Increased raw poultry meat colonization by extended spectrum beta-lactamase-producing Escherichia coli in the south of Spain

International Journal of Food Microbiology 159 (2012) 69–73

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Short communication

Increased raw poultry meat colonization by extended spectrum beta-lactamase-producing Escherichia coli in the south of Spain Pilar Egea a, b, Lorena López-Cerero b,⁎, Eva Torres a, María del Carmen Gómez-Sánchez c, Lara Serrano a, María Dolores Navarro Sánchez-Ortiz b, Jesús Rodriguez-Baño b, d, Alvaro Pascual a, b a

Microbiology Department, School of Medicine, University of Sevilla, Avda. Sánchez Pizjuán s/n. 41009, Sevilla, Spain Microbiology and Infectious Diseases Unit, University Hospital Virgen Macarena, Avda Dr Fedriani s/n. 41009, Sevilla, Spain Hospital Nuestra Señora de la Merced, Osuna, Avda. de la Constitución 2, 41640, Sevilla, Spain d Internal Medicine Department, School of Medicine, University of Sevilla, Avda. Sánchez Pizjuán s/n. 41009, Sevilla, Spain b c

a r t i c l e

i n f o

Article history: Received 8 March 2012 Received in revised form 10 July 2012 Accepted 4 August 2012 Available online 9 August 2012 Keywords: ESBLs E. coli Poultry

a b s t r a c t The present study was conducted to assess the prevalence of retail chicken and turkey meat colonized by extended-spectrum beta-lactamase-producing Escherichia coli (ESBLEC) in Seville, Spain. ESBLEC recovered from meat samples purchased in 2010 were characterized by specific PCR analysis for bla genes, phylogenetic groups and subgroups (genotypes) and O25b/pabB/B2 traits of ST131. Results were compared with those obtained in a previous study in 2007, when a high percentage of retail meat samples were found to be colonized by ESBLEC. The prevalence of retail poultry meat colonized by ESBLEC increased from 62.5% in 2007 to 93.3% in 2010 (p = 0.005). Non-pathogenic B1 and A1 genotypes accounted for more than 60% of the 60 isolates recovered. Sequence type ST131 or B2 phylogroup isolates were not detected. Clonal relatedness was detected in just 2 CTX-M-1-producing isolates from 2 chicken samples belonging to phylogenetic group A, genotype A1. There continued to be a significantly high quinolone resistance, with 85.4% and 32.2% of isolates showing resistance to nalidixic acid and ciprofloxacin, respectively. SHV-12 was the most common ESBL harbored by E. coli, although it has decreased in prevalence since 2007. Meanwhile, CTX-M ESBLs prevalence has increased. We conclude that the trend of colonization by ESBLECs—particularly CTX-M-producing isolates— in raw poultry meat has increased in a short period of time in our area. © 2012 Elsevier B.V. All rights reserved.

1. Introduction In the last few years, extended-spectrum beta-lactamase-producing Escherichia coli (ESBLEC) has emerged in the community causing human infections all over the world (Coque et al., 2008; Rodriguez-Bano et al., 2008a). In ESBL-producers, resistance to third-generation cephalosporins is usually accompanied by resistance to other antibiotics, such as aminoglycosides and fluoroquinolones. The epidemiology of ESBLEC is both evolving and complex. The potential role of food-producing animals as a possible reservoir of ESBLEC is currently being analyzed from different perspectives. Several reports describing the spread of extended spectrum beta-lactamase-producing Enterobacteriaceae mainly in the poultry industry (Blanc et al., 2006; Randall et al., 2011; Smet et al., 2008), but also in wild animals (Costa et al., 2006), pets (Carattoli et al., 2005) and retail meat (Aarestrup et al., 2006; Jouini et al., 2007) have been published. Various authors have also reported a relatively high prevalence in our country of healthy human carriers of faecal ESBLEC (Rodriguez-Bano et al., 2008b; Valverde et al., 2004). In recent years, a

⁎ Corresponding author. Tel.: + 34 955008138; fax: + 34 954377413. E-mail address: [email protected] (L. López-Cerero). 0168-1605/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ijfoodmicro.2012.08.002

few studies have provided evidence that ESBLECs and plasmid-encoded ESBLs can be transmitted between humans who have shared the same meal (Lavilla et al., 2008; Prats et al., 2003). The contamination of meat products by ESBLECs may therefore be contributing to the dissemination of the beta-lactamase encoding genes (bla), such as ESBLs, within the human population. In a previous study carried out by our group in Seville between 2006 and 2007, the prevalence of ESBLEC isolates in food samples of animal origin was analyzed. A high proportion of retail meat samples was found to be colonized by ESBLECs, mostly non-pathogenic (phylogenetic groups A and B1) (Doi et al., 2010). Recently, a group of isolates sharing the same sequence type (ST) by multi locus sequence type (MLST), named as ST131, has emerged as a widespread clonal group causing human infections in our country. ST131 is characterized by serotype O25b:H4, allele 3 of pabB gene and belongs to phylogenetic group B2, genotype B23. Ten percent of these isolates has been found producing an ESBL of the CTX-M family, the CTX-M-15 enzyme, in Spain (Blanco et al., 2011). Besides, ST131 isolates have emerged as a human pathogen producing several types of ESBLs all over the world (Coelho et al., 2011; Kim et al., 2011). Isolates belonging to this clonal group have also been detected among E. coli poultry isolates harbor blaCTX-M-9 in Spain (Mora et al., 2010).

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P. Egea et al. / International Journal of Food Microbiology 159 (2012) 69–73

These results led us to conduct a second study between 2010 and 2011 to assess trends in the prevalence and diversity of ESBLEC in retail chicken and turkey meat samples. We characterized patterns of antimicrobial resistance, type of ESBL, clonal relationships and phylogenetic groups and subgroups of ESBLEC, and compared these findings with the previous results. The spread of the O25b:H4-ST131 clonal group was also specifically investigated in all isolates belonging to phylogenetic groups B2 and D. 2. Materials and methods Thirty raw retail meat samples (15 chicken and 15 turkey breasts) were purchased from 10 local supermarkets in Seville between October and December, 2010. The samples were transported to the laboratory on ice and processed the same day. Twenty-five grams of each sample were homogenized with a Stomacher blender in 225 ml peptone broth. After overnight incubation at 37 °C, 0.1 ml aliquot of the broth was plated onto MacConkey agar with 1 μg/ml of cefotaxime or ceftazidime supplement and incubated overnight. When the culture showed positive on selective media, at least three colonies plus each distinct morphotype were selected for subsequent characterization and further identification using standard biochemical tests. Genetic relationship was determined for all cephalosporin-resistant E. coli isolates recovered from the same sample using repetitive extragenic palindromic (REP)-PCR (Vila et al., 1996). When identical REP-PCR patterns were found, only 1 isolate was selected for further characterization. Antibiotic susceptibility to amoxicillin/clavulanic acid, piperacillin/ tazobactam, cefoxitin, cefuroxime, ertapenem, fosfomycin, nalidixic acid, ciprofloxacin, cefepime, tobramycin, gentamicin and amikacin was performed using the disc diffusion method, following CLSI methodology (CLSI, 2010). ESBL production was confirmed by the double disc synergy test, according to CLSI recommendations (CLSI, 2010). To determine ESBL type, PCR assays were performed using TEM (Rasheed et al., 1997), SHV (Rasheed et al., 1997), CTX-M-9 (Sabate et al., 2000), and CTX-M-1 (Oteo et al., 2006) group-specific primers and both strands of amplicons sequenced further. Isolates were assigned to phylogenetic groups using a previously described multiplex PCR method (Clermont et al., 2000). Genotypes of the corresponding phylogroups were determined according to the scheme by Branger et al. (2005). O25b typing of isolates belonging to phylogenetic groups B2 and D was performed by PCR as previously described (Clermont et al., 2008). Phylogenetic subgroup A1 constituted the largest group in the first study on meat isolates (Doi et al., 2010) and continued to be frequent in the present survey. In 2007, a cluster of 5 A1 isolates were detected in samples of different sources (Lopez-Cerero et al., 2011). To detect other possible clusters or persistence of some strains, subgroup A1 isolates was selected for molecular typing. The genetic relatedness of isolates was determined by XbaI PFGE analysis (http://www.pulsenetinternational.org/protocols/Pages/default.aspx) for genotype A1 isolates. Dendrograms were created using Fingerprinting 3.0 software (BioRad). Cluster analysis was performed using the unweighted pair group method, with the Dice coefficient of similarity and tolerance set at 1%. Isolates were considered as belonging to the same PFGE cluster if the Dice similarity index was >85%. Categorical variables were compared using the chi-squared test (or Fisher's exact test when appropriate). For each comparison, a p value b 0.05 was considered as denoting a significant difference. SPSS software (version 17.0; SPSS, Chicago, Illinois, United States) was used for analyses. 3. Results ESBLECs were detected in 14 (93.3%) of 15 chicken samples, and 14 (93.3%) of 15 turkey meat samples. A total of 62 ESBLEC isolates were recovered from 28 positive samples: 39 ESBLECs (62.9%) were isolated in chicken breast samples and 23 (37.1%) in turkey meat

Table 1 Comparative distributions in 2007 and 2010 of the occurrence of ESBLs and phylogenetic groups/genotypes of E. coli isolates recovered from retail poultry meat samples in Seville, Spain.*This TEM producer was not available for further ESBL characterization by sequencing. Trait

2010

2007

p

Number of isolates (N = 62) (%)

Number of isolates (N = 52) (%)

19 10 2 5 2 0 41 1 1

9 5 0 1 1 2 43 0 0

Type of ESBL

Phylogenetic group A B1 B2 D

CTX-M-group CTX-M-14/17 CTX-M-9 CTX-M-32 CTX-M-1 CTX-M-15 SHV-12 SHV-12 + CTX-M-32 TEM* Genotype A0 A1 B1 B23 D1 D2

Non typeable

10 19 23 0 8 0 2

(30.6) (16.1) (3.2) (8.1) (3.2) (66.1) (1.6) (1.6) (16.7) (30.6) (38.3) (13.3) (3.3)

2 19 15 3 1 12 0

(17.3) (9.6) (1.9) (1.9) (3.8) (82.7)

(3.85) (36.5) (28.8) (5.8) (1.9) (23.1)

0.099

0.055

0.034

0.035 b0.001

samples. Eighteen (64.3%) samples were colonized by more than one ESBL-producing E. coli isolate, with an average of 2.21. SHV-12 was the most frequently detected ESBL (Table 1) and one isolate produced more than one ESBL type. Non-pathogenic A and B1 phylogroups predominated among isolates in 2010 (Table 1), and B2 was not found. The prevalence of resistant isolates is shown in Table 2. These data were compared with the first study, carried out in 2007. ESBLEC was detected in a higher proportion of raw chicken and turkey meat samples in 2010 (93.3%) than in 2007 (62.5%) (p =0.005). In 2010, a higher increase in the rate of colonized turkey meat (from 58% to 93.3%, p= 0.06) was observed than in broiler meat (from 67% to 93.3%, p= 0.13) comparing to 2007. In 2007, 69.5% of poultry samples were colonized by more than 1 isolate, and no isolate produced more than one ESBL type. The prevalence of SHV-12 has significantly decreased

Table 2 Antimicrobial susceptibility of ESBL-producing E. coli isolates recovered from retail poultry meat samples in Seville, Spain, in 2007 and 2010. Inhibition zone diameter (mm) corresponding to resistance breakpoint for tested antimicrobials: Nalidixic acid (NAL; ≤13), Ciprofloxacin (CIP; ≤15), Amoxicillin-clavulanic acid (AMC; ≤13), Piperacillin-tazobactam (TZP; ≤14), Cefuroxime (CXM; ≤14), Cefepime (FEP; ≤14), Cefoxitin (FOX; 14), Ertapenem (ETP; ≤ 19), Amikacin (AK; ≤ 14), Gentamicin (CN; ≤ 12), Tobramycin (TOB; ≤ 12), Fosfomycin (FOT; ≤ 12). Trait

Number (%) of isolates from year: 2010 (N = 62)

Quinolone resistance NAL 53 CIP 20 Beta-lactam/Beta-lactamase AMC 2 TZP 0 Cephalosporin resistance CXM 62 FEP 0 FOX 2 Carbapenem resistance ETP 0 Aminoglycoside resistance AK 0 CN 5 TOB 1 Fosfomycin resistance FOT 0

2007 (N = 52)

(85.4) 41 (78.9) (32.2) 21 (40.4) inhibitor combination resistance (3.2) 1 (1.9) 0 (100)

52 2

(3.2)

(100) (3.8) 0

p 0.35 0.37 0.66

0.21 0.49

0

(8.1) (1.6)

3 3

0 (5.8) (5.8) 0

0.72 0.34

P. Egea et al. / International Journal of Food Microbiology 159 (2012) 69–73

since 2007. However, frequencies of CTX-M type enzymes became higher in 2010 than they were in 2007, except for CTX-M-15, which has not been detected in 2010 (Table 1). Some differences were observed in the phylogenetic subgroup distribution of ESBLEC. There has been a significant increase in isolates belonging to genotypes A0 and D1 since 2007 (p = 0.03), while genotype D2 isolates, which accounted for 23% of total isolates in 2007, were not detected in 2010 (p b 0.001). Group B2 isolates recovered in 2007 were found to be negative for O25b. There were no differences in resistance patterns to the various antimicrobials tested (Table 2). The XbaI-macrorestriction profiles obtained by PFGE showed that only two CTX-M-1-producing isolates from 2 samples of chicken clustered with > 85% similarity in the dendrogram (Fig. 1). These samples did not

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belong to the same batch; they were obtained in different periods of time, one from a retail store and the other from a local supermarket. For 7 isolates a PFGE profile could not be obtained. 4. Discussion We report an increase in the prevalence of retail chicken and turkey meat that has been colonized by ESBLEC in Seville, Spain. In a study carried out in 2007, our group found a high degree of colonization by ESBLEC in these meat types. In this study, we found a rising trend of contaminated raw poultry meat, which was significantly higher in turkey meat. Several studies have suggested that retail meat may be a reservoir for ESBL-producing E. coli (Leverstein-van Hall et al., 2011; Overdevest et

Dice (Opt:0.50%) (Tol 1.0%-1.1% ) (H>0. 0% S>0.0%) [0.0% -100. 0% ]

PFGE

Isolate No. PG

ESBL

Sample

1011

A1

SHV-12

Chicken

2010

93/3

A1

SHV-12

Turkey

2006

100/2

A1

SHV-12

Chicken

2007

6/2

A1

SHV-12

Chicken

2006

87/7

A1

SHV-12

Chicken

2006

113/2

A1

SHV-12

Turkey

2007

1513

A1

SHV-12

Chicken

2010

62/1

A1

SHV-12

Turkey

2006

1122

A1

SHV-12

Turkey

2010

821/C

A1

SHV-12

Turkey

2010

94/7

A1

SHV-12

Chicken

2006

113/1

A1

CTX-M-15 Turkey

2007

1412

A1

SHV-12

Chicken

2010

513

A1

SHV-12

Chicken

2010

522

A1

SHV-12

Turkey

2010

123

A1

SHV-12

Turkey

2010

100/1

A1

SHV-12

Chicken

2007

221

A1

SHV-12

Turkey

2010

1521

A1

SHV-12

Turkey

2010

412

A1

SHV-12*

Chicken

2010

113/3

A1

SHV-12

Turkey

2007

413/C

A1

SHV-12

Chicken

2010

1111

A1

SHV-12

Chicken

2010

1522

A1

SHV-12

Turkey

2010

313

A1

SHV-12

Chicken

2010

112/C

A1

CTX-M-1

Chicken

2010

913/C

A1

CTX-M-1

Chicken

2010

41/2

A1

SHV-12

Chicken

2006

1221

A1

SHV-12

Turkey

2010

87/3

A1

SHV-12

Chicken

2006

46/3

A1

SHV-12

Chicken

2006

Year

100

80

60

PFGE

Fig. 1. XbaI-PFGE dendrogram for phylogenetic group A, genotype A1 E. coli isolates. PG: Phylogroup Genotypes (Clonal isolates are shown inside the red frame). *Isolate number 412 produced SHV-12 and CTX-M-32.

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al., 2011), although there have been differences in the prevalence of meat colonized by ESBLEC, and no temporal studies have been carried out. Our results are consistent with the findings of other investigators. In a recent study, Overdevest et al. (2011) found that 79.8% of retail chicken meat acquired in grocery shops in the Netherlands in 2010 carried ESBLEC. Leverstein-van Hall et al. (2011) reported a similar percentage, also in the Netherlands in 2010. The authors found that 94% of 98 retail samples of chicken meat studied contained at least one E. coli isolate with an ESBL phenotype. In a previous study, carried out in northeast Spain in 2004– 2006, Lavilla et al. (2008) reported a prevalence of colonized chicken meat up to 57.4%. SHV-12 is very widespread among ESBLs harbored by E. coli causing community and nosocomial infections in humans in Spain (RodriguezBano et al., 2009). Our results show that, in Seville, south of Spain, SHV12 continues to be the most frequent ESBL carried by E. coli isolates in raw poultry meat, although there has been a significant decrease since 2007. On the other hand, CTX-M group enzymes, which are also prevalent in human infections in Spain (Mora et al., 2011), were detected in a higher proportion of isolates in 2010. Data on prevalence of ESBLEC in fecal samples from broilers, environmental samples from slaughterhouses, meats transports or from slaughterers are scarce in our country to state the origin of these ESBLEC. Previously, Blanc et al. (2006) have identified SHV-12 in 7.8% and CTX-M-14 in 45.3% of isolates recovered from floor samples of fecal material from 10 farms at the North of Spain, but further studies are needed to determine the risk of colonization at every point of the chain. Resistance to quinolones continues to be very high, with 85.35% and 32.79% of isolates showing resistance to nalidixic acid and ciprofloxacin, respectively. Other authors have reported similar results for isolates taken from fecal samples of food animals. In 2010, Cortes et al. (2010) found a significant presence of resistance to nalidixic acid on poultry farms. The authors reported that 93% and 35% of ESBL- and CMYproducing E. coli isolated from the floor of farms showed resistance to nalidixic acid and ciprofloxacin, respectively. The phylogenetic distribution of isolates in the study by Cortés et al. was also consistent with our results, with phylogroups A (22.8%), B1 (38.6%) and D (31.6%) being the most frequently represented. Increased meat colonization does not appear to be due to clonal spread, since only 2 isolates from the majority group, A1, showed genetic relatedness by PFGE in this study. ST131 was not identified in our meat samples, in agreement to the results of a previous survey in the Netherlands (Overdevest et al., 2011). Overlapping of ESBL types in both human and meat isolates could indicate transfer of these genes in the same area. The spread of SHV-12 or CTX-M-14/17 could be due to one or various successful plasmids or multiple strains are acquiring different plasmids harboring the same enzyme. A further plasmid characterization would be required. Our study presents several limitations. The number of samples processed was small, not randomized, and systematic surveillance was not used; however, we purchased our meat samples from the same local supermarkets as in 2007, for purposes of comparison. Less frequent CTX-M groups than CTX-M-1 or CTX-M-9 have not been investigated. At least one ESBL type was detected in all isolates, but production of non‐studied enzymes could not be discarded. Although CTX-M-2 and CTX-M-25 groups have been found in human and poultry E. coli isolates, CTX-M-25 has not been detected in Spain, and only one human isolate harboring CTX-M-2 has been found in Madrid, Spain (Valverde et al., 2004). The theoretical hazards to human health from food-borne ESBLECs require further assessment. It is generally accepted that thorough cooking destroys bacteria in food, although cross-contamination to uncooked food may occur if hygiene measures are inadequate. In a recent survey in Seville, cooked poultry samples from volunteers were found to be negative, although one ESBL-producing E. coli was isolated in a sample of salad (Egea et al., 2011). The coexistence of the same ESBL types among clinical and food isolates could indicate exchanges of plasmids or mobile elements between E. coli isolates from

animals and humans. These findings are also compatible with transmission of ESBLEC, although there is scant (if increasing) evidence of that. Genotype A1, CTX-M-15-producing E. coli strains belonging to ST410 have been recovered from poultry meat and patients with UTI in Seville (Lopez-Cerero et al., 2011). Other MLST clusters, belonging to ST10 complex as well as ST410, containing CTX-M-1- and TEM-52-producing strains in both humans and meat have been found in the Netherlands (Overdevest et al., 2011). In summary, we found an upward trend for raw poultry meat colonized by ESBLECs in the study area, located in Seville, which attained the high prevalence recently detected in the Netherlands. Funding This study was supported by the Ministerio de Sanidad y Consumo, Instituto de Salud Carlos III-FEDER, Spanish Network for Research into Infectious Diseases (REIPI RD06/0008), Fondo de Investigación Sanitaria (FIS), Instituto de Salud Carlos III (PI070190), Consejerías de Salud e Innovación, Junta de Andalucía (PI-0048/2008 and CTS-5259) and the Ministerio de Educación y Ciencia (AGL-2008-02129). P. E. is supported by the research contract RIO HORTEGA from ISCIII (CM11/00297). Transparency declarations None to declare. References Aarestrup, F.M., Hasman, H., Agerso, Y., Jensen, L.B., Harksen, S., Svensmark, B., 2006. First description of blaCTX-M-1-carrying Escherichia coli isolates in Danish primary food production. The Journal of Antimicrobial Chemotherapy 57, 1258–1259. Blanc, V., Mesa, R., Saco, M., Lavilla, S., Prats, G., Miro, E., Navarro, F., Cortes, P., Llagostera, M., 2006. 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