IDENTIFICATION OF AMPC Β-LACTAMASE-PRODUCING CLINICAL ISOLATES OF ESCHERICHIA COLI

Objective: Indiscriminate use of β-lactam antibiotics has resulted in the emergence of β-lactamase enzymes. AmpC β-lactamases, in particular, confer resistance to penicillin, first-, second-, and third-generation cephalosporins as well as monobactams and are responsible for antibiotic resistance in nosocomial pathogens. Therefore, this study was undertaken to screen nosocomial Escherichia coli isolates for the presence and characterization of AmpC β-lactamases. The study also envisaged on the detection of inducible AmpC β-lactamases and extended-spectrum β-lactamases (ESBLs) in AmpC β-lactamase-producing E. coli. Methods: A total of 102 clinical isolates of E. coli, were subjected to cefoxitin screening, and screen-positive isolates were further subjected to inhibitorbased detection method, phenotypic confirmatory test, disc antagonism test, polymerase chain reaction (PCR), and isoelectric focusing (IEF). Results: In this study, 33% of E. coli were resistant to cefoxitin, of which 35% were found to be positive for AmpC β-lactamase by inhibitor-based phenotypic test. Of the AmpC-positive isolates, 83% were positive for ESBLs, whereas 25% were producing inducible AmpC β-lactamases. PCR and IEF showed CIT and EBC types of AmpC β-lactamases present in the tested isolates. Conclusion: Our study showed the presence of inducible AmpC enzymes and ESBLs in E. coli isolates and PCR identified more isolates to be AmpC producers.


INTRODUCTION
There has been an implausible rise in the incidence of antibiotic resistance, more importantly to cephalosporins in bacterial pathogens [1,2]. Of the various resistance mechanisms, production of β-lactamases is the most widespread. AmpC β-lactamases are clinically significant as they confer resistance to a broad spectrum of antibiotics such as penicillin, first-, second-, and third-generation cephalosporins and monobactams such as aztreonam and are not inhibited by commercially available β-lactamase inhibitors [3,4]. Escherichia coli is a commensal and opportunistic pathogen which can be responsible for intestinal as well as urinary tract infections, and hence, is targets for antibiotic therapies [5]. This can lead, in some cases to a high degree of antibiotic resistance. AmpC β-lactamases are encoded by ampC genes which can be chromosomally or plasmid mediated [6]. In E. coli, ampC is expressed constitutively, but at low level, mutations in the ampC promoter/attenuator region can result in constitutive overexpression [7]. Plasmid-mediated ampC genes are also reported in E. coli, which are expressed constitutively in high levels and are also induced by β-lactams such as ACT-1 β-lactamases [6]. Hence, plasmid-mediated AmpC β-lactamases have considerable clinical significance [8]. Moreover, clinical isolates possessing plasmids encoding AmpC enzymes often are resistant to multiple antibiotics hence, leaving few therapeutic options.
Taking into consideration the significance of AmpC β-lactamases and coexistence of extended-spectrum β-lactamases (ESBLs) in AmpC β-lactamase-producing isolates in clinical world, this study was undertaken to identify and characterize different types of AmpC β-lactamases in E. coli. The present study compared different phenotypic methods with a genotypic method polymerase chain reaction (PCR) to detect AmpC β-lactamases in clinical isolates of E. coli.

Bacterial isolates
A total of 102 isolates of E. coli recovered from a variety of clinical specimens were collected from two different hospitals of Delhi during the period from April 2003 to December 2005. All the isolates were identified by standard biochemical methods. E. coli ATCC 25922 was included as a negative control in all the tests described, whereas E. coli C600 pMG230 and Klebsiella pneumoniae 48188 were used as positive controls in all the experiments.

Screening for AmpC production
The isolates were tested for antimicrobial susceptibility to cefoxitin by Kirby-Bauer disc diffusion method [9]. The inhibition zone sizes were interpreted as per the CLSI (formerly NCCLS) guidelines [10]. All the isolates with an inhibition zone diameter of <18 mm were selected.

Detection of AmpC β-lactamases
The screened positive isolates were tested by an inhibitor (boronic acid)-based detection method to detect AmpC-producing E. coli [11]. Briefly, a stock solution of boronic acid was prepared by dissolving 120 mg of phenylboronic acid (Sigma-Aldrich, St. Louis, USA) in 3 ml dimethyl sulfoxide, to which 3 ml of sterile distilled water was added. Discs were prepared by dispensing 20 µl of the stock solution on 30 µg of cefotetan discs. These were dried for 30 min and used immediately or stored in airtight vials with a desiccant at 4°C. This test was performed by inoculating Mueller-Hinton Agar (MHA) plate with test isolates by the standard disc diffusion method (CLSI/NCCLS) and placing a disc containing 30 µg of cefotetan along with a disc containing 30 µg of cefotetan and 400 µg of boronic acid. The inoculated plates were incubated overnight at 37°C. Organism showing an increase of 5 mm of zone diameter around the disc containing cefotetan and boronic acid as

Gupta et al.
compared to that of zone diameter around the disc of cefotetan alone was considered as AmpC producer.

Detection of inducible AmpC β-lactamases
Disc antagonism test (DAT) was carried out as described by Yan et al., 2002, [12]. Briefly, a disc of cefoxitin (30 µg) was kept in the center of the inoculated (test isolate) MHA plate and discs of ceftazidime (CAZ), cefpodoxime, and cefotaxime (CTX) were placed around it in a way that each of these was 15 mm apart. The plates were incubated overnight at 37°C. Blunting of the zone of inhibition around any of the cephalosporin discs adjacent to the cefoxitin disc was considered as a positive result.

Phenotypic confirmatory test (PCT) for detection of ESBLs
This test was performed by the disc diffusion technique as recommended by CLSI (NCCLS). A ≥5 mm increase in the zone diameter for either CAZ or CTX tested in combination with clavulanic acid (CLA), versus its zone when tested alone, confirmed an ESBL-producing organism.
All the media and antibiotic discs used for phenotypic tests were procured from Becton, Dickinson & Co., USA.

DNA extraction
A few (2-3) colonies of the test isolates were inoculated in 5 ml Luria Bertani broth (Becton, Dickinson & Co., USA) and incubated overnight at 37°C. Boiled lysate of each of the cultures was prepared by boiling the culture at 100°C for 10 min and collecting the supernatant after centrifugation at 9838× g for 5 min. The supernatant was used as a source of template DNA.

Detection of ampC genes by multiplex PCR
Detection of plasmid-mediated AmpC β-lactamase genes (ampC) in all the screened positive isolates was carried out by PCR using the method described by Pérez-Pérez and Hanson 2002, [13]. Family-specific primers, as shown in Table 1, were used to detect different families/ types of AmpC β-lactamases in the isolates. Each 50 µl PCR mixture contained 0.2 mM of each dNTPs (Sigma-Aldrich Chemical Pvt. Ltd, Bengaluru, India), 0.5 µM of forward and reverse primers (Sigma-Aldrich Chemical Pvt. Ltd, Bengaluru, India), 1X reaction buffer (New England Biolabs Inc., USA) with 2 mM of MgCl 2 (New England Biolabs Inc., USA), 1.25 U of Taq polymerase (New England Biolabs Inc., USA), and 2 µl of DNA. Nuclease-free water was used in all the reactions and as a negative control. PCR was carried out in a PTC-100™ Programmable thermal cycler (MJ Research Inc.) using the following conditions: 94°C for 3 min, 94°C for 30 s, 64°C for 30 s, and 72°C for 1 min for 30 cycles followed by a final extension at 72°C for 7 min. PCR products were visualized on a 2% ultrapure agarose (Sigma-Aldrich, St. Louis, USA) gel stained with ethidium bromide (Sigma-Aldrich, St. Louis, USA, 10 mgL −1 ).

Nucleotide sequence analysis of ampC genes
Nucleotide sequencing of PCR amplicons was undertaken on both strands using respective PCR-specific primers (Table 1) by dideoxychain termination method using the services of the commercial vendor (Link Biotech, India). Sequence analysis and comparisons were performed using nucleotide BLAST, and sequences were compared against the database of ampC genes present at the NCBI server (http://www.ncbi. nlm.nih.gov).

Detection and characterization of AmpC β-lactamases by isoelectric focusing (IEF)
Characterization of AmpC β-lactamases was carried out by IEF, following the procedure of Matthew et al., 1975, [14] with few modifications. Briefly, crude enzyme extract from test isolates was prepared by subjecting the cell pellet from overnight grown cultures to 20 cycles of freezing at −20°C for 30 min and thawing at 37°C for 15 min. Nitrocefin sensitivity test was performed by loading 50 µl of nitrocefin solution and 10 µl of crude extract of the test organism into a 96-well microtiter plate. The rate of color change was graded on a scale of 5-1. For the score of 5, 1 µl of crude enzyme extract, and for a score of 1, 20 µl of crude enzyme extract were loaded on commercially prepared polyacrylamide gels with a pH range of 3.5-10. Gel electrophoresis was carried out using on a LKB Bromma 2117 Multiphor II electrophoresis unit (Ampholine PAG plate; GE Healthcare Uppsala, Sweden). The gel was run at 1500V, 50mA, and 30W for 90 min. The bands were analyzed by placing a filter paper soaked in nitrocefin and placing it over the gel for 10 min.

Detection of ampC genes
All the screened positive isolates were subjected to multiplex PCR. Of the 34 screened positive isolates of E. coli, 16 were found to show ampC genes of amplified products of 462 bp (CIT type) and 302 bp (EBC), respectively (Fig. 1). Of these 16 PCR-positive isolates, 13 harbored ampC genes of CIT family (EC 2, 3, 4, 10,11,14,15,20,24,26,29,32, and 34) and 3 (EC 1, 19, and 22) of EBC family ( Table 4). None of the tested isolates harbored genes for MOX, ACC, DHA, and FOX families. CIT-type ampC gene was found to be predominant in the PCR-positive isolates (Table 4). Nucleotide sequencing of the CIT family amplicons was found to have 100% homology to CMY-2 and EBC family amplicons to have 100% homology to ACT-1-type AmpC β-lactamases. PCR detected four additional isolates: EC 14, 15, 32, and 34 to produce AmpC β-lactamases as compared to phenotypic test, and all these isolates produced CITtype AmpC enzyme ( Table 3).   (Fig. 2 and Tables 3 and 5). Interestingly, in 12/34 isolates of E. coli, bands of pI 5.4 could be detected, which did not correspond to any of the known AmpC types (data not shown). In addition, 6 of the isolates showed the presence of multiple β-lactamase bands (data not shown).
Our study showed similar results (35% by phenotypic test) to some of the researchers reporting a high incidence of AmpC β-lactamases in E. coli, indicating a rising trend of resistance, which should be a cause of concern.    Amoxicillin-CLA combination is usually given in treating infection caused by β-lactamase-producing isolates because CLA is known to be an active inhibitor of ESBLs. Since CLA can act as an inducer for AmpC β-lactamases, it becomes imperative to check for the presence of inducible AmpC β-lactamases in the clinical isolates. Studies have reported the presence of inducible AmpC β-lactamases in 4-21% of E. coli isolates [25,29]. Our study showed similar results with 16.6% of the AmpC-positive isolates to be inducible; however, more number of isolates need to be tested to show an increasing trend. Hence, testing for the presence of inducible AmpC producers becomes important, as there can be a therapeutic failure if antibiotics inducing the enzymes are administered for treating the infection empirically.
A multiplex PCR which distinguished plasmid-mediated AmpC enzymes from chromosomally mediated and also differentiated between six different families of plasmid-mediated AmpC β-lactamases was used in this study. A number of workers have used PCR to detect the presence of ampC genes E. coli and have reported PCR to be more sensitive than phenotypic methods to detect AmpC-producing isolates [4,[30][31][32]. Our study showed the presence of ampC gene for plasmid-mediated AmpC enzymes in 47% of the screen-positive isolates of E. coli and was found to detect four additional AmpC producers than inhibitor-based method.
In the present study, a high percentage (81.2%) of the PCR-positive isolates of E. coli were shown to produce CIT type of AmpC β-lactamases, followed by 18.75% producing EBC type. Our study corroborates the findings of other studies, showing a high percentage (88%) of E. coli isolates to produce CIT type of AmpC enzymes [28,33,34]. However, there are studies which reported a lower prevalence, 11%, 34%, and 56%, respectively, of the E. coli isolates to be producing CIT-type AmpC β-lactamases [4,35,36]. The presence of EBC family or ACT-1 subtype of AmpC enzymes in E. coli has been reported in many studies; however, EBC-type AmpC β-lactamases was found to be more prevalent in Klebsiella spp. [37][38][39][40]. In the present study, we found ~19% of the inducible AmpC-producing E. coli isolates to be of EBC (ACT-1) type. In our study, inducible AmpC β-lactamases were detected by DAT, IEF as well as PCR.
The inhibitor-based phenotypic test detected less number of isolates to be AmpC positive as compared to the PCR, and this suggests that PCR should be used as the gold standard method for identification of beta-lactamases. However, more number of isolates need to be tested to confirm this.
Detection and characterization of β-lactamases employing IEF were for the first time carried out by Matthew et al. in 1975 [14]. Subsequently, IEF was used for characterizing AmpC β-lactamases in a number of bacterial pathogens [12,[41][42][43]. In the present study, β-lactamase bands of pI values >8.4, corresponding to CIT type, bands of pI value 8.4 corresponding to EBC type, bands of pI values ranging between 6.3-7.2 corresponding to FOX/DHA, and bands of pI value 7.6 corresponding to ACC type AmpC enzymes were observed. These were further confirmed by PCR to be the respective AmpC enzymes except ACC and FOX or DHA types. According to our PCR results, none of the E. coli isolates harbored ampC genes for ACC and DHA families. In comparison to PCR, IEF detected less isolates to produce CIT type but extraisolates to produce ACC and FOX-/DHA-type AmpC β-lactamases. Moreover, bands of pI value 5.4 were detected which did not correspond to any of the known AmpC β-lactamases. This suggests that there may be a possibility that these pI bands correspond to some ESBLs and that some of the tested isolates coproduced ESBLs. It was found that 83% of the AmpCproducing isolates in this study were also producing ESBLs. Many studies have shown the presence of ESBLs along with AmpC enzymes which also showed bands of different pI values [24,27,[44][45][46][47]. In this study, there was one isolate, EC 22, which was found to be positive for inducible AmpC β-lactamases, coproduced ESBL and was found to harbor genes for ACT-1 subtype of AmpC β-lactamase. This coexistence of multiple enzymes in clinical isolates may restrict the therapeutic options available for treating cases infected with such pathogens.

CONCLUSION
Our findings may suggest that there is a need to initiate steps to rationalize the use of the β-lactams and also to have a more vigorous surveillance for detection of such resistance in clinical isolates both at the hospital as well as at the community level. However, a larger set of E. coli isolates along with other nosocomial pathogens need to be tested to have more substantiate data. This constitutes the part of our future studies.