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The health agenda

The Health Agenda, Volume 3. Issue 2. April, 2015 ORIGINAL ARTICLE
Prevalence of extended spectrum beta-lactamases (ESBLs) among uropathogenes
at a tertiary care hospital in Tripura
Authors: Anup Saha, Tapan Majumdar, Arunabha Dasgupta and Purnima Saumandal
Corresponding author: Dr. Anup Saha
Tutor, Department of Microbiology
Tripura Medical College and Dr. BRAM Teaching Hospital Hapania, Agartala, Tripura 799014 Mail ID: ABSTRACT
Background: Extended spectrum beta-lactamases (ESBLs) are β-lactamases which can hydrolyse the
penicillin and cephalosporin group of antibiotics. Emergence of ESBLs in urinary tract infections threatens to
failure of presumptive therapy. Objectives: To culture, isolate and identify bacterial pathogens causing
urinary tract infection, to evaluate antibiotic sensitivity pattern of isolated bacteria, and to detect ESBLs
among isolated organisms. Methodology: This was a hospital based cross-sectional study in which 260
midstream urine samples were collected from March 2012 to February 2013 from clinically suspected UTI
patients of various departments. All the samples were processed as per standard microbiological protocol
and antimicrobial susceptibility was assessed by Kirby-Bauer method. Further ESBLs was detected by double
disc synergy and combined disc diffusion test. Results: A total of 112 bacterial pathogens were isolated.
Escherichia coli (48.21%) was the predominant bacteria followed by Klebsiella pneumoniae (33.92%).
Nitrofurantoin, ofloxacin, amikacin showed good sensitivity, whereas, amoxycillin showed very poor
sensitivity pattern. Overall sensitivity of third generation Cephalosporins was <50%. Prevalence of ESBLs was
found 56.25%, and Klebsiella pneumoniae (74%) was major ESBLs producers followed by Escherichia coli
(57%). Conclusion: Antibiotic stewardship programme and active surveillance of hospital circulating strains
is need of hour to combat this emergent situation.
Key words: Extended spectrum beta-lactamases, Urinary tract infection, Uropathogenic E. coli
dramatic increase in their prevalence worldwide in a very short span of life. (3) The prevalence of ESBL producing organisms among clinical isolates cephalosporins into clinical practice in the early varies from 20-71% in India and 8-45% 1980s was heralded as a major breakthrough in worldwide. (4,5 ) the fight against beta lactamase-mediated bacterial resistance to antibiotics. However, the Urinary tract infection (UTI) is most common hope did not last long and the emergence of infectious presentation in community and hospital extended spectrum beta-lactamases (ESBLs), was set-up since long time. (6) Many organisms can soon discovered. (1) ESBLs are β-lactamases infect urinary tract, but by far the most common capable of conferring bacterial resistance to the agents are the gram-negative bacilli. Escherichia penicillins, first, second, and third-generation coli cause 80% of acute infections. Other gram- cephalosporins, and aztreonam (but not the negative bacilli, Proteus and Klebsiella spp. and cephamycins or carbapenems) by hydrolysis of occasionally Enterobacter spp. accounts for these antibiotics, and which are inhibited by β- uncomplicated UTI. (2) Nosocomial UTI are most lactamase inhibitors such as clavulanic acid. (2) likely to be caused by Escherichia coli, Klebsiella These enzymes are coded by plasmid and their Staphylococcus ability to spread to other bacteria has led to Pseudomonas aeruginosa, Enterococci spp. (7) The Health Agenda, Online ISSN No: 2320-3749
Saha A, Majumdar T, Dasgupta A, et al.: Prevalence of extended spectrum ESBLs among uropathogenes The rapid increase of resistance to broad nitrofurantoin 300 µg, co-trimoxazole 1.2 µg / spectrum beta-lactams among uropathogens has 23.8 µg, amoxycillin 20 µg, ciprofloxacin 5 µg, recently become a major problem globally. It leads ofloxacin 5 µg, gentamicin 10 µg, amikacin 30 µg, to antibiotic ineffectiveness, increased severity of cephalexin 30 µg, cefotaxim 30 µg, ceftazidime 30 illness and cost of treatment. (8) µg, ceftriaxone 30 µg (Himedia, Mumbai, India). (9) Among gram-negative bacilli, ESBLs producers Though non-responses to beta-lactam group of were suspected as per CLSI guidelines if the zone antibiotics are being reported by clinicians, there sizes for the cephalosporins like cefotaxime (30 is no published data for the state of Tripura. μg) ≤27 mm, ceftazidime (30 μg) ≤22 mm, Therefore, this study was undertaken to evaluate ceftriaxone (30 μg) ≤25 mm. (12) Further the prevalence of ESBLs among bacteria causing confirmation of ESBLs production was done by urinary tract infections with the following double disc synergy test (DDST) and combined objectives: 1. To culture, isolate and identify disc diffusion test. bacterial pathogens causing urinary tract infection; 2. To evaluate antibiotic sensitivity DDST: A disc of amoxyclave (20 μg amoxycillin +
pattern of isolated bacteria; and 3. To detect 10 μg clavulanate) was placed on the surface of ESBLs among isolated organisms. the Mueller-Hinton agar (MHA); then, discs of cefotaxime (30 μg) and ceftazidime (30 μg) was METHODOLOGY
kept 20 mm apart from the amoxyclave disc This was a hospital based cross-sectional study (centre to centre). The plates were incubated at carried out at Agartala Govt Medical College and 350c overnight. The enhancement of the zone of GB Pant Hospital for the period of one year (March inhibition of the cephalosporin disc towards the 2012 to February 2013). Study was undertaken clavulanic acid disc was taken as evidence of after approval of institutional ethical committee. ESBLs production. (13) 260 urine samples were collected from suspected Combined disc diffusion test: A sub-culture of
UTI patients of various clinical departments. the test organism was swabbed onto Mueller- Freshly voided mid stream specimen of urine was Hinton agar plate. Ceftazidime disc containing 30 collected in sterile wide mouthed container. (9) μg and ceftazidime and clavulanic acid (20 + 10 The patients having indwelling catheter, samples μg) were placed at a distance of 30 mm from each were collected from the catheter by aspirating other. Plates were incubated at 370c for overnight with sterile syringe and needle after disinfecting and results were read. Increase in zone diameter the area of the catheter with 70% alcohol. (10) ≥5 mm was inferred as positive and considered as After collection, urine samples were transported ESBL producer. (13) directly to the laboratory and processed immediately. At the end of the study data collection and compilation were done. Recorded data was All the samples were inoculated onto blood agar analysed using SPSS version 15.0. and MacConkey agar media by standard loop method and kept overnight at 370c in the incubator. Next day isolated colony was identified Out of 260 samples processed, 112 organisms by gram stain and conventional biochemical tests were isolated i.e. positivity rate 43.07%. Most as per standard protocol. (11) Antibiotic sensitivity common bacteria isolated were Escherichia coli test of the isolated organisms was performed on followed by Klebsiella pneumoniae, Proteus Mueller Hinton agar (MHA) by Kirby-Bauer disc mirabilis, Enterococcus, Staphylococcus aureus etc. diffusion method following clinical and laboratory (Figure 1). Age and sex distribution of culturally standards institute (CLSI) guidelines with confirmed UTI cases are depicted in Figure 2. The Health Agenda, Volume 3. Issue 2. April, 2015 Figure 1: Spectrum of isolated organisms in UTI patients
Figure 2: Age and sex distribution of UTI patients
Antibiotic susceptibility test of isolated bacteria showed poor sensitivity to all organisms. was done by Kirby-Bauer disc diffusion method and it was found that nitrofurantoin and co- susceptibility pattern but third generation trimoxazole are the two first line drugs which cephalosporins like ceftazidime, cefotaxime, showed highest sensitivity to all bacteria. On the ceftriaxone had poor sensitivity profile. (Table 1 other hand, amoxycillin is the only drug which Saha A, Majumdar T, Dasgupta A, et al.: Prevalence of extended spectrum ESBLs among uropathogenes Table 1: Organism-wise antibiotic sensitivity pattern
Amoxycillin
Ofloxacin
Gentamicin
Amikacin
100% 50% 100% 100% 100% Cephalexin
Ceftazidime
Cefotaxime
Ceftriaxone
Figure 3: Percentage of antibiotic sensitivity pattern
Further, ESBLs production was suspected by (positive for confirmatory test for ESBLs) was study criteria and subsequently confirmed by found to be 56.25% (63/112). Major ESBLs DDST and combined disc diffusion method. producers are Klebsiella pneumoniae (74%) Prevalence of ESBLs producing organisms followed by Escherichia coli (57%). (Figure 4) The Health Agenda, Volume 3. Issue 2. April, 2015 Figure 4: Species-wise percentage of ESBLs
Figure 5: Antibiotic resistance pattern among ESBLs and non-ESBLs uropathogenes
DISCUSSION
In this study, overall culture positivity was found was the second frequent isolate contributing 43.07%, which is very similar to study done by 33.92%, similar to study like Wazait et al.(19) Yengkokpam et al. (14) Percentage of positivity of Nitrofurantoin showed good sensitivity to all the culture isolation is reported to be highly variable major isolates. In present study, more than 50% of as can be seen from study done by Kashef et al. (15) major isolates such as E. coli, Klebsiella and Shalini et al., (16) where culture positivity was pneumoniae, Staphylococcus aureus showed 6.3% and 84.17% respectively. resistance to ciprofloxacin. But, ofloxacin showed a bit lower resistance in comparison to Among the organisms isolated in the study, E. coli ciprofloxacin. Compared to fluroquinolones, was the most frequent pathogen being isolated aminoglycosides' resistance was quite low. All the (48.21%), which is similar to other studies like major pathogens isolated in this study exhibited Das et al, Wattal et al.(17,18) Klebsiella pneumoniae below 50% resistance to amikacin. Saha A, Majumdar T, Dasgupta A, et al.: Prevalence of extended spectrum ESBLs among uropathogenes This study showed high rate of circulating ESBLs antibiotics and active surveillance. (25,26) Further producers in present hospital set-up. The analysis of antibiotic resistance pattern between prevalence of ESBLs shows wide range from 6.6% ESBLs and non-ESBLs isolates, it was found that to 91.6% in different parts of the country which ESBLs producing organisms showed higher was found in studies done by Subha et al. (20) and degree of resistance compared to non-ESBLs. Wattal et al. (18) respectively. Among the 112 tested strains, 56.25% were ESBLs producers, CONCLUSION
which is similar to others studies like Rodrigues et al., Rao et al. and Mathur et al., where, rate of This study showed significant number of ESBLs ESBLs occurrence was found to be 53.3%, 61% producing uropathogenes that might be a matter and 68% respectively. (21-23) Factors which might of concern. To know molecular characterization have led to the high prevalence of the ESBL of ESBLs, further study like DNA probing, producers could be indwelling catheters, invasive polymerase chain reaction, restriction fragment procedures, severity of the illness and excessive length polymorphism and isoelectric focusing is use of cephalosporins. Present study revealed necessary. The knowledge of the resistance highest ESBLs producers are Klebsiella pneumonia pattern of the bacterial strains will help in guiding (74%), followed by Escherichia coli (57%) which an appropriate and judicious antibiotic use. is similar to study done by Kashef et al. (15) Study done by Chaudhuri et al. showed that 79% of E. coli and 70% of Klebsiella spp. were ESBL Authors acknowledge DBT, north-eastern cell for producers, which is in line with our results. (24) In providing necessary fund for the study in form of contrast, studies done outside India like Tsering et MD thesis grant. Authors are very much thankful al. and Nijssen et al. showed lower rates of ESBLs to all the faculty and non-teaching staffs of the in these places which may be due to rational use of department for their valuable support. The Health Agenda, Volume 3. Issue 2. April, 2015 REFERENCES
1. Rich LB, Wiley SH. Outbreak of ceftazidime 10. Bailey and Scott's Diagnostic Microbiology. resistance caused by extended spectrum beta- lactamases at a Massachusetts chronic care Microbiology. p: 12. facility. Antimicrob Agents and Chemother. 11. Mackie and Mc-Cartney. Practical Medical 1990; 34(11):2193-99. Microbiology. 14th ed, Chapter 7, Tests for the 2. Bush K, Jacoby GA, Medeiros AA. A functional
identification of bacteria. P: 131-49. classification scheme for beta-lactamases and its correlation with molecular structure. National Committee for Clinical Laboratory Standards (NCCLS), 2011. Performance information supplement NCCLS document 3. Bradford PA. Extended spectrum beta- M100-S21, 31(1). NCCLS, Wayne, USA, 2011. lactamases in 21th century: characterisation, epidemiology and detection of this resistance 13. Metri BC, Jyothi P, Peerapur BV. The threat. Clin Microb Rev. 2001; 14(4):933-51. prevalence of ESBL among enterobacteriaceae in a tertiary care hospital of north Karnataka, 4. Metri BC, Jyothi P, Peerapur BV. The India. J Clin Diag Res. 2011;5(3):470-75. prevalence of ESBL among enterobacteriaceae in a tertiary care hospital of north Karnataka, India. J Clin Diag Res. 2011;5(3):470-75. al. Antibiotic susceptibility pattern of urinary isolates in Imphal (Manipur), India. Winokur PL, Canton R, Casellas JM, Legakis N. Variations in the prevalence of strains extended-spectrum 15. Antimicrobial lactamase phenotype and characterization of susceptibility patterns of community-acquired isolates from europe, the americas, and the uropathogens in Tehran, Iran. western pacific region. Clin Infect Dis. 2010;4(4):202-6. 2001;32(2):94-103. 16. Shalini, Joshi MC, Rashid MK, et al. Study of
6. Peleg AY, Hooper DC. Hospital-acquired antibiotic sensitivity pattern in urinary tract infections due to gram-negative bacteria. N infection at a tertiary hospital. NJIRM 2011; Engl J Med. 2010; 362:1804-13. 7. Forbes BA, Sahm DF, Weissfeld AS. Infections 17. Das NK, Vaje S. The bacteriology of urinary of urinary tract. In: Bailey and Scotts tract infection and its relationship with diagnostic Microbiology. 12th ed. Mosby Inc. different pathological conditions. J Com USA; 2007:842-55. Disease. 1982;14(4):251-55. 8. Yazdi M, Nazemi A, Mirinargasi M, et al. 18. Wattal C. ESBL - an emerging threat to Genotypic versus phenotypic methods to detect extended spectrum beta lactamases Newsletter 2005. Sir Gangaram Hospital; (ESBLs) in uropathogenic Escherichia coli. Ann Biol Res. 2012;3:2454-58. BW. Management of catheter Colle JG, Dugoid JP, Fraser AG, et al. associated UTI. Curr Opin Infect Dis. Laboratory strategy in the diagnosis of 2010;23(1):76-82. infective syndrome. In: Colle JG, Dugoid JP, Fraser AG, et al (Editors). Mackie and Mc- 20. Subha A, Ananthan S. Extended spectrum beta Cartney Practical Medical Microbiology. 14th lactamase (ESBL) mediated resistance to third ed. Churchill Livingstone. Inc: London; 1996: generation cephalosporin. Ind J Med Micro. 2002;19(2):91-95. Saha A, Majumdar T, Dasgupta A, et al.: Prevalence of extended spectrum ESBLs among uropathogenes 21. Rodrigues C, Joshi P, Jani SH, et al. Detection of 24. Chaudhuri BN, Rodrigues C, Balaji V, et al. β-lactamases in nosocomial gram negative Incidence of ESBL producers amongst gram- clinical isolates. Indian J Med Microbiol. negative bacilli isolated from intra-abdominal 2004;22(4):247-50. infections across India (Based on SMART Study, 2007 Data). J Assoc Phys India. 22. Sridhar Rao PN, Basavarajappa KG, Krishna GL. Detection of extended-spectrum β-lactamase from clinical isolates in Davangere. 25. Tsering DC, Das S, Adhiakari L, et al. Extended Indian J Pathol Microbiol. 2008; 51(4):497-99. spectrum beta-lactamase detection in gram-negative bacilli of nosocomial origin. J Global 23. Mathur P, Kapil A, Das B, et al. Prevalence of Infect Dis. 2009;1:87-92. extended spectrum beta-lactamase producing gram-negative bacteria in a tertiary care 26. Nijssen S, Florijn A, Bonten M, et al. β- Lactam hospital. Indian J Med Res. 2002;115:153-57. susceptibilities and prevalence of ESBL-producing isolates among more than 5000 European enterobacteriaceae isolates. Int J Antimicrob Agents. 2004;24:585-91. Particulars of Contributors:
1. Dr. Anup Saha Tutor, Dept. of Microbiology Source of funding: MD/MS thesis grant
Tripura Medical College and Dr. BRAM Teaching Hospital from DBT, North-eastern cell 2. Dr. Tapan Majumdar Associate Professor, Dept. of Microbiology Conflict of interest: None
3. Dr. Arunabha Dasgupta Associate Professor, Dept. of Medicine Date of Submission: 3 April, 2015
4. Dr. Purnima Saumandal Date of Acceptance: 11 April, 2015
Professor and Head, Dept. of Microbiology Date of Publishing: 12 April, 2015
Agartala Govt. Medical College and GB Pant Hospital

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Effect of tetracycline residues in pig manure slurry on tetracycline-resistant bacteriaand resistance gene tet(M) in soil microcosmsAgersø, Yvonne; Wulf, Gitte; Bräuner, Elvira; Halling-Sørensen, Bent; Jensen, Lars Published in:Environment International Document VersionEarly version, also known as pre-print Citation for published version (APA):Agersø, Y., Wulf, G., Bräuner, E., Halling-Sørensen, B., & Jensen, L. (2006). Effect of tetracycline residues inpig manure slurry on tetracycline-resistant bacteria and resistance gene tet(M) in soil microcosms. EnvironmentInternational, 32, 876-882.