Detection of Beta-Lactamases in Multi-Drug Resistant Uropathogens

Abstract

Multidrug-resistant-ESBL-producing E. coli are on high emergence in UTIs and other infections. Such E. coli are big challenge and responsible for serious threats to healthcare professionals, treatment failures, increased morbidity and mortality, and render beta-lactam antibiotics ineffective. A total of 9391 uropathogenic E. coli (non-repetitive) were isolated and identified on the basis of standard biochemical reactions. The antibiotic susceptibility testing was accomplished by the Kirby-Bauer disc diffusion method by following Clinical Laboratory Standard Institute (CLSI) guidelines. MICs of various antibiotics against ESBL-producing uropathogenic E. coli were evaluated by agar dilution method using guidelines of Clinical Laboratory Standard Institute (CLSI). ESBL production of E. coli was detected by Double Disc Synergy Test (DDST). Among 93901 of uropathogenic E. coli, 5916 (63%) were ESBLs producers. They were 100% resistant to beta-lactams, Ampicillin (AMP), Cefotaxime (CTX), 85% to Ciprofloxacin (CIP), 96% to Nalidixic acid (NA), 81% to Aztreonam (ATM), 81% to Trimethoprim/Sulfamethoxazole (SXT), and 58% to Amoxicillin-Clavulanate (AMC), 22% to Piperacillin-Tazobactam (TZP), 22% to Cefoperazone-Sulbactam (SCF), 21% to Nitrofurantoin (F), 16% to Amikacin (AK), 4% to Fosfomycin (FOS) and 2% to Imipenem (IPM). None of the ESBLs-producing E. coli was found resistant to Polymyxin B (PB). Results of MICs were in agreement with the antibiotic disc diffusion results. The objective of the study was to determine the prevalence of ESBL-producing E. coli, co-existence of antibiotics resistance, and to evaluate antibiotics' effectiveness.

Keywords: ESBLs, MDR, E. coli, UTIs

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Introduction

Multidrug-resistant (MDR) E. coli commonly produce beta-lactamase enzymes to withstand various kinds and generations of beta-lactam antibiotics (1). Extended-spectrum β-lactamase (ESBLs) arbitrated resistance is a rising healthcare issue (2). These enzymes confer resistance to Aztreonam, Oxyimino beta-lactams, and 1st to 4th generations of cephalosporins but can be hindered by the clavulanate, sulbactam, and tazobactam (3). ESBLs are frequently present in both members of Enterobacteriaceae and non- Enterobacteriaceae and are emerged by mutations in plasmid-mediated TEM1, TEM2, and SHV genes (4, 5). However, PER, CTX-M, VEB, and GES are recently evolved ESBLs (6). The ordinary antibiotic sensitivity screening in various clinical labs is unsuccessful to identify the ESBL producers E. coli as they might display false susceptible zone to Ceftazidime, Cefotaxime, and Ceftriaxone (7). The wide distribution of spread of ESBLs producing E. coli strains in hospitals and community reduces the use β-lactam antibiotics, leading to severe treatment failures, obliged to imply extended-spectrum and high-priced antibiotics (3). Co-existence of non-beta-lactams resistance genes (Trimethoprim-Sulfamethoxazole, tetracycline, aminoglycosides, and fluoroquinolones) has been reported in ESBLs producing E. coli which further causes therapeutic failure (6, 7). This study was planned to determine the prevalence of ESBL-producing E. coli, co-existed antibiotics resistance in a hospital, and to evaluate antibiotics' effectiveness against MDR-ESBLs-producing uropathogenic E. coli.

Methodology

During five years, a total of 9391 uropathogenic E. coli (non-repetitive) were isolated and identified on the basis of standard biochemical tests (8). The antibiotic sensitivity test was accomplished by the Kirby-Bauer disc diffusion method by following Clinical Laboratory Standard Institute (CLSI) guidelines (9). The following antibiotic discs were implied; Ampicillin 10µg (AMP), Cefotaxime 30µg (CTX), Amoxicillin+clavulanic acid 20/10µg (AMC), Pipracillin+tazobactam 100/10µg (TZP), Cefoperazone+sulbactam 105µg (SCF), Aztreonam 30µg (AZT), Imipenem 10µg (IPM), Ciprofloxacin 5µg (CIP), Amikacin 30µg (AK), Trimethoprim/sulfamethoxazole 25/23.75µg (SXT) and Polymyxin B 300µg (PB).

Determination of Minimum inhibitory concentrations (MICs)

MICs of Ampicillin (AMP), Amikacin (AK), Ceftazidime (CAZ), Cefotaxime (CTX), Ciprofloxacin (CIP), and Imipenem (IPM) were determined against urinary isolates of E. coli by incorporating twofold increasing concentration of antibiotics in molten Muller Hinton agar by using the guidelines of Clinical Laboratory Standard Institute (CLSI). For the standardization of experiment, quality control strains E. coli ATCC25922 were used (10).

Screening for ESBLs

Third-generation cephalosporin-resistant E. coli were subjected to ESBL screening by double disc synergy test (DDST). Standardized 0.5 McFarland suspension of E. coli was prepared in normal saline. Isolated colonies of previously screened GNB were picked by straight wire and dispensed in normal saline tubes then swabbed on MHA plates to prepare the lawn of culture. Disc of amoxicillin-clavulanate (20/10µg) in the centre, cefotaxime (30µg) disc on one side, and aztreonam (30µg) on the other side were dispensed about 20-25mm apart to each other. Plates were incubated at 37°C for 18-24 hours under aerobic conditions. The expansion of cefotaxime and aztreonam zone adjacent to the amoxicillin-clavulanate disc was considered as ESBLs positive (11). E. coli ATCC 25922 was used as negative controls.

Figure 1: Double disc synergy test showing extension of Cefotaxime and Aztreonam zones toward amoxicillin clavulanate

Results

Among 93901 of uropathogenic E. coli, 5916 (63%) were found ESBLs producers. E. coli resistant to third-generation cephalosporins as determined by disc diffusion method, agar dilution method, and further confirmed by double disc synergy test (DDST) as indicated in Fig. 1 (a and b). Figures show patterns of inhibition zone extension of Cefotaxime (CTX) and Aztreonam (ATM) toward amoxicillin plus clavulanate hence, considered ESBL positive. ESBL-producing E. coli was found highly prevalent in urine specimens (63%) Fig. 2.

Fig. 2 Frequency of ESBLs-producing uropathogenic E. coli versus Non-ESBLs producing E. coli

ESBL-producing E. coli isolates were 100% resistant to beta-lactams, Ampicillin (AMP), Cefotaxime (CTX), 85% to Ciprofloxacin (CIP), 96% to Nalidixic acid (NA) (for only urinary isolates), 81% to Aztreonam (ATM), 81% to Trimethoprim/Sulfamethoxazole (SXT), and 58% to Amoxicillin-Clavulanate (AMC). Decreased resistance rates were noticed as 22% to Piperacillin-Tazobactam (TZP), 22% to Cefoperazone-Sulbactam (SCF), 21% to Nitrofurantoin (F), 16% to Amikacin (AK), 4% to Fosfomycin (FOS) and 2% to Imipenem (IPM). None of the ESBLs-producing E. coli was found resistant to Polymyxin B (PB) as shown in Fig. 3.

Fig. 3 Antibiotic resistance profiles of ESBL-producing uropathogenic E. coli

Representatives of ESBL-producing E. coli isolates (n=50), 10 from each of the 5 years were selected for determination of minimal inhibitory concentrations (MICs) against Ampicillin (AMP), Amikacin (AK), Ceftazidime (CAZ), Cefotaxime (CTX), Ciprofloxacin (CIP), Fosfomycin (FOS) and Imipenem (IPM). Results of MICs were interpreted according to guidelines of CLSI. Fig. 3 depicts lower MICs of Imipenem (IPM), Amikacin (AK), and Fosfomycin (FOS) against isolates of ESBL-producing E. coli, and higher MICs for Ampicillin (AMP), Cefotaxime (CTX), Ciprofloxacin (CIP) and Ceftazidime (CAZ). Accordingly, none of the isolates was found susceptible to Ampicillin (AMP), Cefotaxime (CTX), Ceftazidime (CAZ), and Ciprofloxacin (CIP).

Fig. 3: MICs of various antibiotics against ESBL-producing E. coli

Discussions

Extended-spectrum beta-lactamases (ESBLs) appear frequently in Enterobacteriaceae e.g. Escherichia coli. These enzymes are clinically significant because they inactivate penicillins, monobactams, and cephalosporins; important antibiotics, given as first-line antibiotics to treat many seriously patients of UTIs. Delayed detection and improper treatment of deadly infections generated by ESBL producers with cephalosporin has been related with increased mortality. Many ESBL-producing E. coli are multi-drug resistant to non-beta-lactams like quinolones, Trimethoprim-Sulfamethoxazole, and aminoglycosides reducing the treatment choices (12). The sudden rise of ESBL-producing bacteria is an increasing problem and has been characterized as a pandemic (13). In this scenario, proper and correct detection of ESBL is mandatory in clinical microbiology laboratory (14). According to Kumar et al. 2014, 66% of E. coli in blood and 55% of E. coli in urine were recognized as ESBL-producers (15). In another study accomplished in Islamabad, Pakistan, reported high prevalence rate 53% of ESBL-producing E. coli in urine. While higher frequency of UTI-associated ESBL-producing E. coli (63%) was found in present study. Antibiotic-resistant profiles of ESBL-producing E. coli of urine revealed the instant failure of beta-lactams (Ampicillin and Cefotaxime), quinolones (Nalidixic acid and Ciprofloxacin), Aztreonam and Trimethoprim-Sulfamethoxazole. Hence, these antibiotics are not good choice for the treatment of Uropathogenic ESBLs-producing E. coli. Such antibiotic resistance pattern is due to the co-existence of ESBLs and other antibiotic resistance genes such as quinolones and Trimethoprim-Sulfamethoxazole on plasmid or on E. coli chromosome. The high prevalence of increased emergence and evolution of such MDR-ESBLs producing E. coli is due to the selective pressure of low levels of cephalosporins and other antibiotics, misuse of antibiotics, and natural selection of antibiotic-resistant mutants (16). However, low resistance rates were noticed as 22% to Piperacillin-Tazobactam (TZP), 22% to Cefoperazone-Sulbactam (SCF), 21% to Nitrofurantoin (F), 16% to Amikacin (AK), 4% to Fosfomycin (FOS) and 2% to Imipenem (IPM). None of the ESBLs-producing E. coli was found resistant to Polymyxin B (PB). Piperacillin-Tazobactam and Cefoperazone-Sulbactam are satisfactory but few ESBLs type can hinder their activity. The most effective antibiotic treatment choices for uropathogenic ESBLs-producing E. coli are Polymyxin B, Imipenem, Fosfomycin, and Amikacin in sequence (17, 18, 19). The findings of this study are in agreement with that of Taneja and Sharma, 2008 and Ahmed et al. 2015 (14, 18). The antibiotic resistance patterns of representatives of ESBL-producing E. coli were verified or counter-checked by MICs that displayed the higher MICs values against Ampicillin, Cefotaxime, Ciprofloxacin, and Ceftazidime and lower MICs values against Imipenem, Amikacin and Fosfomycin Fig. 3. The results point out the therapeutic importance of Imipenem, Fosfomycin, and Amikacin against uropathogenic ESBL-producing E. coli (20).

Conclusions

The increase in emergence of MDR-ESBL type uropathogenic E. coli was recognized which could be the consequence of overuse of cephalosporins. The best treatment options for UTIs associated MDR-ESBL producing E. coli are Polymyxin B, Imipenem, Fosfomycin, and Amikacin.

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21. AMP 0.06µg/ml 0.125µg/ml 0.25µg/ml 0.5µg/ml 1 µg/ml 2µg/ml 4µg/ml 8µg/ml 16µg/ml 32µg/ml 64µg/ml 128µg/ml 256µg/ml 512µg/ml 50 50 5050 50 50 50 50 50 50 50 45 35 35 AK 0.06µg/ml 0.125µg/ml 0.25µg/ml 0.5µg/ml 1 µg/ml 2µg/ml 4µg/ml 8µg/ml 16µg/ml 32µg/ml 64µg/ml 128µg/ml 256µg/ml 512µg/ml 50 50 50 45 40 40 25 20 15 5 5 5 5 4 CAZ 0.06µg/ml 0.125µg/ml 0.25µg/ml 0.5µg/ml 1 µg/ml 2µg/ml 4µg/ml 8µg/ml 16µg/ml 32µg/ml 64µg/ml 128µg/ml 256µg/ml 512µg/ml 50 50 50 50 50 50 50 50 40 40 35 30 30 25 CIP 0.06µg/ml 0.125µg/ml 0.25µg/ml 0.5µg/ml 1 µg/ml 2µg/ml 4µg/ml 8µg/ml 16µg/ml 32µg/ml 64µg/ml 128µg/ml 256µg/ml 512µg/ml 50 50 50 50 50 50 50 45 40 40 35 35 30 25 CTX 0.06µg/ml 0.125µg/ml 0.25µg/ml 0.5µg/ml 1 µg/ml 2µg/ml 4µg/ml 8µg/ml 16µg/ml 32µg/ml 64µg/ml 128µg/ml 256µg/ml 512µg/ml 50 50 50 50 50 50 50 50 45 45 45 35 35 30 FOS 0.06µg/ml 0.125µg/ml 0.25µg/ml 0.5µg/ml 1 µg/ml 2µg/ml 4µg/ml 8µg/ml 16µg/ml 32µg/ml 64µg/ml 128µg/ml 256µg/ml 512µg/ml 50 50 48 40 35 30 30 25 25 20 5 2 1 1 IPM 0.06µg/ml 0.125µg/ml 0.25µg/ml 0.5µg/ml 1 µg/ml 2µg/ml 4µg/ml 8µg/ml 16µg/ml 32µg/ml 64µg/ml 128µg/ml 256µg/ml 512µg/ml 50 50 50 35 35 15 5 5 5 5 5 4 2 2 Conc. of antibiotics