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ORIGINAL ARTICLE
Year : 2018  |  Volume : 7  |  Issue : 1  |  Page : 45-50

Comparative analysis of extended-spectrum beta-lactamases producing uropathogens in outpatient and inpatient departments


Department of Microbiology, Dr. Baba Saheb Ambedkar Hospital, New Delhi, India

Date of Web Publication1-Mar-2018

Correspondence Address:
Dr. Renu Gur
Department of Microbiology, Dr. Baba Saheb Ambedkar Hospital, Rohini, New Delhi
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ijhas.IJHAS_33_17

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  Abstract 


INTRODUCTION: Since its discovery in 1980, extended-spectrum beta-lactamases (ESBL) have been prevalent throughout the world. Several new and more complicated resistance mechanisms have emerged and have driven the focus away from ESBLs. The present study was conducted to know the isolated prevalence and resistance profile of ESBL-producing Gram-negative uropathogens and outline an empirical therapy for management of urinary tract infections.
MATERIALS AND METHODS: A study was conducted over a period of 3 months (April to June 2015) in the microbiology department of our 500-bedded tertiary care referral hospital. Urine specimens were processed as per standard guidelines. Antimicrobial susceptibility and ESBL detection were performed by Kirby–Bauer Disc diffusion method and results were interpreted as per CLSI 2015 guidelines. Production of other beta-lactamases was not studied. Klebsiella pneumoniae ATCC 700603 (ESBL positive) and Escherichia coli ATCC 25922 (ESBL negative) strains were used as quality control. P <0.05 was considered statistically significant.
RESULTS: Of the 111 Gram-negative urinary isolates, 60 were ESBL positive (54%). ESBL prevalence was highest in E. coli (73.3%), followed by Klebsiella spp. (11.7%), Morganella spp. (6.7), Proteus mirabilis (5%), Pseudomonas spp. (1.6%), and Citrobacter spp. (1.6%). Most of the ESBL isolates were from outpatient department (OPD) (76.7%) while only 23.3% were from inpatient department (IPD), P < 0.0001, which was extremely significant. When compared to IPD, OPD isolates were found to be more resistant to fluoroquinolones and cotrimoxazole.
CONCLUSION: ESBLs can cause therapeutic failure even when host appears to be susceptible in vitro. Hence, it is important to know its prevalence and antimicrobial susceptibility profile to formulate appropriate antibiotic policy.

Keywords: Antimicrobial susceptibility, extended-spectrum beta-lactamases, urinary tract infection


How to cite this article:
Bharara T, Sharma A, Gur R, Duggal SD, Jena PP, Kumar A. Comparative analysis of extended-spectrum beta-lactamases producing uropathogens in outpatient and inpatient departments. Int J Health Allied Sci 2018;7:45-50

How to cite this URL:
Bharara T, Sharma A, Gur R, Duggal SD, Jena PP, Kumar A. Comparative analysis of extended-spectrum beta-lactamases producing uropathogens in outpatient and inpatient departments. Int J Health Allied Sci [serial online] 2018 [cited 2021 Jan 19];7:45-50. Available from: https://www.ijhas.in/text.asp?2018/7/1/45/226259




  Introduction Top


Urinary tract infection (UTI) is one of the most common reasons why patients seek healthcare.[1],[2] Antimicrobial resistance is a well-recognized global threat to human health. Infections caused by antimicrobial-resistant microorganisms are associated with increased morbidity, mortality, as well as healthcare costs.[3] Extended-spectrum beta-lactamases (ESBLs) are plasmid-mediated enzymes capable of hydrolyzing beta-lactams including oxyimino-cephalosporins and monobactams.[4] The prevalence of ESBL-producing organisms varies widely among different geographic areas.[5],[6],[7] The mortality rate in patients with ESBL-producing UTIs has ranged from 42% to 100%.[8],[9],[10] In India, empirical use and easy availability of antibiotics over the counter has led to widespread resistance among the isolates. Testing for ESBLs will be useful for epidemiological and infection control purposes.[11] Against this background, the present study was conducted to compare the prevalence and resistance profile of ESBL-positive uropathogens among outpatient department (OPD) and inpatient department (IPD) patients of UTI in a tertiary care referral hospital of North Delhi.


  Materials and Methods Top


A study was conducted in the microbiology department of our 500-bedded tertiary care referral hospital. The study was carried out over a period of 3 months (April to June 2015).

Inclusion and exclusion criteria

  1. All consecutive urine culture specimens received in the microbiology laboratory during the study period were included
  2. Any repeat specimen received during the 3-month period was excluded from the analysis
  3. A colony count of ≥105 colony-forming unit/ml was considered significant and processed further. Lesser colony counts were considered only if the clinical history was suggestive.[12]


Specimen processing: Urine specimens received in our microbiology laboratory were inoculated on blood agar and MacConkey agar plates as per se miquantitative method, using standard loop of 4 mm diameter. The culture plates were incubated aerobically at 37°C for 18–24 h. The culture result was interpreted as per standard guidelines. Isolates were examined for colony characteristics, Gram-staining, motility, and biochemical tests.[12]

Antimicrobial susceptibility testing: Antimicrobial susceptibility was performed with commercially available discs (Hi Media, Mumbai) by Kirby–Bauer Disc diffusion method. Results were interpreted as per CLSI 2015 guidelines. All the Gram-negative isolates were included in the study and tested for susceptibility toward nitrofurantoin (300 μg), ampicillin (10 μg), amoxicillin-clavulanic acid (20/10 μg), ceftriaxone (30 μg), cefuroxime (30 μg), cefotaxime (30 μg), ceftazidime (30 μg), cefoperazone-sulbactam (75/10 μg), piperacillin-tazobactam (100/10 μg), chloramphenicol (30 μg), cotrimoxazole (23.75/1.25 μg), ciprofloxacin (5 μg), norfloxacin (10 μg), levofloxacin (5 μg), gentamicin (10 μg), amikacin (30 μg), and imipenem (10 μg). ESBL detection was done as per CLSI guidelines. Klebsiella pneumoniae ATCC 700603 (ESBL positive) and  Escherichia More Details coli ATCC 25922 (ESBL negative) strains were used as controls in this study.[11]

Initial screen test (Disc diffusion)

  • Ceftriaxone (30 μg) ≤25 mm or
  • Ceftazidime (30 μ) ≤22 mm or
  • Cefotaxime (30 μg) ≤27 mm.


Phenotypic confirmatory test (Disc diffusion)

  • Ceftazidime 30 μg
  • Ceftazidime-clavulanic acid 30 μg/10 μg
  • Cefotaxime 30 μg
  • Cefotaxime-clavulanic acid 30 μg/10 μg.


A difference of ≥5 mm between the zone diameters of either of the cephalosporin discs and their respective cephalosporin/clavulanate disc is taken to be phenotypic confirmation of ESBL production [Figure 1].[11]
Figure 1: Phenotypic confirmatory test (disc diffusion) for extended-spectrum beta-lactamase detection

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Statistical analysis

Baseline demographics of the study population have been analyzed using descriptive statistical parameters. Data were expressed by percentages. Fisher's exact test was used to evaluate statistical significance. P < 0.05 was considered as statistically significant.


  Results Top


Out of the 777 urine samples processed for culture, 128 bacteria were isolated, out of which 111 were Gram-negative bacilli. The most common isolate was Escherichia coli (68.5%), followed by Klebsiella spp. (21.6%), Pseudomonas spp. (0.9%), Citrobacter spp. (0.9%), Morganella spp. (3.6%), Proteus mirabilis (2.7%), Acinetobacter (0.9%), and Enterobacter spp. (0.9%). Out of the 111 Gram-negative urinary isolates, 60 were ESBL positive (54%). Among individual Gram-negative isolates, ESBL prevalence was highest in E. coli (73.3%), followed by Klebsiella spp. (11.7%), Morganella spp. (6.7), Proteus mirabilis (5%), Pseudomonas spp. (1.6%), and Citrobacter spp. (1.6%).

Most of the ESBL isolates were from OPD 76.7% while only 23.3% were IPD patients, P < 0.0001 which was extremely significant. Other resistance mechanisms were not tested. Organism-wise prevalence of ESBL isolates between IPD and OPD had no statistically significant difference [Table 1]. There was no statistical significance of ESBL isolates with age and sex of the patients [Table 2].
Table 1: Organism-wise prevalence of extended-spectrum beta-lactamases isolates (n=60)

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Table 2: Age group and sex-wise details of patients with extended-spectrum beta-lactamases-positive and extended-spectrum beta-lactamases-negative isolates

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When the antibiogram of ESBL-producing uropathogens among OPD and IPD patients was compared [Table 3], a higher level of resistance to fluoroquinolones and co-trimoxazole was noticed among OPD patients. Furthermore, chloramphenicol resistance was particularly higher in OPD patients, with no resistance seen in IPD patients. Overall, it was noticed that nitrofurantoin, cefoperazone-sulbactam, imipenem, and aminoglycosides were the least resistant drugs in both IPD and OPD patients.
Table 3: Comparative analysis of extended-spectrum beta-lactamases producing uropathogens from outpatient department and inpatient department patients

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Among the antibiotics tested for E. coli and Klebsiella spp. [Table 4], [Table 5] and [Figure 2], [Figure 3], least resistance was shown by imipenem among both ESBL-producing and non-ESBL-producing isolates. Nitrofurantoin was more resistant in non-ESBL-producing E. coli and Klebsiella spp. ESBL-producing Pseudomonas spp. was sensitive to all antibiotics tested [Table 4]. Only one strain of Acinetobbacter spp. was isolated in our study which was non-ESBL producer and was resistant to all antibiotics tested. Among Proteus mirabilis, all strains were ESBL positive and were 100% sensitive to amikacin, imipenem, piperacillin-tazobactam, and cefoperazone-sulbactam.
Table 4: Percentage resistance of extended-spectrum beta-lactamases-positive isolates toward antimicrobial agents

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Table 5: Percentage resistance of nonextended spectrum beta-lactamases isolates toward antimicrobial agents

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Figure 2: Graphical representation of the antibiotic resistance profile of extended-spectrum beta-lactamase and nonextended-spectrum beta-lactamase uropathogenic Escherichia coli isolates

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Figure 3: Graphical representation of the antibiotic resistance profile of extended-spectrum beta-lactamase and nonextended-spectrum beta-lactamase uropathogenic Klebsiella pneumonia isolates

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{Table 4}


  Discussion Top


In our study, we found 54% of ESBL positivity among Gram-negative urinary isolates. In a previous study conducted in our institute in 2011, prevalence of ESBL-positive isolates was found to be 39.6%.[13] The occurrence of ESBL among clinical isolates vary greatly between different geographical areas and tend to change with time.[14],[15],[16],[17] We observed that among individual Gram-negative isolates, ESBL prevalence was highest in E. coli (73.3%), followed by Klebsiella spp.(11.7%), Morganella spp.(6.75), Proteus mirabilis (5%), Pseudomonas spp.(1.6%), and Citrobacter spp.(1.6%). In another study conducted in Kerala, prevalence of ESBL was found to be 68% among E. coli and 20% in K. pneumoniae isolates.[14] This is in accordance with several other studies conducted over the years.[19],[20],[21] In a study conducted in Nepal, ESBL production was more prevalent in K. pneumoniae strains (16.55%), as compared to that in E. coli strains (13.51%).[15] Most of the ESBL isolates obtained in our study were from OPD (76.7%), which was extremely statistically significant (P=<0.0001). This may be attributed to the observation that E. coli is the most common uropathogen both in the community and hospital setting in our area and with ESBL prevalence also being highest in E. coli (64.8% in OPD patients in our study). However, no statistically significant difference was found between individual ESBL isolates and OPD/IPD patients. Widespread dissemination of ESBL-positive uropathogens in the community is an alarming issue worldwide and needs urgent attention. This situation reflects the impact of indiscriminate use of antibiotics in the form of self-medication and over-the-counter sale of these drugs in the community. Previous use of antimicrobial agents, especially cephalosporins and fluoroquinolones, has been reported as risk factors associated with emergence of ESBL.[22],[23]

In a study conducted by Ogefere et al., the prevalence of ESBL did not differ significantly between Gram-negative bacilli recovered from inpatients and outpatients (P = 0.1833).[24] Antibiotics such as cefuroxime, amoxicillin-clavulanic acid, and ampicillin are 100% resistant to ESBL-positive uropathogens in IPD patients of our study while more than 60% resistance is seen in OPD patients also. These drugs especially amoxicillin-clavulanic acid and cefuroxime are commonly prescribed by unregistered medical practitioners, general practitioners, and pharmacists, not only to treat UTI but several other infections also without following the proper dose and duration of treatment many a times. Even fluoroquinolones and cotrimoxazole have shown high resistance in OPD patients in our study which indicates that these drugs, which are empirically used as first-line drugs to treat community-acquired UTI, should not be used empirically but to be given based on sensitivity report. Drugs such as nitrofurantoin, aminoglycosides, and carbapenems are least resistant drugs in both IPD and OPD patients in our study. Because of the restricted use of aminoglycosides in the community due to their injectable mode of administration, the resistance level is low. Also in hospitals, the carbapenems are reserve drugs and are the last resort for treatment of complicated UTI due to multidrug-resistant pathogens and hence their use is restricted for management of UTI. Nitrofurantoin has specific action in urine as it is concentrated there and therefore is not used to treat other infections except UTI and so resistance level is low.

There was no significant association between ESBL production and age and sex in our study; however, in another study by Chander et al., female showed a higher rate of isolation of ESBL-producing organisms.[15] A higher occurrence of ESBL-producing uropathogens in adult age group has been reported in a few studies.[15],[25]

In our study, we also compared some aspects of ESBL positive isolates with ESBL negative isolates irrespective of whether the patient was from (OPD/IPD) [Table 3] and [Table 4]. Nitrofurantoin was more resistant in non-ESBL-producing E. coli and Klebsiella spp. ESBL-producing Pseudomonas spp. was sensitive to all antibiotics tested [Table 3]. Only one strain of Acinetobacter spp. was isolated in our study, which was non-ESBL producer and was resistant to all antibiotics. Among Proteus mirabilis, all strains were ESBL positive and were 100% sensitive to amikacin, piperacillin-tazobactam, and cefoperazone-sulbactam. In a study conducted in Indore, India, percentage resistance among Klebsiella spp. and E. coli for gentamicin and amikacin was found to be 69% and 38% and 59% and 33%, respectively.[26] The high rate for non-ESBL-mediated ceftriaxone-resistant E. coli and Klebsiella spp. observed in our study may be due to their different mechanisms for resistance such as the production of AmpC beta-lactamase and metallo-beta-lactamase.[27]

None of the E. coli isolates showed resistance to imipenem although 50% non-ESBL-producing isolates of Klebsiella spp. were resistant to imipenem. The high prevalence of other resistance mechanisms including carbapenemases is well known among Klebsiella spp., thus explaining the above findings. Similar results were obtained in other studies as well.[14],[27],[28]

Additional resistance mechanisms among ESBL-producing isolates might lead to masking of ESBL effect and thus its detection in the phenotypic confirmatory test. Genotypic analysis could not be performed in our study, due to limitation of resources; therefore, we might have missed on few ESBL-positive isolates.

According to our national treatment guidelines released in 2016, the carbapenems are currently considered drug of choice for serious infections caused by ESBL-producing organisms.[3]


  Conclusion Top


ESBL are plasmid-mediated beta-lactamases, which can be easily transferred between different organisms and also code for resistant determinants to other antimicrobial agents. Therefore, their prevalence and antimicrobial susceptibility should be monitored at regular intervals to guide treatment and prevent their spread. From our study, we recommend nitrofurantoin with least resistance in OPD patients to be continued as first-line drug for empirical treatment of UTI. For IPD patients, nitrofurantoin and cefoperazone-sulbactam may be considered as first-line empirically, while aminoglycosides and carbapenems being least resistant in both OPD and IPD patients remain reserve drugs and be used for more complicated UTI followed by specific treatment based on culture-sensitivity reports.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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[PUBMED]  [Full text]  


    Figures

  [Figure 1], [Figure 2], [Figure 3]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5]


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