|Year : 2014 | Volume
| Issue : 1 | Page : 38-43
Prevalence of multi, extensively and pan drug resistant uropathogens among the women patients visiting a tertiary care hospital in central India
Trupti Bajpai1, Ganesh S Bhatambare1, Maneesha Pandey2, Meena Varma3
1 Department of Microbiology, Sri Aurobindo Institute of Medical Sciences Medical College and PG Institute, Indore, Madhya Pradesh, India
2 Department of Biochemistry, Indira Gandhi National Open University, New Delhi, India
3 Department of Biochemistry, Sri Aurobindo Institute of Medical Sciences Medical College and PG Institute, Indore, Madhya Pradesh, India
|Date of Web Publication||25-Jun-2014|
Department of Microbiology, Sri Aurobindo Institute of Medical Sciences Medical College, MR.10 Crossing, Indore-Ujjain Road, Indore - 453 555, Madhya Pradesh
Source of Support: None, Conflict of Interest: None
Background: The escalation of drug resistance among the uropathogens poses a global threat. The indiscriminate, inadequate and the irrational usage of antimicrobials have contributed to the emergence of resistant strains, which may turn out to be a leading cause for the morbidity and mortality in the developing countries. Aims: The aim of the study was to provide an outlook on the prevalence of drug resistance among the isolated uropathogens. Subjects and Methods: The present study was carried out in the department of Microbiology of a teaching tertiary care hospital from July 2013 to September 2013. A total of 314 urine samples were tested, out of which 115 samples were culture positive. All the uropathogenic isolates were identified up to species level by conventional methods. Antibiotic sensitivity testing was done through disc diffusion method and all interpretations were done in accordance with the Clinical and Laboratory Standards Institute (CLSI)-2013 guidelines. Simultaneously, the extended-spectrum beta-lactamases (ESBL), Amp C and carbapenemase production mechanisms were also detected among the various isolates. Antibiotic resistance profile of the various Gram negative isolates was evaluated and the percentage prevalence of multi drug resistant(MDR), extensively drug resistant(XDR) and pan drug resistant(PDR) isolates was determined. Results: Out of the 314 urine samples tested, 120 isolates were detected among which 91 isolates were Gram negative bacilli. Among the 91 Gram negative isolates, 69 (75.8%) were MDR, 11 (12%) were XDR whereas 2 (2.1%) were PDR isolates. Conclusions: Before prescribing an empirical anti microbial therapy, an in depth knowledge of the etiology, the predisposing factors, the culture positivity and the continued evaluation of the susceptibility patterns of the uropathogens to the traditional as well as new antimicrobials is essential to avoid irrational drug usage and to ascertain the optimal prophylactic therapy.
Keywords: Multi drug resistant, pan drug resistant, uropathogens, extensively drug resistant
|How to cite this article:|
Bajpai T, Bhatambare GS, Pandey M, Varma M. Prevalence of multi, extensively and pan drug resistant uropathogens among the women patients visiting a tertiary care hospital in central India. Int J Health Syst Disaster Manage 2014;2:38-43
|How to cite this URL:|
Bajpai T, Bhatambare GS, Pandey M, Varma M. Prevalence of multi, extensively and pan drug resistant uropathogens among the women patients visiting a tertiary care hospital in central India. Int J Health Syst Disaster Manage [serial online] 2014 [cited 2021 Jan 22];2:38-43. Available from: https://www.ijhsdm.org/text.asp?2014/2/1/38/135357
| Introduction|| |
Antibiotic resistance is a worldwide problem that can cross international boundaries and spread between continents very easily and speedily. World health readers have described antibiotic resistant organisms as "nightmare bacteria" that pose a "catastrophic threat" to people in every country in the world. The use of antibiotic is the single most important factor leading to antibiotic resistance around the world. 
Urinary tract infections (UTI) form the major bulk of the reason for prescription of antibiotics to public.  However, half of all the antibiotics prescribed are either not needed or are not optimally effective as prescribed.  Studies report that significant amount of antibiotic use is unwarranted and there exists large variation in the antibiotic prescription among the physicians without obvious reasons, even within the similar geographical zones. Unwarranted antibiotic usage contributes to resistance problems, unnecessary side-effects and enormous costs. Avery common condition like UTIs are not managed in general practice,  either due to lack of proper microbiological investigations or due to unwillingness of physicians for routine microbiological investigations. This leads to an incomplete and improper reports being sent to patients, ultimately resulting into treatment failures, increased length of hospital stay, increase in the term and magnitude of morbidity, high rates of mortality, high treatment costs and above all the emergence of drug resistance in the community. 
Emergence of resistance to multiple antimicrobial agents among pathogenic bacteria has become a significant public threat, since there are very few or sometimes even no antimicrobial agents available for infections caused by these bacteria. , Until recently the term like multi-drug resistance (MDR) was being used in biomedical literature to describe Mycobacterium tuberculosis. , A group of international experts came together by a joint initiative of European Center for Disease Prevention and Control (ECDC) and the Centers for Disease Control and Prevention (CDC) to characterize different pattern of resistance found in health care-associated, antimicrobial-resistant bacteria. According to ECDC and CDC, MDR is defined as non-susceptibility to at least one agent in three or more antimicrobial categories, extensively drug resistant (XDR) is defined as the non-susceptibility to at least one agent in all but two or fewer antimicrobial categories while pan drug resistant (PDR) is defined as non-susceptibility to all agents in all antimicrobial categories for each bacterium. ,
During the last decade, the efforts to combat MDR microorganisms mainly focused on Gram positive bacteria. Unfortunately, the growing problems of MDR Gram negative bacteria were not paralleled with the development of adequate therapeutic options. , Thus, to prevent the world from entering into pre-antibiotic era, it a right time to intensify attention towards Gram negative resistance.
The aim of the present study is to describe the common uropathogens existing in community and hospital settings and to evaluate their antimicrobial resistance profile in order to categorize them into MDR, XDR and PDR organisms.
| Subjects and Methods|| |
The present prospective study was carried out from July 2013 to September 2013 in the department of Microbiology of a teaching tertiary care hospital. The study protocol was approved by the institutional ethical committee. A total of 314 non-repetitive, clean catch, midstream urine samples from women patients were collected in sterile containers. Semi-quantitative culture of urine was done by a calibrated-loop method  on a UTI chromogenic media.  The culture plates were incubated at 37 o C for 18-24 hours under aerobic conditions. Identification of bacterial growth was confirmed by standard microbiological and biochemical techniques. ,
Antibiotic sensitivity testing (AST) was performed by the Kirby-Bauer disc diffusion method on Mueller-Hinton agar.  The following antibiotics were tested: Amikacin (30 μg), gentamycin (10 μg), ampicillin (10 μg), piperacillin (100 μg), ticarcillin (75 μg ), levofloxacin (5 μg), norfloxacin (10 μg), nitrofurantoin (300 μg), tetracycline (30 μg), co-trimoxazole (1.25/23.75 μg), aztreonam (5 μg), cefazolin (30 μg), cefotaxime (30 μg), ceftazidime (30 μg), cefepime (30 μg), cefoxitin (30 μg) piperacillin-tazobactum (100/10 μg), colistin (10 μg), imipenem (10 μg) and meropenem (10 μg). Dehydrated media and antibiotic discs were procured from Hi-Media, Mumbai, India. The control strains used during the study were Escherichia More Details coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27853. The selection of antibiotics and interpretation of inhibition zone sizes was done according to Clinical Laboratory Standards Institute (CLSI-2013) guidelines. 
Phenotypic extended spectrum beta lactamase (ESBL) and Amp C confirmatory tests were also included in the routine susceptibility test.  While performing antibiotic sensitivity testing, ceftazidime plus clavulanic acid (30/10 μg) and cefotaxime plus clavulanic acid (30/10 μg) discs were also included along with ceftazidime (30 μg) and cefotaxime (30 μg) discs on Mueller-Hinton agar. Organism was considered as ESBL producer if there was a ≥5 mm increase in the zone diameter of ceftazidime/clavulanic acid disc and that of ceftazidime disc alone and or ≥5 mm increase in the zone diameter of cefotaxime/clavulanic acid disc and that of cefotaxime disc alone. Escherichia coli 25922 and a known in-house ESBL producer were used as negative and positive controls, respectively. , [Figure 1].
|Figure 1: Percentage distribution of various uropathogens among the women patients|
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Phenotypic Amp C screening and confirmatory tests were carried out simultaneously. Since there are no published criteria for phenotypic screening or confirmatory test for Amp C beta-lactamases, isolates showing blunting of ceftazidime or cefotaxime zone of inhibition adjacent to cefoxitin disc or showing reduced susceptibility to either of the above test drugs (ceftazidime or cefotaxime) and cefoxitin were considered as "screen positive" for Amp C detection,  whereas cefoxitin-cloxacillin double-disc synergy test was used to confirm Amp C production by the uropathogenic isolates. These isolates were simultaneously confirmed by placing the cefoxitin-cloxacillin (30 μg/200 μg) and cefoxitin (30 μg) at 15 mm apart on Mueller-Hinton agar plate pre-inoculated with the test strain and incubated at 37 o C for 18-24 hours. A difference in the cefoxitin-cloxacillin inhibition zones minus the cefoxitin zones alone, of ≥4 mm. was considered indicative for Amp C production.  [Figure 2].
Modified Hodge Test was performed simultaneously to detect carbapenemase-producing uropathogens. A lawn culture was prepared on Mueller-Hinton agar by using an overnight culture of E.coli ATCC 25922 adjusted to 0.5 McFarland's standard. The plate was left for drying for at least 15 minutes and then a disc of 10 μg meropenem was applied at the center of the plate. The test isolate was streaked from the edge of the ertapenem disc to the periphery of the plate. Four isolates were tested per plate. After an overnight incubation at 37oC under aerobic conditions, the clover-leaf like indentation between the test streaks near the disc was considered as positive for carbapenemase production.  [Figure 3].
The antibiotic resistance pattern of individual isolates was evaluated and then the isolates were categorized as MDR, XDR and PDR. The isolates that were resistant to at least one agent of ≥3 antimicrobial categories were categorized as MDR. Those that were resistant to at least one agent in all but one or two antimicrobial categories were called as XDR while the isolates that were resistant to all agents in all antimicrobial categories were categorized as PDR. 
| Results|| |
In the present study, a total of 314 urine samples from women patients visiting a teaching tertiary care hospital were studied. Out of 314, a total of 115 (36.6%) samples were found to be culture positive from which 120 uropathogenic isolates were detected. This was because five samples had two pathogens that were confirmed through repeat culture. Out of 120 isolates, 91 (75.8%) were found to be Gram negative organisms, 12 (10%) were Gram positive organisms and 17 (14.1%) were found to be Candida species [Figure 4].
Among the various Gram negative isolates, E. coli were 59 (64.8%) that predominated Klebsiella pneumonie, which were 20 (21.9%) followed by Pseudomonas aeruginosa that were found to be 10 (10.9%). The total ESBL producing isolates were 33 (36.2%), Amp C producers were 9 (9.8%) and carbapenemase producers were found to be 6 (6.5%). Only 01 (1.01) isolate produced both ESBL and Amp C and 01 (1.01%) isolate produced both ESBL and carbapenemase. None of the isolates produced both Amp C and carbapenemase. Also none of them produced all the three mechanisms of drug resistance. The maximum ESBL isolates were found among Klebsiella pneumonie that is 8 (40%) followed by E. coli that is 22 (37.2%) and P. aeruginosa that is 2 (20%) among the various Gram-negative bacilli isolates (GNB) isolates.
The detailed insight of antibiotic resistance pattern has been illustrated in [Table 1]. Out of the total 91 Gram negative bacterial isolates, 69 (75.8%) were found to be MDR, 11 (12%) were detected as XDR whereas 02 (2.1%) were detected as PDR isolates. Only, 9 (9.89%) isolates were categorized as sensitive isolates [Table 2].
|Table 1: Antibiotic-resistance profile of the various uropathogenic isolates|
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|Table 2: Distribution of various drug-resistant Gram negative uropathogenic isolates|
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| Discussion|| |
In the current study, 59 (64.8%) of the total 120 Gram negative isolates were identified as E.coli followed by 20 (21.9%) K. pneumonie and 10 (10.9%) P. aeruginosa. Our results resembled those of Sasirekha B, Ramesh et al., Aruna et al., Gales et al., and Mathur et al. who all reported Escherichia coli as the most frequently isolated uropathogen followed by K. pneumonie. ,, Of these, the maximum number of ESBL isolates were amongst K. pneumonie that is 8 (40%) followed by E. coli that is 20 (37.2%) similar to studies by Ramesh et al., (40.2% and 60.07%), Taneja et al.,(51.2% and 78.7%) and Singhal et al., (73% and 62%), who reported higher percentage of K. pneumonie ESBL isolates as compared to E. coli ,, , while Supriya et al., (49.8% and 37.8%), Alipourfard et al., (60% and 40%) and Behroozi et al., (21% and 12%) reported higher percentage of E.coli ESBL isolates as compared to K. pneumonie, respectively. 
In our study, a total of 33 (36.2%) ESBL isolates were detected among the 91 Gram negative isolates. Our results resembled those of Khurana (36.8%) Aggarwal (36%) and Aruna K et al., (34.8%) [22,23] which were inconsistent with those of Kumar et al. (39%), Baby Padmini (39.4%), Taneja et al.,(40.2%), Gururajan et al., (47%), Aruna K. (49.32%), Supriya et al., (49.8), Mathur et al., (58%), Kesavaram et al., (59.1%), Singhal et al., (62%) and Maya et al. (75.5%). ,,, The reports from different studies are indicative of the fact that ESBL producers among the uropathogenic isolates vary greatly geographically and rapidly changing over time. , This may be due to irrational, repeated or sub lethal use of antibiotics. The increasing levels of ESBL's are reducing the clinical utility of this class of antibiotics due to empirical administration of this class of drugs. Routine urine culture sensitivity tests must be requested by the physicians in order to preserve these effective antimicrobials.
Antibiotic resistance offered by different uropathogens is one of the barricades that might hinder the successful treatment. Widespread antibiotic usage exerts a selective pressure that acts as a driving force in the development of antimicrobial resistance. The antibiograms of our Gram negative isolates reveal that ESBL producers possess a higher degree of resistance towards antibiotics that are routinely prescribed against UTI's as compared to non-ESBL producers. The findings were similar to those reported by other authors like Sasirekha B, Ndugulile et al., and Mehrgan et al.  An individual is at significantly high risk of being infected with ESBL's, if he or she is being exposed to antibiotics for prolonged period, was in intensive care unit for long periods, suffers from severe illness, has underwent instrumentation or catheterization or has been a resident of nursing home or institute that frequently use cephalosporins.  Under such circumstances, physicians opt for fluroquinolenes and aminoglycosides, again resulting into resistance against this group of antimicrobials. In our study, fluoroquinoles have shown resistance in the range of 50 to 91% consistent with the previous studies by Manjunath et al., and Akram et al. Aminoglycosides being injectables are used restrictively in community settings and hence show lesser degrees of resistance. ,
Our study revealed 9.8% Amp C producers, which was slightly on the higher side of the studies made by Singhal et al., who reported E. coli and K. pneumonie Amp C producers as 6.97% and 6.18%, respectively. Other authors reported larger number of Amp C producers ranging from 21.1% (Dalela et al.) to 52.6% (Polsfuss et al.). Sasirekha B et al. reported 19.8% Escherichia and 18.2% Klebsiella Amp C producing isolates. Our study reported 1.01% isolates that produced both ESBL and Amp C but other studies revealed a higher number of isolates having both the mechanisms of resistance like Dalela G et al., (3.5%) Sasirekha B et al. (8.8% Escherichia and 7.7% Klebsiella). ,, The co-existence of ESBL and Amp C might be due to dissemination of plasmid-mediated Amp C enzymes among enterobacteriaceae, sometimes in combination with ESBL's. Plasmid-mediated Amp C beta-lactamases typically produced by GNB isolates are usually associated with multiple antibiotic resistance that leaves very few therapeutic options. ,,
The current study reported 6.5% carbapenemase producers. Though several studies reveal 100% sensitivity to carbapenems, (Aruna K, Padmini et al., Ponnusamykonar et al., Sasirekha et al.) , while some of the studies reveal a very high carbapenemase producers like those of Ramana et al., (35.9%), Petropoulou D et al., (41.8%), Castanheira M et al., (66.6%) and Deshpande et al., (91.6%).  Presence of carbapenemase producing isolates among the various clinical isolates is always alarming.
A very high percentage of MDR uropathogens (75.8%) are existing in our setting with a considerable number of extensively drug resistant pathogens (12%). This is the result of all the three mechanisms of resistance prevailing among the uropathogens capable of transmitting the resistance among the other sensitive isolates. It has also been noticed in our case that not only the ESBL producers, but even the non-ESBL producers bear a considerable range of resistance, in fact non-ESBL producers have shown a greater percentage of resistance towards aminoglycosides, piperacillin/tazobactum and carbapenems. Such findings were consistent with Taneja et al.  Not only this, most of the isolates showed drug resistance due to several different reasons. It is well known that mechanism of antimicrobial resistance could happen by enzymatic inactivation, altered receptors or by altered antibiotic transport. (Koneman et al. 1997).  Though carbapenem producers in our case were few (6.5%), overall resistance towards carbapenem (range 13-85%) was very high. Such isolates must be accompanied with additional resistance mechanisms like efflux pump overproduction, decreased opr D, and production of beta-lactamases that usually block the entry of these antibiotics. ,, However, there is a growing reluctance to depend on carbapenems due to emergence of these unforeseen reasons of resistance.
In the current study, we claim 2.1% isolates as PDR but this may or may not be true since we have not tested these isolates against all the antimicrobials of all the classes (according to definition). Hence this could be the limitation of our study. However, through this study we would like to introduce the concept of various categories of resistances among the clinical isolates. If those isolates were truly PDR, then it is really alarming to initiate the intervention and stewardship programs to save the available antimicrobials. Such multiple resistances and their increasing trends can be prevented by avoiding the misuse of antibiotics by healthcare professionals, non-skilled practitioners and general public and inadequate surveillance due to lack of information arising from routine antibiotic sensitivity testing.
| Acknowledgment|| |
The authors wish to thank the chairman and the dean of the institute for providing the healthy working atmosphere during the endeavour. The authors are also grateful towards the technical staff for providing necessary help during the study period.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4]
[Table 1], [Table 2]