FOOD-06423; No of Pages 5 International Journal of Food Microbiology xxx (2014) xxx–xxx

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Detection of Shiga toxin (Stx)-producing Escherichia coli (STEC) in bovine dairy herds in Northern Italy M. Trevisani a,⁎, R. Mancusi a, G. Delle Donne a, C. Bacci b, L. Bassi b, S. Bonardi b a b

Department of Veterinary Medical Sciences, University of Bologna, via Tolara di Sopra 50, 40064 Ozzano dell'Emilia, Italy Department of Veterinary Science, University of Parma, via Del Taglio10, 43126 Parma, Italy

a r t i c l e

i n f o

Available online xxxx Keywords: EHEC Shiga toxin Italian dairy farms IMS-based detection method Pathogenicity island OI-122

a b s t r a c t The aim of this study was to monitor the presence of Shiga toxin (Stx)-producing Escherichia coli in dairy farms authorized to sell raw milk and other farms, located in the same area, which sell milk to industry or use it to produce Parmesan or Grana cheese. Our research was focused on the serogroups O157 and O26, which are the most common in human cases in Italy and genetic markers that characterize the strains that can cause hemorrhagic colitis and hemolytic uremic syndrome (EHEC) in humans. Overall, 255 bulk-milk and 225 milk filter samples were screened for the presence of Shiga toxin genes (stx1 and stx2), O157 and O26 serogroups by using PCR. The samples were collected in 193 bovine dairy farms located in Northern Italy, including 32 farms selling raw milk to consumers. According to the preliminary PCR screening test, 32 out of 255 (12.5%; CI95%, 8.7% to 17.3%) bulk milk samples and 68 out of 225 (30.2%; CI95%, 24.3% to 36.7%) milk filters were positive for stx genes. Of the 32 milk samples that were stx-positive, 4 (1.6%, CI95%, 0.4% to 4%) were also positive by PCR for the rfbEO157 gene and 6 (2.4%, CI95%, 0.9% to 5.1%) were positive for the wzxO26 gene. The culture detection method, which was based on the immunomagnetic separation, achieved isolation rates of E. coli serogroups O157 and O26 in 25–67% of the milk samples that tested positive by PCR for these serogroups. STEC O26 was detected in one milk filter (1.6%) from a farm that sells raw milk to consumers directly and one sample (1.4%) of bulk milk intended for pasteurization. The presence of STEC O157 was also detected in 2 milk filters (1.7%) from farms that use milk to produce Grana cheese. All the STEC stains O157 and O26 isolated carried the genes eae and espK and genes belonging to the pathogenicity island OI-122 (efa1/2, sen, pagC), which are markers suitable for screening the human virulent EHEC strains. These virulence markers were also detected in the three strains of stx-negative E. coli O157 isolated from two filters and one milk sample. These strains could be therefore EHEC strains that have lost the stx genes (EHEC-derivative strains). Concern arise for the presence of EHEC O26 and E. coli O157 isolates that are suspected to be an EHEC-derivative in the milk filters sampled in farms that are used to sell raw milk to consumers and in other dairy farms. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Ruminants are important reservoir of Shiga toxin-producing Escherichia coli (STEC) strains that represent a significant risk to public health (Caprioli et al., 2005). The strains that are more virulent and can cause haemorrhagic colitis and hemolytic uremic syndrome (HUS) in humans are also known as Enterohemorrhagic E. coli (EHEC) (Bugarel et al., 2011a) and the strains belonging to the serogroups O157, O26, O103, O111 and O145 are the most commonly isolated from human cases (Tozzi et al., 2003; EFSA, 2011). Consumption of unpasteurized milk can pose a serious health risk if milk is contaminated by STEC, therefore requiring the implementation of control measures to manage and minimize the risk associated with sale of raw milk to consumers. Not only farms that sell raw milk directly to consumers, but also farms that sell milk for the production of raw milk cheeses ⁎ Corresponding author. Tel.: +39 0512097330; fax: +39 0512097346. E-mail address: [email protected] (M. Trevisani).

should be monitored in order to assess the infectious status of the herd. In a recent outbreak in South Italy, with 17 cases of HUS due to O26 STEC, the infection was related to a suspect contamination of cheeses or vegetables distributed locally in Apulia Region (Scavia et al., 2013). Epidemiological surveillance data not only reflects the current health status of dairy herds, but must be collected to manage environmental contamination problems (i.e. related to disposal of the effluents). Characterization of virulence is relevant for risk assessment. Recent reports indicate that antimicrobial resistance of STEC is rising (Threlfall et al., 2000; Mora et al., 2005) and other studies highlighted the importance of the study of some E. coli strains that express all features of EHEC strains except for the production of Shiga toxins (Bielaszewska et al., 2007a, 2007b; Bugarel et al., 2011b). This group of E. coli that could have lost the Shiga toxin genes (stx-negative derivative strains) was defined as EHEC-like. They can have great significance for the risk assessment of food and the epidemiological studies and therefore their detection should be included in epidemiological surveys.

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Please cite this article as: Trevisani, M., et al., Detection of Shiga toxin (Stx)-producing Escherichia coli (STEC) in bovine dairy herds in Northern Italy, Int. J. Food Microbiol. (2014), http://dx.doi.org/10.1016/j.ijfoodmicro.2013.12.033

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M. Trevisani et al. / International Journal of Food Microbiology xxx (2014) xxx–xxx

STEC might not be detected in bulk raw milk produced in infected herds, because their number is often very low if dairy farms meet the requirements of food hygiene (Jackson et al., 2012; Trevisani et al., 2013) but periodically cows can excrete high number of STEC with feces and the risk of contamination of milk increases. In addition to milk sampling, periodic monitoring of fecal samples and milk filters is needed to assess the prevalence of infected herds, although the cost of analyses can be very high by using the standard method (ISO/TS, 13136:2012). In EU countries, epidemiological reports showed that serogroups O157 and O26 were detected in 41.1–51.7% and 7.0–5.4% of the confirmed human cases in 2010 and 2009, respectively. In the same period, 33.7–28.2% of the cases were due to not-typeable serogroups, 5.3–4.3% to serogroups O103, O145 and O111, and 13–11% to other serotypes (EFSA/ECDC, 2012). In 2009–2010 2,681 raw cows' milk samples were analyzed in four EU Member States and only for Germany it was clearly indicated that 266 out of 603 samples concerned raw milk intended for direct human consumption and only 88 them were sold at farm with the recommendation for heating. In 2009, 6.8% of the raw milk samples sold in Germany to consumers were positive for STEC isolates, but no STEC O157 was found among positive samples. In the previous two years (2007–2008) only Austria, Czech Republic, Germany, Italy and Latvia reported on the occurrence of STEC in raw milk sold directly to consumers. The aim of this study was to monitor the presence of Shiga toxin-producing E. coli in dairy farms authorized to sell raw milk and compare the data with other farms, located in the same area, which are used to sell milk to industry or use it for cheese making. The study was focused on the serogroups O157 and O26. 2. Materials and methods 2.1. Sample collection A total of 267 bulk milk samples and 218 milk filters were collected at 193 dairy farms located in Northern Italy (Emilia Romagna and Lombardy regions) from April 2009 to June 2010. Farms had different characteristics in relation to the milk quality parameters and use of feed ingredients. Thirty-two farms used to sell raw milk to consumers through automatic dispensers (group 1), 36 of them used to sell milk to industry for pasteurized milk production (group 2), while 119 farms used milk for the production of Parmesan or Grana cheese (group 3). Almost all the farms that sell raw milk in the provinces of Bologna and Parma were included in this study. The other farms were randomly selected. In the farms of the group 1 and 2 bulk milk samples and milk filters were taken twice, in summer and fall seasons, but for 14 farms of groups 2 sampling was done at the arrival of milk at the processing plant and therefore the milk filters were not available. In farms processing milk to produce Parmesan or Grana cheese (group 3) milk and filters were taken only once. Bulk milk samples were aseptically taken by using sterilized bottles from the refrigerated storage tank after mixing the mass for 5 min. Filters of the milking machines were taken with single-use gloves and put in sterile bags. All samples were transported in refrigerated bags to the laboratories and analyzed within 24 h. 2.2. Culture and STEC screening Before testing, milk filters were cut into two equal parts with sterile scissors. In order to detect E. coli O157 half milk filters and 25 ml-aliquots of raw milk were suspended in 225 ml of modified Tryptone Soy Broth (m-TSB, Oxoid, Basingstoke, UK) supplemented with novobiocin 20 mg/l (Sigma-Aldrich, Steinheim, Germany) and enriched overnight at 41.5 ± 1 °C. For detection of non-O157 isolates, half milk filters and 25 ml-aliquots of raw milk were suspended in 225 ml of m-TSB with 12 mg/l acriflavin. Broths were incubated at 37 ± 1 °C overnight.

After incubation, all the cultures were tested by PCR for the detection of the Shiga toxin genes (stx1 and stx2). DNA extracts were obtained with thermal cell lysis using Chelex 100 (Bio-Rad, Munich, Germany). An aliquot (5 μl) of the supernatant was used as template for PCR analysis. The 20 μl PCR mixture consisted of 0.4 μM each of MK1 and MK2 primers (Karch and Meyer, 1989) 0.2 μM dNTPs mix, 1.5 mM Mg2Cl, 2.5 μl 10 × PCR buffer (Platinum Taq, Invitrogen) 1 U Taq (Platinum, Invitrogen). The absence of inhibitors in sample extracts was assessed by repeating the amplification in case of negative results after spiking the samples with DNA extracts of ATCC 35150 O157:H7 or FV4028 O26:H11 STEC cultures (100–300 CFU ml−1) or by using an internal amplification control based on the use of recombinant primers MK1-pUC19 and MK2 pUC19 and 2 pg/μl pUC19 (Sigma Aldrich) as described by Wieczorek and Osek (2004). Samples positive for the stx genes were subsequently screened by using a real-time PCR assay for the detection of O157 and O26 serogroups specific genes. The DNA extracts were analyzed using a 5′ nuclease PCR assay with primers and probes targeting rfbEO157 and wzxO26 genes (Perelle et al., 2004). Real-time PCR was performed with a MiniOpticon thermocycler using iQSupermix, manufacturer's protocol. Final concentrations of 500 nM of each primer and 200 nM of each probes were used. The amplification conditions were 95 °C for 3 min, followed by 40 cycles at 95 °C for 10 s and 63 °C for 45 s. Samples that gave positive results to real-time PCR for O157 or O26 serogroups were subjected to the immunomagneticseparation technique (IMS) by using either Dynabeads ® anti E. coli O157 or EPEC/STEC O26 following the manufacturer's instructions. The target bacteria were isolated on specific selective/differential media, Cefixime-Tellurite Sorbitol MacConkey Agar (CT-SMAC, Oxoid, UK) for STEC O157 and Cefixime-Tellurite Rhamnose MacConkey Agar (CT-RMAC, Lab M, Bury, UK) for STEC O26. In addition, MacConkey agar (Oxoid, UK) plates were used for both serogroups. Plates were incubated at 37 °C ± 1 °C for 18–24 h. Up to ten sorbitol non-fermenting, colorless colonies grown on CT-SMAC/CT-RMAC, or pink colonies grown on MacConkey agar were selected and tested for indole production. Indole-positive cultures were subjected to slide agglutination with E. coli O157 Latex test kit (Oxoid) and E. coli O26 antiserum (Denka Seiken, Tokio, Japan). Agglutinating cultures were confirmed biochemically as E. coli by using the API 20 E® system (bioMérieux). 2.3. Colony lift — Stx DIG labeled probes hybridization Detection of non-O157 or non-O26 E. coli was performed using a colony lift DNA hybridization method. Enrichments of stx1 or stx2 PCRpositive samples, but negative for O157 or O26 specific sequences, were diluted with Buffer Saline solution and filtered through ISO-Grid (Neogen-Acumedia, USA) membranes, then put on MacConkey agar plates in order to allow growth of 300–1000 colonies per filter. These were blotted to Nylon + membranes (Roche, Mannheim, Germany) and hybridization (44 °C) and detection steps were carried out using the DIG High Prime DNA Labelling and Detection Starter Kit I (Roche) following the manufacturer's instructions. The membranes were hybridized with 90 ng of both stx1 and stx2 probes in 50 ml of hybridization buffer. The DNA probes for random prime labeling were derived via PCR from E. coli O157 strain ATCC 35150 by using the primers designed by Arthur et al. (2002). The developed films from the colony blots were aligned with the original plates and up to five suspect colonies per sample were streaked for isolation on MacConkey agar. 2.4. Characterization of isolates and antibiotic resistance E. coli O157, O26 and other stx-putative isolates were subjected to multiplex PCR (mPCR) for the detection of the genes coding for Shigatoxins (stx1, stx2), intimin (eae) and enterohaemolysin (hlyA) as described by Paton and Paton (1998). All STEC isolates were further tested by the EU Escherichia coli Reference Laboratory (Istituto Superiore di Sanità, Rome, Italy) for serogroup and virulence confirmation. Virulence

Please cite this article as: Trevisani, M., et al., Detection of Shiga toxin (Stx)-producing Escherichia coli (STEC) in bovine dairy herds in Northern Italy, Int. J. Food Microbiol. (2014), http://dx.doi.org/10.1016/j.ijfoodmicro.2013.12.033

M. Trevisani et al. / International Journal of Food Microbiology xxx (2014) xxx–xxx

traits of the isolates were further characterized by testing for pathogenic island OI-122 sequences, namely those of genes pagC, sen, efa1 and efa2 as described by Karmali et al. (2003) and for espK gene, which is associated with the type III secretion system and arcA (aerobic respiratory control protein A) allelic type by using the method described by (Bugarel et al., 2011b). Susceptibility to antimicrobial agents was determined by the standardized disk diffusion assay on Iso-Sensitest Agar (Oxoid) with commercial antimicrobial susceptibility disks (Oxoid) according to the recommendations of the Clinical and Laboratory Standards Institute (CLSI, 2006a, 2006b). The antimicrobials tested and their corresponding disk concentrations were as follows: ampicillin (A, 10 μg), amoxicillin and clavulanic acid 2:1 (Amc, 30 μg), cefotaxime (Ctx, 30 μg), ciprofloxacin (Cip, 5 μg), cephalothin (Cf, 30 μg), ceftazidime (Caz, 30 μg), chloramphenicol (C, 30 μg), colistine (Col, 10 μg), enrofloxacin (En, 5 μg), gentamicin (Cm, 10 μg), kanamicin (K, 30 μg), nalidixic acid (Nx, 30 μg), neomycin (N, 30 μg), streptomycin (S, 10 μg), sulphonamide (Su, 300 μg), tetracycline (T, 30 μg), and trimethoprim/sulphametoxazole (Sxt, 1.25/ 23.75 μg). Results were categorized as susceptible, intermediate or resistant according to Clinical and Laboratory Standards Institute guidelines (CLSI, 2006a). Outcomes were dichotomized after aggregating the resistant and intermediate resistance categories together according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST). 2.5. Statistical analysis Prevalence and exact confidence intervals based on probabilities derived from the binomial distribution for proportions were calculated using the software EpiTools (Sergeant, 2013). 3. Results and discussion According to the preliminary PCR screening test, 32 out of 255 (12.5%; CI95%, 8.7% to 17.3%) bulk milk samples and 68 out of 225 (30.2%; CI95%, 24.3% to 36.7%) milk filters were positive by PCR for stx genes. Of the 64 samples collected in the 32 farms of the group 1 (selling raw milk directly to consumers) the number of stx-positive milk and filter samples were 4 (6.2%, CI95%, 8.7% to 17.3%) and 17 (26.6%, CI95%, 16.3% to 39.1%), respectively (Table 1). The proportion of stx-positive samples was also lower in milk than in filter samples collected in the farms of the groups 2 and 3, but statistically significantly differences were observed only for samples of the group 3, where the proportions of stx-positive milk and filter samples were 16% (CI95%, 9.9–23.8%) and 37% (CI95%, 28.3–46.3%), respectively. Of the 32 milk samples that

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were stx-positive, 4 (12.5%, CI95%, 3.5% to 29%) were also positive by PCR for the rfbEO157 gene (overall 4/255, 1.6%, CI95%,0.4% to 4%) and 6 (18.8%, CI95%, 7.2% to 36.4%) were positive for the wzxO26 gene (overall 6/255, 2.4%, CI95%,0.9% to 5.1%). Of the 68 filter samples that were stx-positive, 23 (33.8%, CI95%, 22.8% to 46.3%) were positive for the serogroups O157 and 5 (7.4%, CI95%, 2.4% to 16.3%) for the serogroups O26. Overall, 23/225 filter samples (10.2%, CI95%, 6.6% to 14.9%) were positive for the serogroup O157 and 5/225 (2.2%, CI95%, 0.7% to 5.1%) were positive for the serogroup O26. The IMS-based methods achieved the detection of 6 E. coli O157 isolates, two of which were stx-positive, and 9 E. coli O26 isolates, with two of them stx-positive. In addition, E. coli isolates of the serogroup O157, which were found to be deficient for the stx genes, were recovered in 1 bulk milk and in 3 milk filter samples. E. coli O26 stx-negative were isolated from 5 bulk milk and in 2 milk filter samples (Table 1). Nevertheless, the isolates were recovered only in 6 (22.2%) of the 27 enriched samples positive for the gene rfbEO157 and 9 (81.8%) of the 11 enriched samples positive for the gene wzxO26. The recovery of E. coli isolates of the two serogroups was not consistently different in milk and filter samples, i.e. E. coli O157 in 25% of milk samples (1 of 4) and 21.7% of filter samples (5/23); E. coli O26 in 100% of milk samples (6/6) and 60% of filter samples (3/5). Different hypotheses can been done to explain the lower detection rate of the IMS and culture methods, including the fact that the STEC capture efficiency of immunomagnetic beads is not 100% (Madic et al., 2011; Verstraete et al., 2010) and therefore the detection could be difficult in the milk samples where the number of STEC is very low (Trevisani et al., 2013). Moreover, they could not be isolated for the presence of interfering microorganisms that are not removed during the IMS procedure and prevail in the plating media. Nevertheless, the isolation is required to demonstrate the presence of all the genes identified in the same live bacteria cell. In addition to the E. coli isolates of the serogroups O157 and O26, stx-positive strains of other not-typed serogroups (nonO157, O26, O103, O111, O145) were detected in 4 milk filter samples. The virulence characteristics of the stx-positive and stx-negative isolates are reported in Table 2. All the isolates E. coli O157 and O26 STEC stx-positive, also contained the genetic markers eae (encoding intimin) and espK (a type III secreted effector protein of enterohemorrhagic E. coli) and the marker genes of the pathogenicity island OI-122. Also three isolates E. coli O157 stx-negative were found to be positive for eae, espK and the OI-122 marker genes. The rhamnose-fermenting E. coli O26 isolates that were detected in milk samples also possessed the eae and the putative markers of the OI-122, but did not share the espK gene and allelic genotype arcA2, which are regarded as a unique genetic marker for

Table 1 Results of screening and confirmatory tests to detect STEC positive samples. Group

Samples

PCR on enrichment cultures

Detection of E. coli isolates

No. (%) stx-positive

No. (%) O157b

Stx-positive No. (%) with detection of the serogroupa O157 1 2 3 Total

Milk (N = 64) Filter (N = 64) Milk (N = 72) Filter (N = 42) Milk (N = 119) Filter (N = 119) Milk (N = 255) Filter (N = 225)

4 17 9 7 19 44 32 68

6.2% 26.6% 12.5% 16.7% 16.0% 37.0% 12.5% 30.2%

0 2 4 3 0 18 4 23

Stx-negative No. (%) O26b

O26 0% 3.1% 5.6% 7.1% 0% 15.1% 1.6% 10.2%

1 2 4 0 1 3 6 5

1.6% 3.1% 5.6% 0% 0.8% 2.5% 2.4% 2.2%

0 0 0 0 0 2 0 2

0% 0% 0% 0% 0% 1.7% 0% 0.9%

0 1 1 0 0 0 1 1

0% 1.6% 1.4% 0% 0% 0% 0.4% 0.4%

No. (%) otherc serogroups

No. (%) O157b

0 3 0 0 0 1 0 4

0 1 1 1 0 1 1 3

0% 4.7% 0% 0% 0% 0.8% 0% 1.8%

No. (%) O26b

0% 1.6% 1.4% 2.4% 0% 0.8% 0.4% 1.3%

1 0 3 0 1 2 5 2

1.6% 0% 4.2% 0% 0.8% 1.7% 2% 0.9%

Group 1, farms selling row milk directly to consumers; Group 2, farms selling milk to industry for pasteurized milk production; Group 3, farms using milk for production of Parmesan or Grana cheese; stx, genes encoding shigatoxins. a Detection by real-time PCR in stx-positive samples. b Characterization of E. coli isolates detected in samples positive by PCR for O157/O26 serogroups. c Isolates detected by colony DNA probe hybridization.

Please cite this article as: Trevisani, M., et al., Detection of Shiga toxin (Stx)-producing Escherichia coli (STEC) in bovine dairy herds in Northern Italy, Int. J. Food Microbiol. (2014), http://dx.doi.org/10.1016/j.ijfoodmicro.2013.12.033

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M. Trevisani et al. / International Journal of Food Microbiology xxx (2014) xxx–xxx

Table 2 Virulence characteristics of isolates. Origin of samples

Sample code

Serogroup (phenotype)

vtx1

vtx2

eae

hlyA

OI-122 sen

Cow milk filters

Cow milk Cow milk filters

Cow milk

pagC

efa1a

efa1b

espK

arcA allele

P44 B61 B17 B20 B14 B36

O26 (R−) O157 (S+) NA NA NA O26 (R+)

Farms selling raw milk directly to consumers (group 1) + − + + + − − − + − + + + + + − + + + − + − − − − + − − − − − − + + + −

+ + − − − +

+ + − − − +

+ + − − −

Type 2 nd nd nd nd Type 1

P108 P99 P141 P105 P141 B5N B47H B147H

O157 (S−) O157 (S−) O157 (S−) NA O157 (S−) O26 (R−) O26 (R+) O26 (R+)

Farms selling raw milk to industry (groups 2 and 3) + + + + + + − + + − + + − − + + + + + − + + − − − − + + + + + + + − + − − − + − + − − − + − + −

+ + + − + + + +

+ + + − + + + +

+ + + − + + − −

nd nd nd nd nd Type 2 Type 1 Type 1

NA, not agglutinating; R−, non-fermenting rhamnose; S+, fermenting sorbitol; S−, non-fermenting sorbitol; nd, not detected.

EHEC and EHEC-like strains (EHEC strains that may have derived from EHEC by losing stx-genes) (Bugarel et al., 2011b). Whether the stx-negative strains that contained eae, espK and OI-122 putative marker genes were EHEC-derivative (EHEC-like) strains should be further investigated. Detection of various combinations of non-LEEencoded type III effector genes was explored in some recent papers in order to test their suitability for clearly distinguishing the EHEC from other E. coli pathogroups (Bugarel et al., 2011b; Delannoy et al., 2013). In their very recent paper Delannoy et al. (2013) explored different genetic markers, like espK, ureD, espV, espN, Z2098 and espM1, in 1100 E. coli strains and they reported that detection of espK and/or Z2098 allowed detection of most of the EHEC serotypes associated with human infections, but the association of espK with either espV, ureD or Z2098 proved to be the best combinations for a more specific and sensitive detection of EHEC strains. In the present study, antimicrobial resistance was tested by disk diffusion, which should be refereed as a screening method if compared to the minimum inhibitory concentrations (MIC) determination, which is the reference method for antimicrobial susceptibility testing (EUCAST, 2000). All our stx-positive E. coli isolates isolates were susceptible to the 17 antimicrobials tested. Among the stx-negative E. coli isolates, two E. coli O26 strains showed resistance against streptomycin and tetracycline (R-type: STe) and one E. coli O157 strain was resistant to cephalothin. No isolates were resistant to more than two antimicrobial agents. In other studies antimicrobial resistance in O157 and nonO157 STEC strains was tested by the disk diffusion method, detecting high resistance values against sulfisoxazole (39% of the isolates), tetracycline (32%), streptomycin (29%) and ampicillin (10%) in Spain (Mora et al., 2005) or against tetracycline (71%), chloramphenicol (57%), kanamycin (29%), nalidixic acid and ciprofloxacin (14%) in Ireland (Scott et al., 2009). Other authors tested STEC isolates for their MIC against different antibiotics, detecting the highest resistance values for cephalotin (10%), tetracycline (8%) and ampicillin (5%) in Germany (Klein and Bülte, 2003). In Germany and Bosnia MIC resistance for sulfonamide compounds (100%), trimethoprim/ sulphametoxazole (30%–10%) and ampicillin (13–10%) was reported (von Müffling et al., 2007). The restriction in the use of antimicrobials in lactating cows is important to prevent an increase of resistance to antimicrobials in EHEC, presumptive EHEC derivative strains and other STEC that are present in dairy farms. Characterization of the antimicrobial resistance in STEC isolates acts as an epidemiological marker for Shiga toxin-producing E. coli detected at dairy farms. In human therapy, the use of antibiotics in EHEC infections is controversial, as it could induce expression of Stx through replication of phages carrying stx genes, thus enhancing the

development of HUS (Karch et al., 1986). Some studies have reported protective effect or no association of antimicrobial treatments of E. coli O157:H7 enteritis in increasing HUS risk (Safdar et al., 2002). This negative effect was not observed for rifamixin, which could therefore be used to treat human EHEC gastroenteritis (Ochoa et al., 2007). The presence of putative EHEC-derivative (i.e. serogroup O157) in the milk filters is indicative of a possible contamination of milk with EHEC strains that lost the stx genes and their presence also in one sample taken in a farm that used to sell raw milk to consumers is matter of concern. Legislation issued by the Italian Authority oblige farmers to put on the vending machines a label stating that raw milk must be boiled before consumption (CSR, 2007) and forbid the use of raw milk for production of ice creams (OMH, 2010). Regional legislations (e.g. RER, 2008; RV, 2012) oblige farmers that sell raw milk to consumers to test raw milk for STEC O157, O26, O103, O111, O145 and O104 monthly. Only after repeated negative tests (e.g. three times in Italy) the Official Authority can authorize the dairy farm to sell raw milk to consumers. Official controls consist of periodical hygiene audits and analyses of milk samples (e.g. two times per year in Italy). Apart from hard ripened cheeses, such as Parmigiano–Reggiano and Grana-cheeses, semi-hard ripened cheeses and pasteurized-milk cheeses, dairy products made from raw milk represent a potential threat to human health. Dairy producers must be aware of potential on-farm risk factors and reduce/ prevent food-borne pathogen contamination of dairy products leaving the farm. The presence of infected herds in the same area, which potentially may shed EHEC strains in the environment, should also be considered by the dairy producers that sell raw milk directly to consumers or to industry that use it to make raw milk cheese. Acknowledgments The research leading to these results received funding from the European Union Seventh Framework Programme (FP7/2007–2013) under grant agreement no. 222738. Project BASELINE (selection and improving of fit-for-purpose sampling procedures for specific foods and risks). References Arthur, T.M., Barkocy-Gallagher, G.A., Rivera-Betancourt, M., Koohmaraie, M., 2002. Prevalence and characterization of non-O157 Shiga Toxin-Producing Escherichia coli on carcasses in commercial beef cattle processing plants. Appl. Environ. Microbiol. 68, 4847–4852. Bielaszewska, M., Käch, R., Friedrich, A.W., von Eiff, C., Zimmerhackl, L.B., Karch, H., Mellmann, A., 2007a. Shiga toxin-mediated hemolytic uremic syndrome: time to change the diagnostic paradigm? PLoS ONE (10), e1024 (October, accessed at www.plosone.org).

Please cite this article as: Trevisani, M., et al., Detection of Shiga toxin (Stx)-producing Escherichia coli (STEC) in bovine dairy herds in Northern Italy, Int. J. Food Microbiol. (2014), http://dx.doi.org/10.1016/j.ijfoodmicro.2013.12.033

M. Trevisani et al. / International Journal of Food Microbiology xxx (2014) xxx–xxx Bielaszewska, M., Sonntag, A.K., Schmid, M.A., Karch, H., 2007b. Presence of virulence and fitness gene modules of enterohemorrhagic Escherichia coli in atypical enteropathogenic Escherichia coli O26. Microbes Infect. 9, 891–897. Bugarel, M., Martin, A., Fach, P., Beutin, L., 2011a. Virulence gene profiling of enterohemorrhagic (EHEC) and enteropathogenic (EPEC) Escherichia coli strains: a basis for molecular risk assessment of typical and atypical EPEC strains. BMC Microbiol. 11, 142–152. Bugarel, M., Beutin, L., Scheutz, F., Loukiadis, E., Fach, P., 2011b. Identification of genetic markers for differentiation of Shiga toxin-producing, enteropathogenic, and avirulent strains of Escherichia coli O26. Appl. Environ. Microbiol. 77, 2275–2281. Caprioli, A., Morabito, S., Brugere, H., Oswald, E., 2005. Enterohaemorrhagic Escherichia coli: emerging issues on virulence and modes of transmission. Vet. Res. 36, 289–311. CLSI, 2006a. Performance Standards for Antimicrobial Susceptibility Testing; 16th Informational Supplement, M100-S15. CLSI, Wayne, PA (2006). CLSI, 2006b. Performance Standards for Antimicrobial Disk and Dilution Susceptibility Tests for Bacteria Isolated from Animals, Tentative Guideline, M31-A3, 3rd edn. CLSI, Wayne, PA (2006). CSR, 2007. [State-Regions Conference agreement of the 25 January 2007 on the direct selling of raw milk for human consumption] Intesa tra il Governo, le Regioni e le Province autonome di Trento e di Bolzano in materia di vendita diretta di latte crudo per l'alimentazione umana. Punto 3/B-Repertorio Atti n. 5/CSR del 25 gennaio 2007. Delannoy, S., Beutin, L., Fach, P., 2013. Discrimination of enterohemorrhagic E. coli (EHEC) from non-EHEC strains based on detection of various combinations of non-LEEencoded type III effector genes. J. Clin. Microbiol. http://dx.doi.org/10.1128/ JCM.01471-13 (published online ahead of print on 24 July 2013). EFSA, 2011. The European Union Summary Report on Trends and Sources of Zoonoses, Zoonotic Agents and Food-borne Outbreaks in 2009. EFSA J. 9, 2090 [378 pp.]. EFSA/ECDC, 2012. The European Union Summary Report on Trends and Sources of Zoonoses, Zoonotic Agents and Food-borne Outbreaks in 2010. EFSA J. 10, 2597 [442 pp.]. EUCAST, 2000. EUCAST Definitive Document E.DEF 3.1, June 2000: Determination of Minimum Inhibitory Concentrations (MICs) of Antibacterial Agents by Agar Dilution. Clin. Microbiol. Infect. 6, 509–515. ISO/TS 13136:2012, 2012. Microbiology of food and animal feed – real-time polymerase chain reaction (PCR)-based method for the detection of food-borne pathogens – horizontal method for the detection of Shiga toxin-producing Escherichia coli (STEC) and the determination of O157, O111, O26, O103 and O145 serogroups. International Organization for Standardization. Jackson, E.E., Erten, E.S., Maddi, N., Graham, T.E., Larkin, J.W., Blodgett, R.J., Schlesser, J.E., Reddy, R., 2012. Detection and enumeration of four foodborne pathogens in raw commingled silo milk in the United States. J. Food Prot. 75, 1382–1393. Karch, H., Meyer, T., 1989. PCR amplification of Shiga-like toxin genes. J. Clin. Microbiol. 27, 2751–2757. Karch, H., Strockbine, N.A., O'Brien, A.D., 1986. Growth of Escherichia coli in the presence of thrimethoprim-sulfamethoxazole facilitates detection of Shiga-like toxin producing strains by colony blot assay. FEMS Microbiol. Lett. 35, 141–145. Karmali, M.A., Mascarenhas, M., Shen, S., Ziebell, K., Johnson, S., Reid-Smith, R., IsaacRenton, J., Clark, C., Rahn, K., Kaper, J.B., 2003. Association of genomic O Island 122 of Escherichia coli EDL 933 with Verocytotoxin-producing Escherichia coli seropathotypes that are linked to epidemic and/or serious disease. J. Clin. Microbiol. 41, 4930–4940. Klein, G., Bülte, M., 2003. Antibiotic susceptibility pattern of Escherichia coli strains with verocytotoxic E. coli-associated virulence factors from food and animal faeces. Food Microbiol. 20, 27–33. Madic, J., Vingadassalon, N., de Garam, C.P., Marault, M., Scheutz, F., Brugère, H., Jamet, E., Auvray, F., 2011. Detection of Shiga toxin-producing Escherichia coli serotypes O26: H11, O103:H2, O111:H8, O145:H28, and O157:H7 in raw-milk cheeses by using multiplex real-time PCR. Appl. Environ. Microbiol. 77, 2035–2041. Mora, A., Blanco, J.E., Blanco, M., Alonso, M.P., Dhabi, G., Echeita, A., González, E.A., Bernárdez, E.I., Blanco, J., 2005. Antimicrobial resistance of Shiga toxin (verotoxin)producing Escherichia coli O157 and non-O157 strains isolated from humans, cattle, sheep and food in Spain. Res. Microbiol. 156, 793–806.

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Ochoa, T.J., Chen, J., Walker, C.M., Gonzales, E., Cleary, T.G., 2007. Rifamixin does not induce toxin production or phage-mediates lysis of Shiga toxin-producing Escherichia coli. Antimicrob. Agents Chemother. 51, 2837–2841. OMH, 2010. [Decree of the Ministry of Health on urgent measures on the production, distribution and retail of raw milk for human consumption] Proroga dell'ordinanza del Ministro del lavoro, della salute e delle politiche sociali 10 dicembre 2008, concernente: «Misure urgenti in materia di produzione, commercializzazione e vendita diretta di latte crudo per l'alimentazione umana» e integrazioni per la produzione di gelati in imprese registrate ai sensi del Regolamento (CE) n 852/ 2004. Gazzetta Ufficiale N. 11 del 15 Gennaio 2011.. Paton, A.W., Paton, J.C., 1998. Detection and characterization of Shiga toxigenic Escherichia coli by using multiplex PCR assays for stx1, stx2, eaeA, enterohaemorrhagic E. coli hlyA, rfbO111, and rfbO157. J. Clin. Microbiol. 36, 598–602. Perelle, S., Dilasser, F., Grout, J., Fach, P., 2004. Detection by 5′-nuclease PCR of Shiga-toxin producing Escherichia coli O26, O55, O91, O103, O111, O113, O145 and O157:H7, associated with the world's most frequent clinical cases. Mol. Cell. Probes 18, 185–192. RER, 2008. Regione Emilia-Romagna. [Order of Regional Veterinary Public Health Authority concerning direct selling of raw milk from farmers to consumers] Determinazione del Responsabile del Servizio Veterinario e Igiene degli Alimenti 21 aprile 2008, n. 4418. Vendita diretta al consumatore di latte crudo vaccino, ovi-caprino, bufalino e asinino dell'azienda di produzione. Bollettino Ufficiale della Regione EmiliaRomagna numero 74 anno 2008. RV, 2012. [Legislative provisions concerning direct selling of raw milk from farmers to consumers]. Disposizioni per la disciplina della vendita diretta di latte crudo dal produttore agricolo al consumatore finale. ALLEGATO A alla Dgr n. 513 del 03 aprile 2012. Bollettino Ufficiale della Regione Veneto numero 32 del 24 Aprile 2012. Safdar, N., Said, A., Gangnon, R.E., Maki, D.G., 2002. Risk of hemolytic uremic syndrome after antibiotic treatment of Escherichia coli O157:H7 enteritis. J. Am. Med. Assoc. 288, 996–1001. Scavia, G., Germinario, C., Giordano, M., Carbone, V., De Paolo, T., Gianviti, A., Ardissino, G., Chironna, M., Mongelli, O., Ricci, M., Minelli, F., Maugliani, A., Caprioli, A., 2013. Focolaio epidemico di infezione da E. coli produttore di verocitotossina (VTEC) O26 in Puglia, agosto 2013: il punto della situazione. Epicentro. (http://www.epicentro. iss.it/problemi/seu/Puglia2013.asp accessed 20 October 2013). Scott, L., McGee, P., Walsh, C., Fanning, S., Sweeney, T., Blanco, J., Karczmarczyk, M., Earley, B., Leonard, N., Sheridan, J.J., 2009. Detection of numerous verotoxigenic E. coli serotypes, with multiple antibiotic resistance from cattle faeces and soil. Vet. Microbiol. 134, 288–293. Sergeant, E.S.G., 2013. Epitools epidemiological calculators. AusVet Animal Health Services and Australian Biosecurity Cooperative Research Centre for Emerging Infectious Disease. Available at: http://epitools.ausvet.com.au. Threlfall, E.J., Ward, L.R., Frost, J.A., Willshaw, G.A., 2000. The emergence and spread of antibiotic resistance in food-borne bacteria. Int. J. Food Microbiol. 62, 1–5. Tozzi, A.E., Caprioli, A., Minelli, F., Gianviti, A., De Petris, L., Edefonti, A., Montini, G., Ferretti, A., De Palo, T., Gaido, M., Rizzoni, G., Hemolytic Uremic Syndrome Study Group, 2003. Shiga toxin-producing Escherichia coli infections associated with hemolytic uremic syndrome, Italy, 1988–2000. Emerg. Infect. Dis. J. 9, 106–108. Trevisani, M., Mancusi, R., Riu, R., Serraino, A., 2013. Quantitative detection of Shigatoxinproducing and enteropathogenic Escherichia coli serotypes O157 and O26 in bulk raw milk. Food Anal. Methods 6, 1750–1758. Verstraete, K., De Zutter, L., Messens, W., Herman, L., Heyndrickx, M., De Reu, K., 2010. Effect of the enrichment time and immunomagnetic separation on the detection of Shiga toxin-producing Escherichia coli O26, O103, O111, O145 and sorbitol positive O157 from artificially inoculated cattle faeces. Vet. Microbiol. 145, 106–112. Von Müffling, T., Smaijlovic, M., Nowak, B., Sammet, K., Bülte, M., Klein, G., 2007. Preliminary study of certain serotypes, genetic and antimicrobial resistance profiles of verotoxigenic Escherichia coli (VTEC) isolated in Bosnia and Germany from cattle or pigs or their products. Int. J. Food Microbiol. 117, 185–191. Wieczorek, K., Osek, J., 2004. Development of a PCR internal amplification control for the detection of shiga toxin-producing Escherichia coli. Bull. Vet. Inst. Pulawy 48, 397–401.

Please cite this article as: Trevisani, M., et al., Detection of Shiga toxin (Stx)-producing Escherichia coli (STEC) in bovine dairy herds in Northern Italy, Int. J. Food Microbiol. (2014), http://dx.doi.org/10.1016/j.ijfoodmicro.2013.12.033

Detection of Shiga toxin (Stx)-producing Escherichia coli (STEC) in bovine dairy herds in Northern Italy.

The aim of this study was to monitor the presence of Shiga toxin (Stx)-producing Escherichia coli in dairy farms authorized to sell raw milk and other...
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