant Pseudomonas aeruginosa strains. Microb Drug Resist (2003) 9:323–8.[CrossRef][Web of Science][Medline]

37 Pai H, Kim J, Kim J, et al. Carbapenem resistance mechanisms in Pseudomonas aeruginosa clinical isolates. Antimicrob Agents Chemother (2001) 45:480–4.[Abstract/Free Full Text]


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