Low intestinal colonization of Escherichia coli clone ST131 producing CTX-M-15 in Jordanian infants

Transcription

1 Journal of Medical Microbiology (2016), 65, DOI /jmm Low intestinal colonization of Escherichia coli clone ST131 producing CTX-M-15 in Jordanian infants E. F. Badran, 1 R. A. Qamer Din 2 and A. A. Shehabi 2 Correspondence A. A. Shehabi ashehabi@ju.edu.jo Received 9 September 2015 Accepted 17 December Department of Pediatrics, Faculty of Medicine, University of Jordan, Amman, Jordan 2 Department of Pathology-Microbiology, Faculty of Medicine, University of Jordan, Amman, Jordan Over a period of 3 years study ( ), a total of 518 faecal samples were collected and cultured to isolate Escherichia coli. Of these, 338 (65.3 %) E. coli isolates were recovered from infants, and 142/338 (42 %) were multidrug-resistant (MDR) to $3 drug classes using the antimicrobial susceptibility disc diffusion method. A total of 125/142 (88 %) of E. coli isolates were extended-spectrum b-lactamase (ESBL) producers. blactx-m-15 types were observed in 80/125 (64 %) of the isolates, and 60/80 (75 %) were positive for blactx-m-15. Out of 338 E. coli isolates, 9 (2.6 %) were positive for ST131/O25b clone and each isolate was associated with several plasmids of different sizes ( kb). The identities of these nine isolates were confirmed by sequencing for presence of pabb (347 bp) and trpa (427 bp) genes. This study demonstrates low prevalence rate of the highly virulent E. coli ST131 clone producing blactx-m-15 in the intestines of Jordanian infants. INTRODUCTION Recently, Escherichia coli ST131 clone has been isolated from a diverse range of infections caused in community and hospitalized patients across the world (Dahbi et al., 2014; Papagiannitsis et al., 2015; Peirano et al., 2014; Wu et al., 2014). A recent French study showed the proportion of E. coli ST131 as an intestinal colonizing organism increased significantly over time, from 8 % in 2002 to 50 % in 2004 (Han et al., 2014). The pandemic spread of this clone around the world since 2000 was recognized by utilizing multilocus sequence typing (MLST) of CTX- M-15 extended-spectrum b-lactamase (ESBL)-producing E. coli strains from three different continents (Rogers et al., 2011). A study from Egypt indicated that 75 % of clinical CTX-M-15-producing E. coli group B2 isolates belonged to clone ST131 (Fam et al., 2011). Recent studies also reported high prevalence rates of faecal E. coli ST131 among fluoroquinolone-resistant isolates in geriatric patients and residents of long-term care facilities (Burgess et al., 2015; Dahbi et al., 2014; Ho et al., 2015). E. coli ST131 causes a wide variety of infections ranging from cystitis to life-threatening sepsis. Fluoroquinolones and trimethoprim-sulfamethoxazole are no longer effective for empiric therapy of infections caused by this clone (Can et al., 2015; Qureshi & Doi, 2014). Abbreviations: ESBL, extended-spectrum b-lactamase; MDR, multidrug-resistant. The rapid global emergence of E. coli ST131 clone producing blactx-m-15 is explained by carrying a broad range of distinct virulence factor and resistance genes on a transferable plasmid, especially its acquisition of fluoroquinolone resistance genes (Can et al., 2015; Petty et al., 2014). In addition, this clone has also been detected in domestic and wild animals, and foods (Rogers et al., 2011). However, there are still limited data on geographical distribution of this clone or intestinal colonization with multidrug-resistant (MDR) E. coli in Middle East countries and elsewhere (Dashti et al., 2014; Fam et al., 2011). Generally, intestinal colonization of infants with MDR E. coli strains should be considered an alarming risk factor for possible development of serious infection in the future, and it would reflect the status of antimicrobial resistance in the community (Petty et al., 2014). This study investigated the colonizing rate of E. coli ST131 clone producing CTX-M-15 in the intestines of Jordanian infants. METHODS Study design and population. This prospective convenience sampling study included 518 infants v1 year of age, who were investigated at the Paediatric Department, The Jordan University Hospital, Amman, Jordan, from July 2012 through March Biographical data of each infant were obtained and recorded on special forms as part of routine clinical investigation. The form included age, gender, name, duration of hospitalization, disease history, and antibiotics being taken at the time of sampling. Ethical G 2015 The Authors Printed in Great Britain 137

2 E. F. Badran, R. A. Qamer Din and A. A. Shehabi approval was obtained from the Institutional Ethical Review Boards at Jordan University Hospital and the Deanship of Scientific Research at the University of Jordan. Additionally, informed consent was obtained from the mother of each infant investigated. Culture and identification. Faecal/rectal samples were collected from all infants examined by using wet sterile cotton swabs with 0.85 % normal saline and Amies transport media without charcoal (Oxoid), and these were sent within 1 h to the microbiology research laboratory. Swabs were cultured on MacConkey (Oxoid) agar and incubated for h at 37 uc. Five lactose-positive colonies that were morphologically identical to E. coli were picked up and subcultured on MacConkey agar to obtain pure E. coli growth. The isolates were primarily identified as E. coli by standard biochemical characteristics, including citrate utilization, lactose and glucose fermentation in tubes with Kligler iron agar, and being urease-negative and indole-positive. Representative E. coli isolates were also confirmed by commercial Remel RapID ONE test. All confirmed E. coli isolates were kept in cryotubes containing Mueller Hinton agar (Oxoid, England) with 15 % glycerol at 270 uc for further investigation. Antimicrobial susceptibility testing. Antimicrobial susceptibility testing was performed according to the recommendation of the Clinical and Laboratory Standards Institute (CLSI, 2013) using the disc diffusion method. Antimicrobial discs and E-test strips for amikacin, ceftazidime, cefuroxime ciprofloxacin and imipenem (biomérieux) were used to determine MICs of E. coli ST131 isolates. The results of susceptibility tests were interpreted according to the guidelines of CLSI. E. coli ATCC and E. coli ATCC (a b-lactamase producer) were used as control strains. Table 1. Distribution of E. coli isolates and hospitalization in neonates and infants Age group No. (%) E. coli isolates No. (%) hospitalized patients* 1 day 30 days 125/251 (49.8) 55/251 (21.5).1 month.1 year 213/267 (79.8) 22/267 (8.3) Total no. (%) 338/518 (65.3) 77/518 (14.9) *Only one hospitalized infant developed septicaemia due to infection with E. coli, but his faecal E. coli isolate was negative for ST (46.1 %) were females with a mean age of months. The mean age of all infants was months, and 35.9 % of these were neonates. A total of 77 (14.9 %) infants were hospitalized and were on antibiotic treatment. Out of the 518 faecal samples, 338 (65.3 %) were positive for E. coli growth. Of these, 125 (125/251; 49.8 %) were recovered from neonates. The remaining 213 (213/267; 79.8 %) were obtained from infants aged between w1 month and w1 year (Table 1). DNA extraction and PCR procedures. The DNA of 338 E. coli isolates was extracted using a Wizard Genomic DNA Purification kit according to the manufacturer s manual procedures (Promega). The quantity of DNA from each E. coli isolate extract was tested to ensure that the DNA yield was sufficient for PCR (concentration w50 mg ml 21 ) with a purity at A 260 /A , using a spectrophotometer (Bio-Rad). All E. coli isolates were screened, firstly, for the presence of CTX-M groups as described by Mirzaee et al. (2009) and CTX-M-15 as reported by Leflon-Guibout et al. (2004). Secondly, the isolates were investigated for detection of the O25b-ST131 clone using the primers O25pabBspe.F (59-TCCAGCAGGTGCTGGATCGT-39) and O25pabBspe. R (59-GCGAAATTTTTCGCCGTACTGT-39) to amplify a 347 bp fragment of the pabb gene specifically belonging to this clone. A control trpa gene (427 bp fragment) was amplified with primers trpa.f (59-GCTACGAATCTCTGTTTGCC-39) and trpa2.r (59-GCAACGCGGCCTGGCGGAAG-39) as reported by Clermont et al. (2009). Plasmid isolation. Nine E. coli isolates positive for the PabB gene were investigated for the presence of plasmids using a Zyppy Plasmid Minipreparation kit. Statistical analysis. All data analyses were carried out using SPSS version 17. x 2 -test was used for statistical analysis and Pj0.05 was considered statistically significant. RESULTS Patient demographics and E. coli isolates A total of 518 faecal samples were obtained from infants over the 3-year study period. Of these, 279 (53.9 %) were males with a mean age of months, and 138 Journal of Medical Microbiology 65 Antimicrobial susceptibility Table 2 shows the antimicrobial resistance rates among the 338 E. coli samples as the following: ampicillin (99.1 %), nalidixic acid (63.9 %), co-trimoxazole (58 %), tetracycline (55.6 %), then ceftazidime and cefotaxime at nearly the same percentage (47 %) followed by cefuroxime (43.5 %), Ciprofloxacin (24.9 %), imipenem (22.5 %), piperacillin/ tazobactam (18.9 %), amikacin (20.1 %), ertapenem Table 2. Antimicrobial susceptibility patterns of 338 E. coli isolates* Antibiotic Ampicillin 335 (99.1) Nalidixic acid 216 (63.9) Co-trimoxazole 196 (58.0) Tetracycline 188 (55.6) Ceftazidime 159 (47.0) Cefotaxime 158 (46.7) Cefuroxime 147 (43.5) Ciprofloxacin 84 (24.9) Imipenem 76 (22.5) Piperacillin/tazobactam 64 (18.9) Ertapenem 68 (20.1) Amikacin 68 (20.1) Gentamicin 48 (14.2) *42 % were MDR to $3 antibiotic classes. No. (%) resistant isolates

3 Low prevalence of E. coli ST131 in Jordanian infants Table 3. Association of nine E. coli ST131 producers with MDR ESBL phenotype among 125 isolates examined ESBL phenotype No. (%) MDR E. coli No. positive ST131* CTX-M-15** 41 (32.8) 3 CTX-M-I 14 (11.2) 1 CTX-M-II 24 (19.2) 4 CTX-M-I and 20 (16.0) Null CTX-M-II CTX-M-9 12 (9.6) 2 CTX-M 8 and 25/26 4 (3.3) 2 CTX-M-II and 4 (3.2) Null CTX-M-9 CTX-M-I and 4 (3.2) Null CTX-M-9 CTX-M-I, CTX-M-II 2 (1.6) Null & CTX-M-9 Total no. (%) 125 (100) 9 (7.2) *CTX-M phenotypes were found in one or more of the ST131 isolates, and one isolate was detected in a hospitalized infant. **Significant incidence (P,0.05) compared with other CTX-M types. (20.1 %) and gentamicin (14.1 %). A total of 142/338 (42 %) E. coli isolates were MDR(i3 antibiotics). Detection of ST131 and CTX-M-ESBL-producing E. coli among MDR isolates A total of 125/338 (36.9 %) of E. coli isolates were ESBL producers; of those, 121/125 (96.8 %) isolates were MDR (resistant to i3 antibiotic classes). The distribution of CTX-M types is shown in Table 3. CTX-M-15 accounted for 41/125 (32.8 %) of the isolates, while CTX-M-II and CTX-M-I were represented by 19.2 % and 11.2 %, respectively. Only 9/125 (7.2 %) of E. coli isolates were positive for ST131, and their association with various types of CTX-M is shown in Table 3. All nine E. coli isolates were resistant to ciprofloxacin [MIC 90 (mg l 21 ) i16], eight each were resistant to ceftazidime (5.7) and cefotaxime (6.3), seven to gentamicin (16) and five to amikacin (6.4). Distribution of plasmids and pabb and trpa among E. coli isolates Table 4 shows the distribution of plasmids, their numbers and sizes in the nine E. coli ST131 isolates. All isolates carried between one and eight plasmids of different sizes including a larger plasmid of 21.2 kb size. The identity of these nine isolates was confirmed by sequencing and comparison against sequences registered in a pabb gene database with an identity of approximately % (Genewiz); the results were analysed at DISCUSSION This study showed a rapid intestinal colonization of Jordanian infants with E. coli (50 %) within the first month of their life. The study demonstrated that 42 % of their faecal E. coli isolates were MDR to i3 antibiotic classes, including carbapenem-resistant isolates (20 %). These results are slightly higher than a similar study carried out 2 years ago which showed that MDR E. coli isolates were detected only in 30.6 % of the total faecal isolates from infants in the same age group and hospital (Abu Salah et al., 2013). Additionally, there was no significant difference between total incidence of MDR E. coli isolates among infants hospitalized or not. The human intestine is a major reservoir for extra-intestinal pathogenic E. coli strains; especially, urinary tract infections are mostly caused by patients own faecal E. coli (Ejrnæs, 2011; Nielsen et al., 2014). Faecal E. coli is also a frequent cause of extra-intestinal infections in infants in the form of meningitis and blood sepsis (Korhonen et al., 1985). However, over the 3-year period of our study, there was only Table 4. Distribution of plasmid sizes, numbers and antimicrobial resistance factors in nine E. coli ST131 isolates E. coli isolate Plasmid size (kb) No. of plasmids Antimicrobial resistance profile 70C AP, CXM, CAZ, CTX, NA, GM, CIP, SXT, TE 50B(2) AP, CXM, CAZ, CTX, NA, GM, CIP, SXT, TE 57C(2) AP, CXM, CAZ, CTX, NA, GM, AK, CIP, SXT, TE, ETP, IMP 50C 1.4, 1.6, 1.9, 2.0, 3.5, 4.9, 5.1, AP, CXM, CAZ, CTX, NA, GM, AK, CIP, TE 44A 1.4, 2.0, 3.5, AP, NA, AK, CIP, SXT, TE 44B 1.4, 2.5, 3.5, 4.9, AP, CXM, CAZ, CTX, NA, CIP, SXT 29A AP, CXM, CAZ, CTX, NA, GM, AK, CIP, TE 70C(2) 5.1, AP, CXM, CAZ, CTX, NA, GM, CIP, SXT, TE D AP, CXM, CAZ, CTX, NA, GM, AK, CIP, SXT, TE AK, amikacin; AP, ampicillin; CAZ, ceftazidime; CIP, ciprofloxacin; CTX, cefotaxime; CXM, cefuroxime; ETP, ertapenem; GM, gentamicin; IMP, imipenem; NA, nalidixic acid; SXT, co-trimoxazole; TE, tetracycline

4 E. F. Badran, R. A. Qamer Din and A. A. Shehabi one case of septicaemia due to E. coli among hospitalized infants, but his faecal E. coli isolate was negative for ST131. The present study demonstrated that 51.7 % of MDR faecal E. coli isolates were ESBL producers. Of these, 66.1 % MDR isolates carried CTX-M genes, and 75 % were positive for blactx-m-15. Similar results were reported recently from the study of Abu Salah et al. (2013), and higher rates of ESBL-producing E. coli were also reported in India (i80 %) (Johnson & Woodford, 2013) and China (i60 %) among clinical and community isolates (Chong et al., 2011). In recent years, a new E. coli clone called O25:H4-ST131 producing blactx-m-15 has been increasingly detected worldwide, and this clone was mostly associated with the community and caused extra-intestinal infections. The clone is MDR, including resistance to fluoroquinolones and third-generation cephalosporins (Burgess et al., 2015; Han et al., 2014; Ho et al., 2015; Lanza et al., 2014; Peirano et al., 2014). The present study identified only nine (2.6 %) of E. coli faecal isolates as type ST131, and all isolates were producers of various blactx-m types, including significant incidence of CTX-M-15 (Pv0.05), and they were resistant to fluoroquinolones. Moreover, all nine isolates of E. coli ST131 carried a large plasmid of 21.2 kb. Further genetic characterization of their plasmids, especially the larger one might reveal its genetic composition and function, since it has been reported that E. coli ST131 clone harbours a broad range of virulence and resistance genes on a transferable plasmid (Lanza et al., 2014). Recently, plasmid-mediated resistance to b-lactam antibiotics and fluoroquinolones has become increasingly prevalent in community-acquired E. coli infections (Rogers et al., 2011). A recent study by Dashti et al. (2014) in Kuwait reported similar results to our study. Their study examined 832 clinical E. coli isolates, and found 83 (10 %) of those isolates were positive for O25b-B2-ST131, and harboured at least one bla gene with blactx-m-15 being the most prevalent. Infections caused by E. coli O25b/ST131 clone have been also reported to be commonly associated with treatment failure of urinary tract infections, longer hospital stays, and greater hospital expenses (Can et al., 2015). Despite the presence of many epidemiological studies, the true prevalence of ST131 and its association with risk factors in extra-intestinal infection or colonization in various population groups and regions is still unknown. It is likely that intensive misuse of antimicrobial drugs in our country and neighbouring countries is an important factor for the rapid development of MDR in intestinal enteric bacteria, especially E. coli. In conclusion, this study demonstrated a high percentage of MDR E. coli, but lower than expected percentage of E. coli ST131 isolates in the intestines of Jordanian infants. It would be important to carry out further studies to understand the epidemiology of this organism in the intestines of infants and their mothers over the same period. ACKNOWLEDGEMENTS This study has been supported financially by the Dean of Research, the Jordan University, and the Jordanian Research Fund, Amman, Jordan. REFERENCES Abu Salah, M., Badran, E. & Shehabi, A. (2013). High incidence of multidrug resistant Escherichia coli producing CTX-M-type ESBLs colonizing the intestine of Jordanian infants. Int Arab J Antimicrob Agents 3, 4,3. Burgess, M. J., Johnson, J. R., Porter, S. B., Johnston, B., Clabots, C., Lahr, B. D., Uhl, J. R. & Banerjee, R. (2015). Long-term care facilities are reservoirs for antimicrobial-resistant sequence type 131 Escherichia coli. Open Forum Infect Dis 2, ofv011. Can, F., Azap, O. K., Seref, C., Ispir, P., Arslan, H. & Ergonul, O. (2015). Emerging Escherichia coli O25b/ST131 clone predicts treatment failure in urinary tract infections. Clin Infect Dis 60, Chong, Y., Ito, Y. & Kamimura, T. (2011). Genetic evolution and clinical impact in extended-spectrum b-lactamase-producing Escherichia coli and Klebsiella pneumoniae. Infect Genet Evol 11, Clermont, O., Dhanji, H., Upton, M., Gibreel, T., Fox, A., Boyd, D., Mulvey, M. R., Nordmann, P., Ruppé, E. & other authors (2009). Rapid detection of the O25b-ST131 clone of Escherichia coli encompassing the CTX-M-15-producing strains. J Antimicrob Chemother 64, CLSI (2013). Performance Standards for Antimicrobial Susceptibility Testing; 21st Informational Supplement M100-S23 (M100). Clinical and Laboratory Standards Institute: Wayne, PA. Dahbi, G., Mora, A., Mamani, R., López, C., Alonso, M. P., Marzoa, J., Blanco, M., Herrera, A., Viso, S. & other authors (2014). Molecular epidemiology and virulence of Escherichia coli O16:H5-ST131: comparison with H30 and H30-Rx subclones of O25b:H4-ST131. Int J Med Microbiol 304, Dashti, A. A., Vali, L., El-Shazly, S. & Jadaon, M. M. (2014). The characterization and antibiotic resistance profiles of clinical Escherichia coli O25b-B2-ST131 isolates in Kuwait. BMC Microbiol 14,214. Ejrnæs, K. (2011). Bacterial characteristics of importance for recurrent urinary tract infections caused by Escherichia coli. Dan Med Bull 58, B4187. Fam, N., Leflon-Guibout, V., Fouad, S., Aboul-Fadl, L., Marcon, E., Desouky, D., El-Defrawy, I., Abou-Aitta, A., Klena, J. & Nicolas- Chanoine, M. H. (2011). CTX-M-15-producing Escherichia coli clinical isolates in Cairo (Egypt), including isolates of clonal complex ST10 and clones ST131, ST73, and ST405 in both community and hospital settings. Microb Drug Resist 17, Han, J. H., Johnston, B., Nachamkin, I., Tolomeo, P., Bilker, W. B., Mao, X., Clabots, C., Lautenbach, E., Johnson, J. R. & CDC Prevention Epicenters Program (2014). Clinical and molecular epidemiology of Escherichia coli sequence type 131 among hospitalized patients colonized intestinally with fluoroquinoloneresistant E. coli. Antimicrob Agents Chemother 58, Ho, P. L., Chu, Y. P., Lo, W. U., Chow, K. H., Law, P. Y., Tse, C. W., Ng, T. K., Cheng, V. C. & Que, T. L. (2015). High prevalence of Escherichia coli sequence type 131 among antimicrobial-resistant E. coli isolates from geriatric patients. J Med Microbiol 64, Johnson, A. P. & Woodford, N. (2013). Global spread of antibiotic resistance: the example of New Delhi metallo-b-lactamase (NDM)- mediated carbapenem resistance. J Med Microbiol 62, Journal of Medical Microbiology 65

5 Low prevalence of E. coli ST131 in Jordanian infants Korhonen, T. K., Valtonen, M. V., Parkkinen, J., Väisänen-Rhen, V., Finne, J., Orskov, F., Orskov, I., Svenson, S. B. & Mäkelä, P. H. (1985). Serotypes, hemolysin production, and receptor recognition of Escherichia coli strains associated with neonatal sepsis and meningitis. Infect Immun 48, Lanza, V. F., de Toro, M., Garcillán-Barcia, P., Mora, A., Blanco, J., Coque, T. M. & de la Cruz, F. (2014). Plasmid flux in Escherichia coli ST131 sublineages, analyzed by Plasmid Constellation Network (PLACNET), a new method for plasmid reconstruction from whole genome sequences. PLoS Genetics 10, e Leflon-Guibout, V., Jurand, C., Bonacorsi, S., Espinasse, F., Guelfi, M. C., Duportail, F., Heym, B., Bingen, E. & Nicolas-Chanoine, M. H. (2004). Emergence and spread of three clonally related virulent isolates of CTX-M-15-producing Escherichia coli with variable resistance to aminoglycosides and tetracycline in a French geriatric hospital. Antimicrob Agents Chemother 48, Mirzaee, M., Owlia, P. & Mansouri, S. (2009). Distribution of CTX-M b-lactamase genes among Escherichia coli strains isolated from patients in Iran. Lab Med 40, Nielsen, K. L., Dynesen, P., Larsen, P. & Frimodt-Møller, N. (2014). Faecal Escherichia coli from patients with E. coli urinary tract infection and healthy controls who have never had a urinary tract infection. J Med Microbiol 63, Papagiannitsis, C. C., Študentová, V., Jakubů, V., Španělová, P., Urbášková,P.,Žemličková, H. & Hrabák, J. (2015). High prevalence of ST131 among CTX-M-producing Escherichia coli from communityacquired infections, in the Czech Republic. Microb Drug Resist 21, Peirano, G., van der Bij, A. K., Freeman, J. L., Poirel, L., Nordmann, P., Costello, M., Tchesnokova, V. L. & Pitout, J. D. (2014). Characteristics of Escherichia coli sequence type 131 isolates that produce extendedspectrum b-lactamases: global distribution of the H30-Rx sublineage. Antimicrob Agents Chemother 58, Petty, N. K., Ben Zakour, N. L., Stanton-Cook, M., Skippington, E., Totsika, M., Forde, B. M., Phan, M. D., Gomes Moriel, D., Peters, K. M. & other authors (2014). Global dissemination of a multidrug resistant Escherichia coli clone. Proc Natl Acad Sci U S A 111, Qureshi, Z. A. & Doi, Y. (2014). Escherichia coli type 131. Epidemiology and challenges in treatment. Exper Rev Anti Infect Ther 12, Rogers, B. A., Sidjabat, H. E. & Paterson, D. L. (2011). Escherichia coli O25b-ST131: a pandemic, multiresistant, community-associated strain. J Antimicrob Chemother 66, Wu, Y. H., Cheng, M. F., Lai, C. H., Lin, H. H., Hung, C. H. & Wang, J. L. (2014). The role of Sequence Type (ST) 131 in adult communityonset non-esbl-producing Escherichia coli bacteraemia. BMC Infect Dis 14,