European Journal of Microbiology and Immunology 3 (2013) 4, pp. 267–271 DOI: 10.1556/EuJMI.3.2013.4.5

PREVALENCE OF PLASMID-MEDIATED AMPC β-LACTAMASES IN ESCHERICHIA COLI AND KLEBSIELLA PNEUMONIAE AT TERTIARY CARE HOSPITAL OF ISLAMABAD, PAKISTAN Muhammad Shafiq1, Hazir Rahman2,*, Muhammad Qasim2, Najma Ayub3, Shagufta Hussain1, Jafar Khan2 and Madiha Naeem2 1

Department of Microbiology, Quaid-e-Azam University, Islamabad, Pakistan Department of Microbiology, Kohat University of Science and Technology, Kohat, Pakistan 3 Department of Microbiology, Pakistan Institute of Medical Sciences, Islamabad, Pakistan 2

Received: August 4, 2013; Revised: September 8, 2013; Accepted: September 8, 2013 Enterobacteriaceae produces AmpC β-lactamases that make them resistant to commonly used antibiotics. AmpC β-lactamases can be chromosomal-mediated or plasmid-mediated AmpC β-lactamases (PABLs). The present study was undertaken to determine the occurrence of PABLs production in clinical isolates in Escherichia coli and Klebsiella pneumoniae. Among 1328 culture positive samples, 511 isolates were identified as E. coli (81.02%, n = 414) and K. pneumonia (18.98%, n = 97). Cefoxitin resistance was observed in E. coli (19.57%, n = 81) and K. pneumoniae (22.68%, n = 22). Out of these cefoxitin resistant isolates, 40.74% (n = 33) E. coli and 54.55% (n = 12) K. pneumoniae came out to be PABL producers. Prevalence of both PABLs and ESBLs in E. coli and K. pneumoniae was 29.24% (n = 8) and 47% (n = 5), respectively. Isolates coproducing PABLs and ESBL exhibited increased minimum inhibitory concentrations (MICs) for selected cephalosporins. This study documented a high frequency of PABLs producing isolates from hospital which may lead to serious therapeutic problem. Keywords: E. coli, K. pneumoniae, PABLs, ESBLs, antibiogram

Introduction Enterobacteriaceae is the major cause of nosocomial infections [1]. Drug resistance is a serious threat to antimicrobial chemotherapy interventions [2, 3]. The major mechanism of resistance is the production of beta (β)-lactamases including AmpC β-lactamases (AmpC) and extended-spectrum β-lactamases (ESBLs) [3–5]. The genes for AmpC β-lactamases production are chromosomal mediated; however, plasmid-mediated AmpC β-lactamases (PABLs) have arisen through the chromosomal genes transfer to plasmids and can lead to dissemination of antimicrobial resistance to diverse bacterial population including Escherichia coli, Klebsiella spp., Salmonella spp., and Proteus mirabilis [6]. PABLs and ESBLs producing isolates pose diagnostic and therapeutic challenges for health care professionals. Determination of PABLs prevalence is important for surveillance and epidemiological studies and for infection control as these genes can spread to other organisms within the hospital settings [7]. The present study was undertaken to report the prevalence of PABLs producing isolates of E. coli and Klebsiella pneumoniae in tertiary care hospital of Islamabad. PABLs

production was determined by using specific AmpC disk test. Furthermore, PABLs producing isolates were tested for ESBLs production and for the antibiotic susceptibility. The study will be helpful to adopt a judicious hospital antibiotic policy against PABLs producing pathogens.

Materials and methods Samples The study was carried out in an 822-bed tertiary care hospital of Islamabad, Pakistan, from February 2008 to June 2008. A total of 3036 samples were obtained from surgical, nonsurgical and out-door patients, and then inoculated on blood agar and MacConkey agar except urine, which were inoculated on CLED agar using bacteriuria strips (MediTest, UK). These plates were incubated at 35 ± 2 °C for 18 h aerobically. Only those samples were further processed for PABLs and ESBLs production which showed significant growth and identified as E. coli and K. pneumoniae on the basis of culture, Gram staining, and biochemical characteristics

* Corresponding author: Dr. Hazir Rahman; Department of Microbiology, Kohat University of Science and Technology, Kohat, Pakistan; E-mail: [email protected] ISSN 2062-509X / $ 20.00 © 2013 Akadémiai Kiadó, Budapest

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using API 20E (bioMerieux, Germany). This research has been approved by the Research Ethics Committee of the hospital.

at concentration higher than the range seen for wild-type strain. Minimum inhibitory concentrations (MICs) were determined using two-fold agar dilution plate method for ceftazidime, cefoxitin, cefepime, and imipenem as previously described [13].

Detection of PABLs production Bacterial susceptibility to cefoxitin (30 μg) was tested on Mueller–Hinton agar (MHA) plate according to the standard disk diffusion method [8], and isolates showing 512 μg/ml. PABLs and ESBLs producing isolates exhibited increased MICs for cefoxitin. Both PABLs E. coli and K. pneumo-

Fig. 3. Antimicrobial resistance in PABLs producing E. coli: percent (%) antibiotic activity against E. coli is shown in the figure. The antibiotics used were CPD (cefpodoxime), CAZ (ceftazidime), CRO (ceftriaxone), ATM (aztreonam), FEP (cefepime), CIP (ciprofloxacin), AK (amikacin), IPM (imipenem), TZP (piperacillin/tazobactam), SCF (cefoperazone/sulbactam), AMC (amoxicillin/clavulanic acid), SXT (trimethoprim–sulfamethoxazole), TE (tetracycline), C (chloramphenical), PRL (piperacillin), and TGE (tigecycline) European Journal of Microbiology and Immunology 3 (2013) 4

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Fig. 4. Antimicrobial resistance in PABLs producing K. pneumoniae: percent (%) antibiotic activity against E. coli is shown in the figure. For abbreviations of antibiotics see legend to Fig. 3

niae had very high MICs for ceftazidime, >512 μg/ml. MICs of cefepime for PABLs E. coli were observed as 4 to 256 μg/ml, and 4 to128 μg/ml for K. pneumoniae. PABLs and ESBLs coproducing E. coli and K. pneumoniae exhibited higher MICs against cefepime. Two of each coproducing PABLs and ESBL E. coli and K. pneumoniae exhibited MICs of cefepime in intermediate range of ≥16 μg/ml, whereas only two PABLs non-ESBLs E. coli exhibited MICs ≥32 μg/ml of cefepime. MICs of 0.5 to 2 μg/ml of imipenem were determined against E. coli and K. pneumoniae (Table 1). Table 1. Minimum inhibitory concentration (MIC) in E. coli and K. pneumoniae with plasmid-mediated AmpC β-lactamase MICs (μg/ml) Antibiotics

E. coli

K. pneumoniae

Cefoxitin

256 to >512

256 to >512

Ceftazidime

512 to >512

512 to >512

Cefepime

4 to 256

4 to 128

Imipenem

0.5 to 2

0.5 to 2

Discussion The Enterobacteriaceae are important causes of nosocomial infections in humans, and resistance to antimicrobial agents in these species is a challenge to health care professionals [7]. Emerging resistance mechanism is due to the production of ESBLs and PABLs [14]. In the present study, PABLs producing E. coli and K. pneumoniae were 7.97% and 12.37%, respectively. A similar study in Pakistan reported PABL producers E. coli (18%) and K. pneumonia (14%) [15]. Another study reported different results which might be due to regional differences and different detection methods for PABLs production [16]. The PABLs producing E. coli and K. pneumoniae were relatively more commonly isolated from surgical European Journal of Microbiology and Immunology 3 (2013) 4

sites [17]. About 54.54% PABLs producing E. coli and 66.67% PABLs producing K. pneumoniae were isolated from surgical sites. In outpatients, the incidence of PABLs producing isolates (E. coli, 18.88%, and K. pneumoniae, 8.33%) were observed. This prevalence can be due to referral cases from other hospitals which might indicate the penetration of PABLs producing strains in community as ESBL producing strains [18]. In this study, we found coproduction of PABLs and ESBLs in E. coli (24.24%), and K. pneumoniae (47%) These observations are consistent with a previous study, which reported 60% coproducing ESBLs [15]. The cephalosporin resistance was also documented among the isolates. A previous study by the SENTRY antimicrobial surveillance program reported more than 3000 ceftazidime-resistant gram-negative bacilli between 1998 and 2004 and found 90% isolates sensitive to cefepime [19]. In this study, PABL producers E. coli (69.7%) and K. pneumoniae (58.33%) were sensitive to cefepime. All isolates coproducing ESBLs and AmpC β-lactamase were resistant to cefepime. Two isolates of E. coli producing only AmpC β-lactamase were also resistant to cefepime, which is in line with a previous study [20]. Moreover, in our study, the bacterial isolates exhibit variable degree of resistance to broad spectrum antibiotics including trimethoprim–sulfamethoxazole, amikacin, chloramphenicol, and ciprofloxacin, which is consistent with previous reports [10, 21]. Quite variable results might be due to multiple resistant mechanisms used by isolates against antibiotics at different regions of the world [16]. In addition, 42.42% and 30.3% PABLs E. coli and 41.66% and 33.3% K. pneumoniae were susceptible to piperacillin/tazobactam and cefoperazone/sulbactam, respectively, as reported earlier [22]; however, all the isolates were sensitive to imipenem, a broad spectrum β-lactam antibiotic [23]. For PABLs producing E. coli and K. pneumonia, the MICs for ceftazidime, cefoxitin, cefepime, and imipenem were 512 to ≥512 μg/ml, 256 to ≥512 μg/ml,

Prevalence of plasmid-mediated AmpC β-lactamases in Escherichia coli and Klebsiella pneumoniae

4 to 256 μg/ml, and 0.5 to 2 μg/ml, respectively, which are similar to previous findings [14, 21]. High MICs were observed in PABLs producing strains for third generation cephalosporin, making it inappropriate for empirical therapy.

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Conclusions This study shows a high frequency of PABLs producing isolates from hospital, which are likely to be overlooked. The present study will be helpful to highlight both the judicious use of broad-spectrum antimicrobial agents and rigorous attention to infection control procedures to reduce the prevalence of multidrug-resistant microorganisms.

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Acknowledgements

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We thank the technical staff at PIMS Islamabad for expert assistance. 16.

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