Is Escherichia coli urinary tract infection a zoonosis? Proof of direct link with production animals and meat

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1 Eur J Clin Microbiol Infect Dis (2012) 31: DOI /s ARTICLE Is Escherichia coli urinary tract infection a zoonosis? Proof of direct link with production animals and meat L. Jakobsen & P. Garneau & G. Bruant & J. Harel & S. S. Olsen & L. J. Porsbo & A. M. Hammerum & N. Frimodt-Møller Received: 17 June 2011 / Accepted: 3 September 2011 / Published online: 28 October 2011 # Springer-Verlag 2011 Abstract Recently, it has been suggested that the Escherichia coli causing urinary tract infection (UTI) may come from meat and animals. The purpose was to investigate if a clonal link existed between E. coli from animals, meat and UTI patients. Twenty-two geographically and temporally matched B2 E. coli from UTI patients, community-dwelling humans, broiler chicken meat, pork, and broiler chicken, previously identified to exhibit eight virulence genotypes by microarraydetection of approximately 300 genes, were investigated for clonal relatedness by PFGE. Nine isolates were selected and tested for in vivo virulence in the mouse model of ascending UTI. UTI and community-dwelling human strains were closely clonally related to meat strains. Several human L. Jakobsen (*) : S. S. Olsen : A. M. Hammerum : N. Frimodt-Møller Department of Microbiological Surveillance and Research, Statens Serum Institut, Build. 47/213, Ørestads Boulevard 5, 2300 Copenhagen S, Denmark lja@ssi.dk L. J. Porsbo National Food Institute, Technical University of Denmark, Copenhagen, Denmark P. Garneau : J. Harel Groupe de Recherche sur les Maladies Infectieuses du Porcine, Faculté de médecine vétérinaire, Université du Montreal, Montreal, QC, Canada G. Bruant Biotechnology Research Institute, National Research Council of Canada, Montreal, QC, Canada N. Frimodt-Møller Department of Clinical Microbiology, Hvidovre Hospital, 30 Kettegård alle, DK-2650 Copenhagen, Hvidovre, Denmark derived strains were also clonally interrelated. All nine isolates regardless of origin were virulent in the UTI model with positive urine, bladder and kidney cultures. Further, isolates with the same gene profile also yielded similar bacterial counts in urine, bladder and kidneys. This study showed a clonal link between E. coli from meat and humans, providing solid evidence that UTI is zoonosis. The close relationship between community-dwelling human and UTI isolates may indicate a point source spread, e.g. through contaminated meat. Introduction Escherichia coli exists as a common commensal in the intestines of humans and mammals. Apart from various toxin-related diarrheal infections E. coli also causes extraintestinal infections, including urinary tract infections (UTI). The disease burden of UTI is enormous. UTI is believed to be the most common bacterial infection with an estimated 150 million cases globally [1, 2]. In addition, a considerable number of E. coli UTI cases results in bacteremia and consequently deaths. It has been estimated that one of every three women will experience a case of treatment-requiring UTI before the age of 26 and 40 50% of women will experience at least one UTI during their lifetime [3]. About 25 30% of, otherwise healthy, women with UTI will develop a recurrent infection [4]. In the U.S. alone, the estimated cost burden is approaching 1 billion dollars yearly [5]. Extraintestinal pathogenic E. coli, designated ExPEC, is responsible for 80 90% of the UTI cases [1, 4]. Most often the E. coli belongs to phylogroup B2, characterized by possessing numerous virulence genes allowing the isolate to cause infection outside the intestinal tract, e.g. by

2 1122 Eur J Clin Microbiol Infect Dis (2012) 31: adhering to host surfaces, scavenge iron or other micronutrients, or evade host response [6, 7]. UTI is most often caused by enteric E. coli. This transmission route is widely referred to as the feacal-perineal-urethral route [8, 9]. It has been suggested that the exterior source of ExPEC to the intestine may be food and animals [10]. However, studies that link UTI isolates with food and animal isolates due to clonal relatedness using highly discriminative typing methods, like pulsed-field gel electrophoresis, are lacking. In this study, we investigate the clonal relatedness of geographically and temporally matched E. coli from UTI patients, community-dwelling humans, broiler chicken meat, broiler chickens, pork, and pigs as well as the virulence of non-uti isolates in a mouse UTI model which is representative of human UTI [11]. Materials and methods Bacterial strains A strain collection at Statens Serum Institut previously described with respect to phylogroups and virulence genes were used for this study [12, 13]. In short, E. coli isolates were collected in Denmark in 2004 from community-dwelling humans (n=102), Danish (n=197) and imported broiler chicken meat (n=86), broiler chickens (n=138), Danish (n=177) and imported pork (n=10), and pigs (n=145) by using a stratified sampling scheme as part of the Danish Integrated Antimicrobial Resistance Monitoring and Research Programme (DANMAP) [14]. Communitydwelling humans were chosen by a selection algorithm so that they represented the age and gender distribution of the total Danish population taking the differential participation rates of various demographic groups into account (scientific ethical committee approval (KF) /02). A total of 102 UTI isolates were collected later from November 2005 to April 2006 in a general practitioner clinic (serving approximately 10,800 persons) south of Copenhagen from patients with community-acquired uncomplicated or complicated UTI [12]. UTI isolates were deliberately sampled later than animal, meat, and community-dwelling human isolates to allow time for consumption of the meat and possible colonization and invasiveness of the E. coli isolates. Previously, isolates belonging to phylogroup B2 were identified among all origins and 161 B2 isolates were investigated for more than 300 virulence genes and 80 resistance genes using a microarray [12, 13]. A number of strains within and across isolate origins shared identical gene profiles and was used for this study [13]. We analyzed a total of 22 B2 E. coli strains from UTI patients (n=10), community-dwelling humans (n=6), Danish (n=3) and imported broiler chicken meat (n=1), broiler chickens (n=1), and Danish pork (n=1). These 22 isolates exhibited eight different virulence gene profiles according to the microarray data (Table 1). The eight gene profiles represented either (i) isolates from several origins, i.e. both animal and meat origin, or meat and human origin, or from both human origins (gene profile 1 5) or (ii) they consisted of UTI isolates only (gene profile 6 8) (Table 1). All 22 isolates, based on their microarray virulence gene profile, were previously designated to belong to the pathotype ExPEC [13]. The E. coli species identity was confirmed using API20E (Biomérieux, France) according to the manufacturer s instructions. Dietary habits, contact with animals, travel and occupation of the communitydwelling humans were obtained by questionnaire. Address and family names were obtained for UTI patients where possible. Pulsed-field gel electrophoresis To investigate the clonal relatedness between isolates of the same gene profile, we subjected the 22 strains to pulsedfield gel electrophoresis (PFGE) with XbaI using the protocol made available by CDC s PulseNet [15, 16]. Salmonella Braenderup H9812 was used as molecular size standard [16]. All strains were processed on one gel. The PFGE profiles were analyzed using the Bionumerics software, version 6.01 (Applied Maths, Sint-Martens- Latem, Belgium) resulting in a dendrogram (1% tolerance, 1% optimization). The PFGE patterns were interpreted according to Tenover et al. [17]. Accordingly, PFGE profiles with four to six bands difference were considered to be possibly related and profiles with two to three bands difference were considered closely related. Mouse model of ascending urinary tract infection To investigate virulence of the non-uti isolates, nine strains from community-dwelling humans, Danish and imported broiler chicken meat, broiler chickens, and Danish pork, selected among isolates with gene profiles 1 5, were tested in a mouse model of ascending UTI (animal inspectorate approval no. 2004/ ). The E. coli O rough:h- D1923 was used as negative control in the mouse experiments. The mouse model has been described previously [18, 19]. In short, mice bladders were emptied by gently pressing their abdomen and 50 μl (10 8 CFU) of each bacterial suspension was inoculated slowly transurethrally in six outbred female albino OF1 mice (28 32 gram, Charles River Laboratories, France) by use of plastic catheters. Ninety-six hours post inoculation urine was collected from each mouse by carefully pressing the abdomen. The mice were then euthanized by cervical dislocation, and bladder and kidneys were removed and

3 Eur J Clin Microbiol Infect Dis (2012) 31: Table 1 Virulence gene profiles of B2 Escherichia coli isolates from animals, meat, community-dwelling humans, and UTI patients that shared gene profiles Characteristics Gene profiles Isolate origins Broiler chicken, n=1; Danish b.c. meat, n=1; Imp. b.c. meat, n=1 Adhesins csga, csge, fima, fimh Colicins and microcins cei, cia, cvac, mcja Danish b.c. meat, n=2; Comm. human, n=1; UTI, n=1 csga, csge, f165 (1)A, fima, fimh, papa (11), papc, papgii Danish pork, n=1; Comm. human, n=1 csga, csge, fima, fimh, papa(48), papc, papgiii Comm. humans, n=3; UTI, n=1 csga, csge, sfad, sfad, fima, fimh, papa(12), papc, papgiii, pixa Comm. humans, n=1; UTI, n=1 csga, csge, foca, focg, sfad, fima, fimh, papa(10), iha UTI, n=2 UTI, n=3 UTI, n=2 csga, csge, f165 (1)A, fima, fimh, papa (11), papc, papgii csga, csge, faca, sfad, sfahii, fima, fimh cia, cvac ce1a, cia, cvac ce1a, mchb Ce1a, mchb cba, cia, cma, cvac Toxins cdtb-1, cdtb-4 asta, cnf1 sat cdtb-1 sat Iron acqusition or transport systems Capsular and somatic antigens Haemolysins and hemaglutins Various functions Newly recognized or putative virulence genes Total no. of virulence genes sita, kpsm-ii, neua, iucd, iuta, sita, kpsm-ii, neua, iucd, iuta, sita, sita, kpsm-ii kpsm-ii, neua, hlye, vat hlye, vat hlye, hra hlya, hlye, hra1, vat flic, ibea, ibeb, ompa, ompt, trat tspe4. C2, yjaa, mvim, mvin, usp deok, flic, flic (H7), ibeb, trat gimb(orf1), tspe4.c2, yjaa, mvim, mvin, usp agn43, deok, flic, ibea, ibeb, iss,, malx, ompa, ompt, trat tspe4.c2, yjaa, mvim, mvin, usp agn43, flic, ibeb, ompa, ompt, tspe4. C2, yjaa, mvim, mvin, usp iucd, iuta,sita, kfib, kpsm-ii, wzx (O6) chua, fepc, fyua, irp1, irp2, sita, kpsm-ii, neua, iucd, iuta, sita, kpsm-ii, neua, hlye, vat hlye, vat hlye, hra1, tsh, vat agn43, deok, flic, ibeb, pic, senb, trat b1432, tspe4.c2, yjaa, mvim, mvin, usp deok, flic, flic (H7), ibeb, gimb(orf1), tspe4.c2, yjaa, mvim, mvin, usp ccdb, flic, flic (H7), ibea, ibeb, trat gimb (orf1), tspe4.c2, yjaa, mvim, mvin, usp csga, csge, fima, fimh, papa(8), papa(40), papc, papgii, iha cvac iucd, iuta, sita, kfib, kpsm-ii hlya, hlye, vat agn43, ccdb, flic, ibeb, malx, senb, trat tspe4. C2, yjaa, mvim, mvin, usp Danish b.c. meat Danish broiler chicken meat, Imp. b.c. meat imported broiler chicken meat, Comm. humans community-dwelling humans, UTI UTI patients For description of microarray analysis including probes and specific genes consult Jakobsen et al. [13]

4 1124 Eur J Clin Microbiol Infect Dis (2012) 31: stored in Eppendorf tubes. All urine samples were processed the same day by spotting (20 μl) of a series of tenfold dilutions ( ) in duplicates on bromthymol blue agar plates (SSI Diagnostika). The bladder and kidneys were stored in 0.9% saline solution and kept at 80 C until processing. They were then incubated at room temperature for 1 h and subsequently homogenized using a TissueLyser (Qiagen, Ballerup, Denmark). Plates for bacterial counting were processed as described above. The detection limit was 25 CFU/ml. Bacterial counts for the different treatment groups were compared using the Mann-Whitney test (two-tailed) with a significance level of p 0.05 (GraphPad Prism 5, GraphPad Software, San Diego, CA, USA). Results Bacterial strains A total of 22 phylogroup B2 E. coli isolated from UTI patients, community-dwelling humans, Danish and imported broiler chicken meat, broiler chickens, and Danish pork were analyzed. Gene profile overview, isolation date, number of virulence genes, gender and age for human isolates are summarized in Table 2. Two strains were obtained from a gardener (isolate 2030c04, gene profile 3) and a house wife (isolate 2174c04, gene profile 4) who reported eating pork and broiler chicken on a regular basis, and having been in contact with dogs and cats but not with food-producing animals over a 7-day period prior to the sampling date, and had not travelled within the last three months. The gardener further reported being in contact with captive frogs and the house wife had been in contact with wild rabbits. Isolate 2571c04 (gene profile 5) was obtained from a 5-year-old kindergarten pupil. He was eating pork and broiler chicken on a regular basis, had been in contact with dogs and cats (but not production animals) over a 7-day period prior to the sampling date, and had been on a 5-day trip to Sweden within the last three months. The other three community-dwelling humans (all women, aged 3 (2180c04), 44 (2235c04), and 60 (2168c04), respectively) did not complete the questionnaire. None of the UTI patients shared address or family name which indicated that they seemingly may be unrelated. PFGE typing In the absence of alternative analysis methods, we used Tenover s interpretation criteria in our study, which were not intended for independently collected isolates from humans, meat, and animals [17]. The 22 isolates yielded 13 PFGE types of closely related (n=11), possibly related (n=5), or unrelated isolates (n=6) (Table 2). Only isolates with identical virulence gene profiles were possibly or closely related and no isolates from different gene profiles shared related PFGE profiles. Two broiler chicken meat isolates were closely related to a UTI isolate (a 3-band difference, 92% similarity, gene profile 2) and a pork isolate was closely related to a community-dwelling human isolate (a 2-band difference, 96% similarity, gene profile 3) (Fig. 1). Virulence in mouse UTI model The bacterial counts in urine, bladder, and kidneys of the nine strains from animals, meat and community-dwelling humans are shown in Fig. 2. Results show that all nine isolates had positive urine, bladder, and kidney cultures ranging from 10 4 to 10 7 CFU in the urine, CFU in the bladder and CFU in the kidneys (Fig. 2). Isolates with identical gene profiles mostly (profiles 1, 3, and 4) had statistically similar bacterial counts. Isolates and 2235c04 (gene profile 2) had similar bacterial kidney counts, but not urine (p 0.037) or bladder (p 0.026) counts. The negative control strain failed to produce any bacterial counts above the detection limit except for one positive bladder culture. Discussion Outbreaks of community-acquired UTI have been reported in Europe and Northern America, and closely related or indistinguishable UTI isolates from unrelated women have suggested point source dissemination [20 24]. Although transmission routes were not investigated food may have been a possible source, as an epidemiological study has previously suggested that frequent pork and chicken consumption were related to multidrug-resistant UTI [25]. Similarity between animal, food, and human ExPEC isolates with respect to phenotypic antimicrobial resistance and virulence genotypes, and other molecular characteristics have been demonstrated by several researchers, including us, supporting the hypothesis that UTI may be a zoonosis [12, 13, 26 31]. This was substantiated recently by the first study demonstrating virulence of E. coli isolates from healthy production animals and fresh retail meat in a mouse model of ascending UTI [19]. However, studies providing evidence of a clonal link between UTI and animals and meat E. coli using a highly discriminative method like PFGE are few. Ramchandani et al. investigated if E. coli strains belonging to clonal group A (CGA) could be traced to food animals and identified one E. coli strain from a cow isolated in 1988 with a 94% similar PFGE pattern to a human CGA isolate from 1999 [10]. However, no epidemiological connection was known, neither did the

5 Eur J Clin Microbiol Infect Dis (2012) 31: Table 2 Background information on E. coli B2 strains from different origins sharing identical microarray virulence gene profile Strain Isolate origin a Isolate obtained (day-month-year) PFGE type b Virulence genes shared Background information Gender Age (years) Gene profile Broiler chicken (feces) A a Danish broiler chicken meat A a Imported broiler chicken meat B Gene profile Danish broiler chicken meat C a Danish broiler chicken meat C a 2235c04 Comm. humans (feces) D F a UTI patient (comp., recurrent) C a F 62 Gene profile Danish pork E a c04 Comm. humans (feces) E a M 47 Gene profile c04 Comm. humans (feces) F 47 F c04 Comm. humans (feces) G b F c04 Comm. humans (feces) G b F a UTI patient (uncomp.) G b F 38 Gene profile c04 Comm. humans (feces) H a 51 M a UTI patient (uncomp.) H a F 28 Gene profile 6 69-a UTI patient (comp., elderly) I 38 F a UTI patient (uncomp.) J F 38 Gene profile a UTI patient (uncomp.) K b 52 F a UTI patient (comp., elderly) K b F a UTI patient (comp., recurrent) L F 45 Gene profile a UTI patient (comp., elderly) M a 46 F a UTI patient (comp., pregnancy) M a F 27 Comm. humans community-dwelling humans; Comp. complicated UTI due to (i) recurrent, recurrent case of UTI, (ii) elderly, elderly UTI patient, or (iii) pregnancy, pregnant UTI patient; Uncomp. uncomplicated UTI a Closely related isolates (2 3 PFGE bands difference) b Possibly related isolates (4 6 PFGE bands difference) isolates share antibiotic susceptibility patterns nor virulence gene profiles. Still, PFGE is a highly discriminating method and with the high diversity within the E. coli species, finding indistinguishable or related isolates are remarkable [10]. However, the pathogenecity of the cow isolate was not investigated and thus the zoonotic potential is unknown. Another study by Johnson et al. demonstrated two PFGE profiles with a 3-band difference of two faecal E. coli isolates from a chicken (from 1997) and a human volunteer (sampled during ) [32]. Both isolates were 076 antigen positive and contained fimh, iuta, trat, and ompt when screened for 35 virulence genes and 13 papa alleles. Even though no evident epidemiological connection was shown, the strength of the study by Johnson et al. was the analysis of isolates from the same locale and the 3-yearsampling period as compared to the study by Ramchandani et al. Like in the study by Ramchandani et al. the pathogenecity of the chicken isolate and thus the zoonotic potential also remained uninvestigated [10, 32]. Finally, recent studies reported clonal connections between a retail

6 1126 Eur J Clin Microbiol Infect Dis (2012) 31: Fig. 1 PFGE profiles of selected E. coli B2 isolates from broiler chicken meat, pork, community-dwelling humans and UTI patients. Lanes with meat isolates are underlined. Lanes 1 2 broiler chicken meat isolates ( and , respectively), lane 3 UTI isolate (357-a), lane 4 pork isolate ( ), and lane 5 community-dwelling human isolate (2030c04). The relatedness of all strains within the individual gene profiles are indicated in Table 2 chicken and UTI isolates, and between APEC isolates and human ExPEC isolates (from asymptomatic bacteruria, cystitis, urosepsis, intestinal sepsis, and respiratory sepsis) [33, 34]. In our study, we selected 22 B2 isolates with eight different gene profiles identified previously by microarray analysis of more than 300 virulence genes. With such an extensive array of traits finding isolates with identical gene profiles sharing genes was very unlikely even though the isolates were geographically and temporally matched. The identical gene profiles in themselves suggest a connection between the isolates [13]. We therefore investigated the PFGE profiles to explore any clonal relatedness. This study revealed the association of (i) two broiler chicken isolates and one UTI isolate with closely clonally related PFGE profiles showing a 3-band difference and (ii) one pork isolate and one community-dwelling human isolate with closely related PFGE profiles (2-band difference). This finding provides evidence of a clonal link between E. coli from meat, humans and UTI thus suggesting that UTI is a zoonosis. Although we did not investigate directionality of the transmission but a possible link exclusively, it is much more likely that the UTI patient acquired the isolate from animals through the food chain than a UTI patient transmitting isolates to food and animals, hence the term zoonosis. We also identified closely and possibly clonally related isolates from community-dwelling humans and UTI patients as well as from UTI patients only. To our knowledge, these isolates were epidemiologically unrelated. Clonally related UTI isolates from otherwise unrelated UTI patients have been demonstrated in studies which suggested, based on previous investigations, that it could be related to a point-source spread of these E. coli from, e.g., pork and chicken [25, 35]. This could also be the case in this study as the meat products in Denmark are distributed nationwide and the community-dwelling humans, who completed the questionnaire, all reported eating chicken and pork several times a week. Unfortunately, no dietary information could be collected from the UTI patients. Further, as mentioned previously, outbreaks of community-acquired UTI have been reported previously which substantiates the theory of point source spread [20 23]. From a food safety perspective, foodborne UTI isolates are of great concern as both broiler chicken meat and pork are frequently consumed many places. In comparison, fewer people are exposed to livestock why the overall risk of acquiring UTI isolates through direct contact with animals is lower. Some isolates with identical gene profiles did not have clonally related PFGE profiles, e.g. isolates with gene profile 1, 2, and 4. Obviously, this may be due to the fact that the strains are unrelated. It may also be explained by the time elapsed between the sampling of isolates allowing for the genome to change (e.g. gene profiles 2 and 4). Due to the plasticity of the E. coli genome, differences may likely arise on a PFGE profile. Further, we have no knowledge of which isolate is the original isolate (outbreak isolate) or if it is present among the isolates in our collection. We may therefore be comparing isolates not to the parental strain but to other diverted isolates. A second finding is the demonstration of in vivo virulence of E. coli isolates from a broiler chicken, meat and community-dwelling humans in the mouse model of ascending UTI. The mouse model is representative of human UTI and is thus a very important tool for investigating the hypothesis of UTI being a zoonosis, which molecular characterization cannot replace [11]. This further supported a previous study, where we demonstrated the virulence of B2 animal and meat isolates in the mouse UTI model [19]. Interestingly, isolates with identical gene profiles had also identical in vivo virulence profiles. This was consistent for isolates tested in vivo from gene profile 1, 3, and 4 even though isolates from gene profiles 1 and 4 had different PFGE types, respectively. This clearly demonstrated that meat isolates are similar to human isolates and that isolates from a broiler chicken, fresh meat as well as community-dwelling humans can cause UTI. Taken together, the above findings provide solid typing and in vivo evidence that UTI is at times a zoonosis. Limitations of the study include the limited knowledge of epidemiological information on UTI patients, e.g. dietary habits, as well as uncompleted questionnaires from three of

7 Eur J Clin Microbiol Infect Dis (2012) 31: Fig. 2 Bacterial counts in urine, bladder, and kidneys of mice killed 72 hours after inoculation with the different B2 E. coli strains from healthy production animals or meat. Each point represents the colony counts from one mouse. The small horizontal line represents the median bacterial count. The dotted line indicates the detection limit the six community-dwelling humans. Further, we do not have information on any epidemiological link between the meat isolates and UTI patients although consumption is likely. Finally, the UTI isolates were from one Danish region only. Strengths include the geographically and temporally matched isolates from animals, meat, and humans, the knowledge of the extensive genotypic background of the isolates, the epidemiological information on community-dwelling human isolates, and the in vivo model of UTI. In conclusion, we have demonstrated closely clonally related meat, community-dwelling human and UTI isolates by the highly discriminating typing method, PFGE. In addition, we have clearly demonstrated that a broiler chicken isolate, meat and community-dwelling human isolates are virulent in the mouse model of ascending UTI. This is, to our knowledge, the first study to show possible clonally related isolates from UTI patients, community-dwelling humans, animals and meat by virulence gene profile, PFGE types and in vivo virulence characteristics, thus providing solid evidence that UTI is at times a zoonosis. The possible and closely related community-dwelling human and UTI isolates may indicate

8 1128 Eur J Clin Microbiol Infect Dis (2012) 31: a point source spread, e.g. through contaminated meat. Further studies determining the proportion of animal attributed UTIs are essential to understand the actual impact of these isolates. Depending on the impact, different measures might be necessary, e.g. application of hazard analysis and critical control point (HACCP) in the animal production and slaughter, guidelines for handling and separation of specific food items to avoid cross-contamination of ready-to-eat food, and consumers implementing stricter hygiene routines in kitchens or elsewhere. Acknowledgments Frank Hansen, Karin S. Pedersen, Frederikke R. Petersen, Leila Borggild, Jytte M. Andersen, and Dorte Truelsen are thanked for excellent technical assistance. This study was supported by The Danish Research Council (grant no ). 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