Extended-spectrum and CMY-type b-lactamase-producing Escherichia coli in clinical samples and retail meat from Pittsburgh, USA and Seville, Spain

Extended-spectrum and CMY-type b-lactamase-producing Escherichia coli in clinical samples and retail meat from Pittsburgh, USA and Seville, Spain Y. Doi 1 , D. L. Paterson 1,2 , P. Egea 3 , A. Pascual 3 ,L.Lo ´pez-Cerero 3 , M. D. Navarro 4 , J. M. Adams-Haduch 1 , Z. A. Qureshi 1 , H. E. Sidjabat 1,2 and J. Rodrı ´guez-Ban ˜o 4 1) Division of Infectious Diseases, University of Pittsburgh Medical Center, Pittsburgh, PA, USA, 2) University of Queensland Centre for Clinical Research and Royal Brisbane and Women’s Hospital, Brisbane, Qld, Australia, 3) Servicio de Microbiologı ´a and 4) Seccio ´n de Enfermedades Infecciosas, Hospital Universitario Virgen Macarena, Seville, Spain Abstract Infections due to Escherichia coli producing extended-spectrum b-lactamase (ESBL) or CMY-type b-lactamase (CMY) are increasingly observed in non-hospitalized patients. The origin of these organisms is uncertain, but retail meat contaminated with E. coli may be a source. In the present study, clinical information and strains collected from patients infected or colonized with ESBL-producing and CMY-producing E. coli at hospitals in Pittsburgh, USA and Seville, Spain were investigated. Retail meat purchased in these cities was also studied for the presence of these organisms. Twenty-five and 79 clinical cases with ESBL-producing E. coli and 22 cases and one case with CMY-producing E. coli were identified in Pittsburgh and Seville, respectively. Among them all, community-acquired and healthcareassociated cases together constituted 60% of the cases in Pittsburgh and 73% in Seville. Community-acquired cases were more common in Seville than in Pittsburgh (49% vs. 13%; p <0.001). ESBL-producing and CMY-producing E. coli isolates were commonly recovered from the local retail meat. In particular, 67% (8/12) of retail chickens in Seville and 85% (17/20) of those in Pittsburgh contained ESBL- producing and CMY-producing E. coli isolates, respectively. Among the ESBL-producing isolates, CTX-M and SHV were the most common ESBL types in both clinical and meat isolates. Approximately half of the ESBL-producing and CMY-producing E. coli isolates from meat belonged to phylogenetic groups associated with virulent extra-intestinal infections in humans. Community and healthcare environments are now significant reservoirs of ESBL-producing and CMY-producing E. coli. Retail meat is a potential source of these organisms. Keywords: Antimicrobial resistance, clinical epidemiology, CMY-type b-lactamases, Escherichia coli, extended-spectrum b-lactamases, food, molecular epidemiology, b-lactamases Original Submission: 29 April 2009; Revised Submission: 6 July 2009; Accepted: 7 July 2009 Editor: M. Paul Article published online: 22 July 2009 Clin Microbiol Infect 2010; 16: 33–38 Corresponding author and reprint requests: J. Rodrı ´guez-Ban ˜o, Seccio ´n de Enfermedades Infecciosas, Hospital Universitario Virgen Macarena, Avda Dr Fedriani 3, 41009 Seville, Spain E-mail: [email protected]m Introduction Escherichia coli is a leading cause of community-acquired infections, especially urinary tract infections. In E. coli, resistance to oxyimino-cephalosporins, such as ceftriaxone and ceftazidime, occurs by production of extended-spectrum b-lactamases (ESBLs) and plasmid-mediated AmpC-type b-lactamases [1]. ESBLs and plasmid-mediated AmpC-type b-lactamases in E. coli were first noted in the 1980s [2,3], and have been associated with hospital-acquired infections. In E. coli, CMY-type b-lac- tamase (CMY) is the most commonly encountered plasmidmediated AmpC-type b-lactamase worldwide [4]. Although it is conventionally perceived as a nosocomial pathogen, ESBL-producing E. coli has emerged as a community pathogen in many parts of the world [5]. Communityacquired cases are largely caused by E. coli that produces CTX-M-type ESBLs [5]. Recent reports suggest that CMY- producing E. coli may cause community-acquired infections as well [6–8]. The spread of ESBL-producing and CMY-produc- ing E. coli outside of the hospital environment may compromise the usefulness of penicillins and cephalosporins for infections such as complicated urinary tract infections and bacteraemia [7–12]. In addition, these E. coli isolates often have co-resistance to other classes of antimicrobials, mediª2009 The Authors Journal Compilation ª2009 European Society of Clinical Microbiology and Infectious Diseases ORIGINAL ARTICLE 10.1111/j.1469-0691.2009.03001.x ated by other resistance mechanisms (e.g. aminoglycosides, trimethoprim–sulphamethoxazole and fluoroquinolones) [1]. One potential source of resistant E. coli isolates causing infection in the community is food that we eat. Some food animals, including chickens, turkeys, pigs and cattle, may be colonized by ESBL-producing and CMY-producing E. coli and have been considered as potential sources of these organisms that cause community-acquired diseases [13]. In the present study, we sought to systematically evaluate clinical infection with these organisms, using rigorous definitions of the place of acquisition, and to identify these organisms from retail meat purchased locally during the same time period. Patients and Methods Laboratory surveillance of ESBL-producing and CMY-producing E. coli Prospective laboratory-based surveillance was conducted from September 2006 to March 2007 to identify all cases of ESBL-producing and CMY-producing E. coli infection or colonization at the University of Pittsburgh Medical Center (Presbyterian-Shadyside Campus), Pittsburgh, USA and Hospital Universitario Virgen Macarena, Seville, Spain. The University of Pittsburgh Medical Center (Presbyterian-Shadyside Campus) is a 1300-bed tertiary teaching hospital with affiliated outpatient clinics. Hospital Universitario Virgen Macarena is a 950-bed tertiary teaching hospital with affiliated outpatient clinics and primary-care centres. ESBL production was screened for and confirmed by the disk diffusion method defined by the CLSI [14]. For isolates that were non-suscep- tible (intermediate or resistant) to ceftriaxone and had negative confirmatory test findings for ESBL production, PCR analysis was conducted to identify plasmid-mediated AmpC- type b-lactamases, including CMY [15]. Study sample and data collection The study was approved by the Institutional Review Boards of the University of Pittsburgh and Hospital Universitario Virgen Macarena, which waived the need to obtain written informed consent, owing to the observational nature of the study. Computerized medical and laboratory records were reviewed to document the place of acquisition of infection [16]. The same patient could be re-enrolled only when a positive culture was identified ‡30 days from the initial enrolment. A hospital-acquired case was defined as a positive culture obtained from a patient who had been hospitalized for ‡48 h. A healthcare-associated case was defined as a positive culture obtained from a patient at the time of hospital admission or <48 h after admission if the patient fulfilled any of the following criteria: (i) received intravenous therapy at home, received wound care or specialized nursing care through a healthcare agency, family, or friends, or had selfadministered intravenous medical therapy £30 days before enrolment; (ii) attended a hospital or haemodialysis clinic or received intravenous chemotherapy £30 days before enrolment; (iii) was hospitalized in an acute-care hospital for ‡2 days in the 90 days before enrolment; or (iv) resided in a nursing home or long-term-care facility [17]. A communityacquired case was defined as one in which a positive culture was obtained from a patient at the time of hospital admission or <48 h after admission and that did not meet the criteria for a healthcare-associated case. Complementary clinical data for patients from Pittsburgh have been published elsewhere [7]. Statistical analysis Categorical variables were compared using the chi-squared test. SPSS software (version 13.0; SPSS, Chicago, IL, USA) was used for the analyses. Culturing of retail meat Chicken, turkey, pork and ground beef were purchased from local supermarkets at least every 2 weeks in metropolitan Pittsburgh and Seville during the study period. After immediate transport of the samples to the research laboratory on ice, approximately 25 g of each sample was suspended in buffered peptone broth with the use of a sterile technique, and homogenized manually or with the use of a stomacher. After overnight incubation at 37C, an aliquot of the broth was plated on MacConkey agar plates containing 1 mg/L cefotaxime 0% 10% 20% 30% 40% 50% 60% Pittsburgh Seville Pittsburgh Seville Pittsburgh Seville CMY ESBL Hospitalacquired Healthcareassociated Communityacquired FIG. 1. Distribution of extended-spectrum b-lactamase (ESBL)-pro- ducing or CMY-type b-lactamase (CMY)-producing Escherichia coli cases according to the place of acquisition at both study sites. 34 Clinical Microbiology and Infection, Volume 16 Number 1, January 2010 CMI ª2009 The Authors Journal Compilation ª2009 European Society of Clinical Microbiology and Infectious Diseases, CMI,16, 33–38 or ceftazidime and further incubated overnight. Lactose-fer- menting colonies were identified as E. coli using standard biochemical tests. Production of ESBL was confirmed by observing ‡5 mm of growth inhibition around a ceftazidimecontaining or cefotaxime-containing disk in the presence of clavulanic acid on a Mueller–Hinton agar plate [14]. The colonies with negative confirmatory test findings for ESBL production were tested for the presence of plasmid-mediated AmpC-type b-lactamase genes as described above. Molecular typing and genetic analyses For all ESBL-producing and CMY-producing isolates of both clinical and food origin, PCR analyses to determine the type of ESBL gene carried by each isolate were performed. Primer sets to detect TEM-type, SHV-type and CTX-M-type ESBL genes as well as CMY genes were used [15,18]. Positive results were confirmed by sequencing of the products. In addition, we determined the phylogenetic groups of the E. coli clinical isolates and those of food origin by multiplex PCR analysis [19]. To determine the genetic relatedness of the study isolates, pulsed-field gel electrophoresis analysis was performed, using XbaI as a restriction endonuclease, and electrophoresing the genome on a CHEF III DR system (Bio- Rad, Hercules, CA, USA), on all isolates, as described previously [7]. Cluster analysis was performed by using the unweighted pair group method based on Dice coefficients. The results were interpreted according to the criteria proposed by Tenover et al. [20]. Results In Pittsburgh, 2583 E. coli isolates were identified during the study period. Of these, 1183 (46%) were obtained from outpatient locations. In Seville, 2606 E. coli isolates were identified. Of these, 2062 (79%) were obtained from outpatient locations. In Pittsburgh, 47 cases with ESBL-producing and CMY- producing E. coli were identified and enrolled. Eighty cases were identified and enrolled in Seville. They represented cases from a total of 125 patients, as two patients were enrolled for two separate episodes. Urinary tract and blood were the most common sites, accounting for 95 cases (75%) and 21 cases (14%), respectively. Other less frequent sites of infection included the respiratory tract (six; 5%), wounds (three; 2%), and others (two; 2%). The places of acquisition and types of b-lactamase produced by the E. coli isolates are summarized in Table 1 and Fig. 1. At both sites, among the hospital-acquired and healthcare-associated isolates, the majority of E. coli isolates produced either CTX-M-type or SHV-type ESBLs or CMYs. In Pittsburgh, the communityacquired isolates produced CTX-M-type ESBLs or CMYs. In Seville, most community-acquired isolates produced either CTX-M-type or SHV-type ESBLs. We conducted 20 and 12 independent meat samplings in Pittsburgh and Seville, respectively. These involved seven local supermarkets in Pittsburgh and ten in Seville. Fig. 2 summarizes the results. ESBL-producing E. coli was frequently isolated from retail meat purchased in Seville. In Seville, eight (67%), seven (58%), three (25%) and one (9%) chicken, turkey, pork and ground beef samples, respectively, yielded a total of 55 clonally unrelated ESBL-producing E. coli isolates. They produced either CTX-M-type ESBLs (CTX-M-9 group, six isolates; CTX-M-1 group, four isolates; overall, 18%) or SHV-type ESBLs (SHV-12, 45 isolates; 82%). No CMY-pro- ducing E. coli isolate was identified from retail meat in Seville. In contrast, in Pittsburgh, only one chicken sample (and none of the other meat types) grew ESBL-producing E. coli, which TABLE 1. Distribution of extendedspectrum b-lactamase (ESBL)-pro- ducing and CMY-type b-lactamase (CMY)-producing Escherichia coli according to the place of acquisition and the b-lactamase types Seville Pittsburgh No. of isolates ESBL/CMY families No. of isolates ESBL/CMY families Hospital-acquired 22 CTX-M-1 group: 9 18 CTX-M-9 group: 5 CTX-M-9 group: 5 SHV-7: 1 a SHV-12: 8 CMY-2/32/33: 11 Unidentified: 2 Healthcare-associated 19 CTX-M-1 group: 4 22 CTX-M-1 group: 5 CTX-M-9 group: 8 CTX-M-2 group: 1 SHV-12: 6 CTX-M-9 group: 2 Unidentified: 1 SHV-7/12: 6 TEM-137: 1 CMY-2: 7 Community-acquired 39 CTX-M-1 group: 7 6 CTX-M-1 group: 3 CTX-M-9 group: 15 CMY-2: 3 SHV-12: 16 CMY-4: 1 Data are expressed as number of cases. The site of acquisition could not be determined for one CMY case in Pittsburgh. a This isolate also produced CTX-M-type ESBL. CMI Doi et al. ESBL-producing and CMY-producing E. coli in patients and meat 35 ª2009 The Authors Journal Compilation ª2009 European Society of Clinical Microbiology and Infectious Diseases, CMI,16, 33–38 produced CTX-M-1 group ESBLs. However, CMY-2-produc- ing E. coli isolates were identified in 17 (85%), 14 (70%), two (10%) and one (5%) chicken, turkey, pork and ground beef samples, respectively, in Pittsburgh. Clonality was not observed among isolates from patients and retail meat at either site, except for the ESBL-producing E. coli isolate identified in a retail chicken product in Pittsburgh. This isolate showed a pulsed-field gel electrophoresis pattern indistinguishable from that of an isolate identified in a healthcare-associated patient. Among the E. coli clinical isolates and those of food origin from both sites, membership in a virulence-associated phylogenetic group (group B2 or D) of available isolates was similar for ESBL-producing E. coli (54/103 (52%) vs. 25/56 (45%), respectively). For CMY-pro- ducing E. coli, this was higher in clinical isolates than in meat isolates (21/23 (91%) vs. 17/34 (49%), respectively). Discussion In this analysis of ESBL-producing and CMY-producing E. coli, we observed a predominance of community-acquired and healthcare-associated cases at teaching hospitals in Pittsburgh and Seville. The occurrence of infections due to ESBL-pro- ducing and CMY-producing E. coli among community patients has serious implications for the empirical management of common infections, and warrants continued surveillance. The predominance of healthcare-associated acquisition of ESBL- producing and CMY-producing E. coli in Pittsburgh could be attributed, at least in part, to recent hospital admission and residence in nursing homes. Data regarding the prevalence of colonization with ESBL-producing Enterobacteriaceae among nursing home residents are scarce. Reports from the 1990s document colonization rates of 15% at baseline to as high as 46% in an outbreak setting [21,22]. A better understanding of the transmission dynamics of ESBL-producing and CMY- producing E. coli within these facilities and between them and acute-care hospitals is vital for the formulation of effective infection control strategies. We isolated E. coli producing ESBLs and CMYs from a large proportion of retail meat samples in Seville and Pittsburgh, respectively. The ESBL and CMY gene contents of the E. coli isolates of food origin correlated well with the genes that were locally prevalent in clinical isolates, although we were surprised by the fact that SHV enzymes were more frequent than CTX-M enzymes. The importance of SHV-pro- ducing E. coli isolates as community pathogens has been recently analysed in Spain [23]. Contamination of retail meat, especially poultry, with E. coli producing CMYs is well documented [13,24]. Recovery of ESBL-producing E. coli from retail meat is less common, but has also been reported [25– 28]. Numerous studies have reported the isolation of E. coli producing ESBLs and CMYs from food-producing animals [13,24]. Less is known about whether these animals actually serve as the source of the organisms causing disease in humans. Recent studies suggest that antibiotic-resistant E. coli strains isolated from retail poultry share similar phylogenetic and virulence markers with contemporary human strains [29], and that poultry isolates have higher virulence scores than beef and pork isolates [30]. Also, certain food habits have been found to be associated with faecal carriage of ESBL-producing E. coli [31]. Our study has several limitations. First, because the study was performed at single centres in Pittsburgh and Seville, the national trend could not be captured at either country to allow the drawing of strong conclusions of broader public health relevance. Second, the Pittsburgh site had limited numbers of specimens from outpatient locations. Third, retail foods other than meat were not cultured. Finally, we provided an ecological relationship between contamination of retail meat and colonization or infection due to ESBL-pro- ducing and CMY-producing E. coli, but we could not demonstrate a direct causal relationship. In summary, the majority of infections due to ESBL- producing and CMY-producing E. coli were acquired in the community or in association with the healthcare system but outside hospitals, both in Pittsburgh and Seville. The majority of retail poultry samples contained E. coli producing ESBLs or CMYs at both locales. Our findings provide further circum- 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% Pittsburgh Seville Pittsburgh Seville Pittsburgh Seville Pittsburgh Seville CMY ESBL Chicken Turkey Pork Ground beef FIG. 2. Prevalence of extended-spectrum b-lactamase (ESBL)-pro- ducing or CMY-type b-lactamase (CMY)-producing Escherichia coli in retail meat at both sites. The retail chicken product with ESBL-pro- ducing E. coli in Pittsburgh also grew CMY-producing E. coli (*). 36 Clinical Microbiology and Infection, Volume 16 Number 1, January 2010 CMI ª2009 The Authors Journal Compilation ª2009 European Society of Clinical Microbiology and Infectious Diseases, CMI,16, 33–38 stantial evidence that retail meat may serve as a source of ESBL-producing and CMY-producing E. coli isolates, which may then colonize the human intestine and cause infections, or serve as donors of ESBL and CMY genes to the human strains by means of conjugal transfer of resistance plasmids. Acknowledgements We thank A. O’Keefe, D. L. Pakstis, L. G. Clarke and the clinical microbiology laboratory staff at both study sites for their assistance. The authors are grateful to J. R. Johnson for critical reviews of the early drafts of the manuscript. This work was presented in part at the 48th Interscience Conference on Antimicrobial Agents and Chemotherapy (ICAAC) and the 46th Annual Meeting of the Infectious Diseases Society of America (IDSA), Washington DC, 2008. Transparency Declaration This study was supported by a joint fellowship from the National Foundation of Infectious Diseases and the Infectious Diseases Society of America. It was also partly supported by grants from the Ministerio de Sanidad y Consumo (PI070190), Spanish Network for the Research in Infectious Diseases, REIPI (RD06/0008), Junta de Andalucı ´a (PI0048/ 2008), and National Institute of Allergy and Infectious Diseases, National Institutes of Health (T32AI007333). The authors declare no conflicts of interest. References 1. Paterson DL, Bonomo RA. Extended-spectrum b-lactamases: a clinical update. Clin Microbiol Rev 2005; 18: 657–686. 2. Bauernfeind A, Horl G. Novel R-factor borne b-lactamase of Escherichia coli confering resistance to cephalosporins. 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