PHENOTYPIC DETECTION OF FAECAL CARRIAGE EXTENDED SPECTRUM BETA LACTAMASE PRODUCING KLEBSIELLA PNEUMONIAE IN HILLA CITY Dr. FATIMA MOEEN ABBAS* *Dept. of Biology, College of Sciences for Women, University of Babylon, Iraq ABSTRACT This study investigates the prevalence of intestinal carriage extended spectrum beta lactamases producing Klebsiella pneumoniae in Hilla city. During the period from April to June 2014, a total of 109 faecal samples were collected from women attending Marjan Teaching Hospital. Twenty-three (21.1%) isolates were detected as K.pneumoniae.Antimicrobial susceptibility testing was performed for all isolates by Kirby-Bauer disk diffusion method. Higher resistant rate was observed for ampicillin 21(91.3%),followed by carbencillin 19(82.6%),the lowest rate 2(8.7%) for imipenem antibiotic. ESBLs were detected by phenotypic confirmatory disc diffusion test using ceftazidime alone and in combination with clavulanic acid. Twenty (86.9%) isolates were ESBL producers. Plasmid profile analysis of four ESBLs producing isolates revealed the presence of three plasmid bands, one large plasmid and two small plasmid bands. KEYWORDS: Klebsiella Pneumoniae, Faecal Carriage, Extended Spectrum Beta Lactamase, Disk Combination Method, Plasmid Profile. INTRODUCTION Multidrug-resistant Gram-negative bacteria are a major public health threat. However, despite intense efforts to limit their spread, the number of multidrug resistant Gram-negative bacteria continues to increase globally (Villar et al.,2013). Bacteria from the genus Klebsiella causes numerous infections in human, which are often treated with β lactam antibiotics. The fundamental mechanism of Klebsiella resistance to penicillin or cephalosporin involves the production of enzymes called extended spectrum β- lactamases (ESBLs). In addition to ß-lactam antibiotics, ESBL producing isolates, often exhibit resistance to other classes of drugs such as aminoglycosides, cotrimoxazole, tetracycline and fluoroquinolones ((Sekosawa et al., 2002; Dinesh et al., 2011). Resistant organisms are now a major problem in the world. Their number is continuously increasing and the therapeutic options are limited (Al-Mayahie, 2013). 53
Extended-spectrum β-lactamases are the enzymes, mostly encoded by plasmids in result of mutation due to which bacteria show resistance to various β-lactam antibiotics including cephalosporins and monbactams(ummadevi et al.,2011). As a results of mutations, numerous types of ESBLs have been described in various species of the Enterobacteriaceae family and other non enteric organisms, including Pseudomonas aeruginosa and Acinetobacter sp. TEM- and SHV-type β- lactamases, mainly produced by Klebsiella pneumoniae, have spread throughout hospital settings, and CTX-M enzymes, mainly produced by Escherichia coli, have become predominant in the community (Mirelis et al.,2003; Pitout and Laupland.,2008). The present study was undertaken to detect intestinal carriage ESBLs producing K.pneumoniae by disk combination method and determining plasmid profile of these isolates in Hilla. Materials and Methods Patients and bacterial isolates During the period of three months from April to June 2014, a total of 109 faecal samples were collected from women aged 30-80 years with gastrointestinal complaints attending Marjan Teaching Hospital in Hilla city / Babylon Province. All samples were cultured on MacConkey's agar (Himedia) and incubated at 37 C for 24 hrs. Bacterial isolates of K. pneumoniae were identified using the standard biochemical tests according to Holt et al (1994), Collee et al (1996) and MacFaddin (2000). Confirmatory identification was carried out by VITEK 2 system following manufacturers instructions. Antimicrobial susceptibility test Antimicrobial susceptibility testing of all isolates was peformed using the standard method, Kirby-Bauer disk diffusion on Mueller- Hinton agar (Oxiod). Antibiotic discs included were Ampicillin (10µg), Carbenicillin (100 µg),cefaclor (10µg), Ceftazidime (30 µg), Ceftriaxone (30 µg), Cefoxitin (30 µg), Imipenem (10 µg), Gentamicin (10 µg), Nalidixic acid (30 µg), Levofloxacin (5 μg) and Tetracycline (30µg). The cultures were incubated at 37 C o for 18 hrs under aerobic conditions and bacterial growth inhibition zones diameter were measured and interpreted in accordance with the Clinical and Laboratory Standards Institute (CLSI) guidelines (CLSI, 2010). Escherchia coli ATCC 25922 was used as the standard strain for antibiotic susceptibility testing. 54
Phenotypic detection of ESBLs This was performed by phenotypic confirmatory test as per the recommendations of CLSI. The ceftazidime (30 µg) discs alone and in combination with clavulanic acid (ceftazidime + clavulanic acid, 30/10 µg discs) were used. An increase of 5mm in zone of inhibition of the combination discs in comparison to the ceftazidime disc alone was considered to be ESBL producer (Wadekar et al.,2013). Plasmid profile Four ESBLs producing K.pneumoniae were selected for plasmid profile. Plasmid DNA of clinical isolates was extracted by salting out method as described by Pospiech and Neuman (1995) with some modification. Plasmids were separated on a 1.5 % agarose gel and electrophorsed at 70 volts for 3hrs. Results and Discussion Bacterial isolates Results of the present study revealed the presence of 23(21.1%) K.pneumoniae isolates recovered from 109 feacal samples. This result is in line with that recorded by Doi et al(2009) who identified 20% of isolates as K.pneumoniae obtained from stool samples.in another study, Taher et al (2015) characterized 35(16.8%) K.pneumoniae isolated from stool and urine samples of patients and 24(19.2%) from stool samples of healthy individuals. Antibiotic susceptibility test In the present study higher resistant rate was recorded for ampicilin 21 (91.3%), the next most resistance antibiotic was carbenicillin 19 (82.6%), followed by ceftazidime and ceftriaxone 18(78.3%) each, 16(69.6%) for cefaclor and cefoxitin, 15(65.2%) for tetracycline, 11(47.8%) for nalidixic acid, 10(43.5%) for gentamicin, 8(34.8%) for levofloxacin and 2(8.7%) for imipenem (table-1). In India, Khadri et al (2007) reported (95%) resistant rate for ampicillin antibiotic among K. pneumoniae strains isolated from patients suffering from intestinal and extra intestinal infection. The higher resistance to ampicillin and carbencillin antibiotics may be due to widespread use of these antibiotics in Hilla hospitals. 55
Table (1): Susceptibility profile of clinical isolates of K. pneumoniae (n =23). Type of antibiotic No. (%) of resistant isolates No. (%) of sensitive isolates Ampicillin 21(91.3) 2(8.7) Carbenicillin 19(82.6) 4(17.4) Cefaclor 16(69.6) 7(30.4) Ceftazidime 18(78.3) 5(21.7) Ceftriaxone 18(78.3) 5(21.7) Cefoxitin 16(69.6) 7 (30.4) Imipenem 2(8.7) 21(91.3) Gentamicin 10(43.5) 13(56.5) Nalidixic acid 11(47.8) 12 (52.2) Levofloxacin 8(34.8) 15(65.2) Tetracycline 15(65.2) 8(34.8) Phenotypic detection of ESBLs producing K.pneumoniae Phenotypic detection of ESBLs was performed by disk combination method. Results revealed that 20(86.9%) isolates were ESBL producers, these isolates exhibited zones enhancement with clavulanic acid, indicating their ESBL production (Figure -1). Results of the present study revealed high prevalence of ESBLs producers among clinical isolates of K.pneumoniae. A research work conducted in Pakistan characterized 213 (99.5%) ESBL producing K.pneumoniae among paediatric patients (Ejaz et al.,2013). In another study, Wadekar et al (2013) reported 9(37.5%) of Klebsiella spp. as ESBLs producer by this method. The early detection of beta lactamase producing isolates would be important for the reduction of mortality rates for patients and also to avoid the intra hospital dissemination of such strains. Simple phenotypic screening tests are proved to be rapid and convenient for their detection in the clinical laboratory.to overcome the problem of emergence and the spread of multidrug resistant organisms, a combined interaction and cooperation between the microbiologists, clinicians and the infection control team is needed (Wadekar et al., 2013). 56
Figure (1):Disk combination test for identification potential ESBLs producing K.pneumoniae.CZA: Ceftazidime ; CZC: Ceftazidime- clavulanic acid. Plasmid profiles of ESBLs-positive K.pneumoniae In the current study, four ESBLs producing K.pneumoniae were tested to determine their plasmid profiles patterns. Since standard plasmid of known molecular weight (size marker) was unavailable, the molecular weights of plasmids isolated in this study were not determined. DNA samples were subjected to gel electrophoresis, which involved separation of small and large plasmids according to their molecular weights. The results showed that all isolates have the same plasmid profiles,one large plasmid (mega plasmid) and two small plasmid bands (Figure-2).This finding similar to a previous study in Hilla by Al-Charrakh (2005) who found that two clinical isolates of K.pneumoniae,6 C and 22 C harbored one large plasmid and two small plasmid bands. In China, another study conducted by Wei et al (2005) revealed that the plasmid patterns of six clinical isolates of K.pneumoniae were identical, exhibiting two plasmids of approximately 95 kb and 200 kb. 1 2 3 4 Mega plasmid Small plasmid bands Figure (2):The plasmid profile of clinical isolates of K.pneumoniae determined by agarose gel electrophoresis. 57
References 1. Al- Charrakh, A. H. (2005). Bacteriological and genetic study on extended- spectrum - lactamases and bacteriocins of Klebsiella isolated from Hilla city. Ph. D. Thesis. College of Sciences, Baghdad University. 2. Al-Mayahie, S.M.(2013). Phenotypic and genotypic comparison of ESBL production by vaginal Escherichia coli isolates from pregnant and non-pregnant women. Ann. Clin. Microbiol.Antimicrob.12:7. 3. Clinical and Laboratory Standards Institute (CLSI). (2010). Performance standards for antimicrobial susceptibility testing; 20 th. Informational Supplement. Approved standard M07-A8. Clinical and Laboratory Standards Institute. 4. Collee, J. G.; Fraser, A. G.; Marmiom, B. P. and Simmon, A. (1996). Mackie and McCarteny Practical Medical Microbiology. 4th ed. Churchill Livingstone Inc., USA. 5. Dinesh, S.; Chandel, Judith A. ;Johnson, Rama Chaudhry et al. (2011). Extended spectrum betalactamase-producing Gram-negative bacteria causing neonatal sepsis in India in rural and urban settings. J. Med. Microbiol.60: 500 507. 6. Doi,M.Y.;Adams-Haduch,J.M.;Shivannavar,C.T.;Paterson,D.L. and Gaddad,S.M. (2009).Raecal carriage of CTX-M-15-producingn Klebsiella pneumoniae in patients with acute gastroenteritis.indian.j.med.res.129.599-602. 7. Ejaz,H.;Ul-Hag,I.Mahmood,S.;Zafar,A.and Javed,M.M.(2013). Detection ofextended-spectrum β- lactamases in Klebsiella pneumoniae: Comparison of phenotypic characterization methods. Pak.J. Med. Sci.29(3).768-772. 8. Holt, J. G.; Krieg, N. R.; Sneath, H. A. Stanley, J. T. and Williams, S. T. (1994). Bergeys manual of determinative bacteriology. 9 th. Ed., Baltimore; Wiliams and Wilkins, USA. 9. Khadri.C; Surekha, S.; Lakshmi, S and G. Narasimha. (2007). Multi drug resistance and lactamase production by Klebsiella pneumoniae. Afr. J. Biotechnol. 6 (15): 1791-1793. 10. MacFaddin, J. F. (2000). Biochemical tests for identification of medical bacteria. 3 rd ed. Lippincott Williams and Wilkins, USA. 11. Mirelis, B.; Navarro, F.; Miro, E. ;Mesa, R.J; Coll, P. and Parts,G. (2003) Community transmission of extended-spectrum beta-lactamase. Emerg. Infect. Dis. 9:1024 1025. 12. Pitout,J.D.D.and Laupland,K.B (2008). Extended-spectrum beta-lactamase-producing Enterobacteriaceae: an emerging public-health concern. Lancet Infect. Dis. 8: 159 166. 13. Pospiech, T. and Neumann, J. (1995). In genomic DNA isolation Kieser eds. John Innes Center. Norwich NR4 7UH. U.K. 14. Sekowska, A., Janika, G., Klyszejko, C., Wojda, M., Wroblewskiand, M and Szymankiewicz, M. (2002). Resistance of K. pneumoniae strains producing and not producing ESBL (extended spectrum β- lactamase) type enzyme to selected non β-lactam antibiotics. Medical Science Monitor. 8: 100-104. 15. Taher,I.A ; Abukhres, O.M. and Shahlol,A.M. (2015): Prevalence and Characterization of Extended- Spectrum β-lactamase-producing Enterobacteriaceae in Brack-Alshati, Fezzan, Libya. EC Microbiology 1: 23-32. 16. Ummadevi, S.; Joseph, N.M.; Kumari, K.; Easow, J.M.; Kumar, S.; Stephen, S. et al.(2011). Detection of ESBLs, AmpC beta lactamase and metallo-beta-lactamases in clinical isolates of ceftazidime resistant P. aeruginosa. BJM. 42(1):1284-1288. 17. Villar,H.E.;Baserni,M.N. and Jugo,M.B.(2013).Faecal carriage of ESBL producing Enterobacteriaceae and carbapenem-resistant Gram negative bacilli in community setting.j.infect.dev.ctries 7(8):630-634. 18. Wadekar,M.D.;Anuradha,K.and Venkatesh,D.(2013). Phenotypic detection of ESBL and MBL in clinical isolates of Enterobacteriaceae.Int. J. Curr. Res. Aca. Rev. 1 (3):89-95. 19. Wei,Z-Q ; Chen,Y-G ; Yu,Y-S.; Lu,W-X and Li,L-J. (2005). Nosocomial spread of multi-resistant Klebsiella pneumoniae containing a plasmid encoding multiple lactamases. J. Med. Microbiol. 54:885 888. 58