Screening and Identification of Extended Spectrum β-lactamase (ESBL) Pathogens in Urine Sample of UTI Patient’s

Extended spectrum β-Lactamases (ESBL) are enzymes produced by pathogens belonging to Enterobacteriaceae, most commonly Escherichia coli and Klebsiella pneumoniae [1]. They cause serious infections and have high mortality rates. Multidrug-resistant ESBL E. coli (CTX-M enzyme type) has emerged as an important cause of UTI [2]. ESBL strains are capable of efficiently hydrolyzing many beta-lactam antibiotics including 3rd generation cephalosporins and monobactams. β-lactamase inhibitors (clavulanic acid, sulbactam, and tazobactam) generally inhibit ESBL producing strains [3]. It is increasingly being reported that they are acquiring a transmissible form of antibiotic resistance. This indicate that penicillins and cephalosporins which have been used for many years are no more effective against ESBL positive [4]. β-lactamase enzymes undergo changes at nucleotide and amino acid sequences, accordingly, four classes A, C and D (serine based mechanism) and class B (metallo β-lactamase) are recognized. This can be correlated to ESBL genes CTX-M, TEM, SHV, OXA and AMP-C types [5]. ESBL producing pathogenic enterobacteria poses a serious antibiotic management problem, as these genes are easily transferred from one organism to the other via plasmids [6]. ESBL-containing organisms, which were hitherto found mainly in hospitals, are now becoming fairly common as community acquired infections, especially those of the urinary tract [7]. Findings from a south Indian study documents high prevalence of multidrug resistant CTX-M type ESBL among nosocomial isolates [8]. A report from north Indian tertiary referral hospital documents the presence of blaNDM-1 gene from clinical isolates of Acinetobacter baumannii [9]. Their importance of causing nosocomial and community acquired infections draws our attention even more. ESBL producers do cause UTI, respiratory tract infections, endocarditis etc. resulting in increased treatment cost, morbidity and mortality. In public health, recently, fecal carriage, intestinal colonization, international travel, and household member transmission were also determined to be contributing factors to the spread of ESBL-producing organisms [10-13]. A study from Coimbatore, Tamil Nadu, showed the presence of ESBLs to be 40% and from Nagpur 50% in urinary isolates [14,15]. In another comparative study it was reported that 68.78% of gram negative bacteria was found to be ESBL producers [16]. Carbapenems are considered mainstay antibiotics to treat drug-resistant infections [17]. New antibiotics or new combinations of antibiotics would be required to overcome the current threat posed by ESBL pathogens [18]. At present the spread of multidrug-resistant Gram-negative bacteria in the community is a serious problem which needs to be curtailed. In this study, an attempt was made to screen for the presence of ESBL pathogens and confirm their resistant characteristics by phenotypic detection method.


Introduction
Extended spectrum β-Lactamases (ESBL) are enzymes produced by pathogens belonging to Enterobacteriaceae, most commonly Escherichia coli and Klebsiella pneumoniae [1]. They cause serious infections and have high mortality rates. Multidrug-resistant ESBL E. coli (CTX-M enzyme type) has emerged as an important cause of UTI [2]. ESBL strains are capable of efficiently hydrolyzing many beta-lactam antibiotics including 3 rd generation cephalosporins and monobactams. β-lactamase inhibitors (clavulanic acid, sulbactam, and tazobactam) generally inhibit ESBL producing strains [3]. It is increasingly being reported that they are acquiring a transmissible form of antibiotic resistance. This indicate that penicillins and cephalosporins which have been used for many years are no more effective against ESBL positive [4]. β-lactamase enzymes undergo changes at nucleotide and amino acid sequences, accordingly, four classes A, C and D (serine based mechanism) and class B (metallo β-lactamase) are recognized. This can be correlated to ESBL genes CTX-M, TEM, SHV, OXA and AMP-C types [5]. ESBL producing pathogenic enterobacteria poses a serious antibiotic management problem, as these genes are easily transferred from one organism to the other via plasmids [6]. ESBL-containing organisms, which were hitherto found mainly in hospitals, are now becoming fairly common as community acquired infections, especially those of the urinary tract [7]. Findings from a south Indian study documents high prevalence of multidrug resistant CTX-M type ESBL among nosocomial isolates [8]. A report from north Indian tertiary referral hospital documents the presence of blaNDM-1 gene from clinical isolates of Acinetobacter baumannii [9]. Their importance of causing nosocomial and community acquired infections draws our attention even more. ESBL producers do cause UTI, respiratory tract infections, endocarditis etc. resulting in increased treatment cost, morbidity and mortality. In public health, recently, fecal carriage, intestinal colonization, international travel, and household member transmission were also determined to be contributing factors to the spread of ESBL-producing organisms [10][11][12][13]. A study from Coimbatore, Tamil Nadu, showed the presence of ESBLs to be 40% and from Nagpur 50% in urinary isolates [14,15]. In another comparative study it was reported that 68.78% of gram negative bacteria was found to be ESBL producers [16]. Carbapenems are considered mainstay antibiotics to treat drug-resistant infections [17]. New antibiotics or new combinations of antibiotics would be required to overcome the current threat posed by ESBL pathogens [18]. At present the spread of multidrug-resistant Gram-negative bacteria in the community is a serious problem which needs to be curtailed. In this study, an attempt was made to screen for the presence of ESBL pathogens and confirm their resistant characteristics by phenotypic detection method.

Sample collection
Urine samples were collected from human volunteers having urinary tract infections visiting Health Center, VIT University, Vellore, Tamil Nadu, India. Samples were collected in a appropriately labeled sterile disposable containers following stringent precautions.

Abstract
Extended Spectrum β-Lactamases (ESBL) exhibit most important resistant mechanism challenging broad spectrum of antibiotics, ESBL's hamper current treatment strategies limiting therapeutic options. Hence, it is important to screen those organisms using standard techniques to fix the spread and outcome consequences. Aim and objective of our perspective cohort study was to isolate and to identify the resistant ESBL pathogens consecutive ESBL positive strains were recovered by Disc Diffusion Test (DDT) and 90% of the isolates were E. coli and Klebsiella pneumoniae. Further, antibiotic susceptibility assay against third line cephalosporins like cefotaxime (30 g), cefotaxime/clavulanic acid (30 µg/10 µg), ceftazidime (30 µg), ceftazidime/clavulanic acid (30 µg/10 µg), ampicillin (30 µg) and amikacin (30 µg) were tested by CLSI guidelines from NCCLS (National Committee for Clinical Laboratory Standards). Minimum Inhibitory Concentration (MIC) was determined against E-test ESBL strips containing cefotaxime (CT), cefotoxime/clavulanate (CTL), ceftazidime (TZ), ceftazidime/Clavulanate (TZL). E. coli (ATCC 25922) was used as negative control and (ATCC 700603) as a positive control strain. Controls were satisfactory in comparison to standard chart. Our study results unveil a very high spread of such super bugs in health centers. The empiric use of 3 rd generation cephalosporins should be curtailed, as it is associated with increased risk of ESBL production. The necessity for continued antimicrobial resistance surveillance to prevent higher degree of resistance is justified in the light of our findings.

Screening and Identification of Extended Spectrum β-lactamase (ESBL) Pathogens in Urine Sample of UTI Patient's
Volunteers were instructed for clean catch mid-stream urine before collection [19]. Pus samples were collected using sterile swabs from human volunteers with genitourinary problems. The collected swabs were transferred in to nutrient broth within 45 minutes and brought to the laboratory.

Isolation and identification of bacterial isolates
The transported nutrient broth was incubated at 37°C for 24 hours. After incubation, one loopful of culture was inoculated into macconkey agar plates for further studies. Simultaneously, 0.01 µl of each urine sample was directly streaked on nutrient broth and incubated at 37°C for 24 hours. Morphologically distinct colonies were selectively sub-cultured on mac-conkey agar and incubated at 37°C for 24 hours to obtain isolated colonies. After incubation period the plates were observed and identified for the bacterial strains. Gram negative bacteria were taken for biochemical test including mannitol motility, triple sugar ion, indole, citrate, MR, VP and carbohydrate utilization tests for identification [20].

Detection of ESBL isolates
Confirmation of ESBL producing strains was made through E-test ESBL strips (AB Biodisk) impregnated with cefotaxime (CT), cefotaxime/clavulanate (CTL) and ceftazidime (TZ), ceftazidime/ clavulanate (TZL) according to manufacturer's instructions. The presence of ESBL was confirmed by the appearance of a phantom zone or deformation of the CT or TZ ellipse or either CT or TZ. MIC is reduced by ≥ 3 log2 dilutions in the presence of clavulanic acid [23].

ESBL confirmation E-strips test (n=10)
E. coli (n=44) 2 Others (n=36) --   Colonies suspected to be coliform based on their gram reaction and cultural morphology (macConkey agar) correlation was identified by cultural and biochemical tests. Culturing of urine sample resulted in isolation of 195 coliform bacteria. All coliform bacteria (n=195) belonging to family Enterobacteriaceae were screened for β-lactamase production. ESBL positives (n=10) were confirmed from collected urine samples by Disc Diffusion Test (DDT). Other gram negative profile includes E. coli (n=44), K. pneumoniae (n=113), Citrobacter sp (n=2), other gram negatives (n=36). Gram negative isolates showed positive for ESBL production is shown in Table 1. Out of 44 E. coli isolates 2 were found to be positive for ESBL production, out of 113 K. pneumoniae isolates 7 were positive for ESBL production and out of 2 Citrobacter sp. one isolate was found to be positive for ESBL production. The growth of K. pneumoniae ATCC 700603 (positive control) and the test isolate (ESBL positive) on MacConkey agar is shown in figure 1A respectively. Large encapsulated colony morphology of K. pneumoniae on MacConkey agar media was observed. Primary screening by Kirby-bauer method for ESBL strains were further confirmed with disc diffusion test. The positive ESBL isolates showed the inhibition zone by disc diffusion is shown in figure 2. ESBL production by positive isolates was further confirmed by E-test ESBL strips and is shown in figure 3. E-test ESBL strips and antibiotic sensitivity profile for positive ESBL isolates using disc diffusion test and E-test are given in table 2. All the ESBL producing isolates showed varying sensitivity towards different third generation antibiotics tested. The present study highlights the importance of routine screening and alsoexplicitly reports the presence of ESBL producers, Multi Drug Resistance (MDR) isolates in small health center setups. and Proteus sp [24,25]. As per these guidelines the ESBL producing organism should be resistant to all cephalosporins except cephamycins and monobactams. β-lactam/β-lactamase inhibitor combinations such as piperacillin/tazobactam and cefaperazone/sulbactam may be reported as sensitive. Non-ESBL-producing organism E. coli ATCC 25922 and a positive ESBL organism Klebsiella pneumoniae ATCC 700603 may be used as controls to perform the tests. ESBL's are mostly detected by disc approximation and double disc synergy methods. For the double-disc synergy test, the antibiotic impregnated discs were placed 30 mm away from each other. ESBL production was considered positive when an enhanced zone of inhibition of β-lactamase inhibitorcontaining disc, ceftazidime/clavulanic acid (30 µg/10 µg) was ≥ 5 mm than the antibiotic containing ceftazidime (30 µg) alone [26,27].

Discussion
However, phenotypic confirmation test should be carried out to ascertain the diagnosis.
Urinary tract infection is one of the commonest bacterial infections in adults. Current update on antimicrobial resistant pathogens is mandatory [28]. The production of beta-lactamase may be of chromosomal or plasmid origin. Plasmid mediated production is often acquired by transfer of genetic information from one organism to another. Such transferable plasmid also codes for resistant determinants to other antimicrobial agents. Hence multidrug resistance is expected to be more common in ESBL producing organisms [29]. In this study ESBL producing urinary pathogens were found to be resistant to third generation cephalosporins, evidencing the worsened situation in health centers due to increased spread of multidrug resistance. An updated standard technique for routine screening of ESBL uropathogens along with conventional antibiogram useful to all health centers is recommended. Carbapenems are the mainstay of therapy but, they are expensive and require prolonged intravenous administration. The circumstances have changed and growing carbapenem resistance is a major clinical concern [30]. Fosfomycin, and tigecycline, colistin and gentamycin have shown to be effective in most cases [31]. The degree of resistance is highly variable among ESBL enzymes [32]. Improved techniques like PCR and DNA sequencing need to be used for detection and typing of different ESBL enzymes. Considering the complicated task of ESBL identification due to their competing resistance over broad spectrum β-lactam antibiotics and increased spread via plasmid mediated transfer there is an urgent need for standard routine screening and detection in all the health centers.

Conclusion
In our cohort study an ESBL producing Citrobacter species resistant to 3 rd -line cephalosporin's, ceftazidime (30 µg), ceftazidime/ clavulonic acid (30 µg/10 µg) were isolated and identified. The results of our study further confirmed that ESBL producing pathogens were more prevalent in urinary tract infections. Degree of resistance is highly variable among ESBL enzymes, hence molecular methods like PCR and DNA sequencing need to be used for detection and typing of different ESBL enzymes.   Antibiotic sensitivity assay of ESBL pathogens isolated from urine samples.