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Journal of Environmental Science and Health, Part B: Pesticides, Food Contaminants, and Agricultural Wastes Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/lesb20
Resistance and virulence factors of Escherichia coli isolated from chicken a
a
Silvie Pavlickova , Magda Dolezalova & Ivan Holko
ab
a
Faculty of Technology, Department of Environmental Protection Engineering, Tomas Bata University in Zlin, Zlin, Czech Republic b
Vetservis s.r.o., Nitra, Slovak Republic Published online: 06 Apr 2015.
Click for updates To cite this article: Silvie Pavlickova, Magda Dolezalova & Ivan Holko (2015) Resistance and virulence factors of Escherichia coli isolated from chicken, Journal of Environmental Science and Health, Part B: Pesticides, Food Contaminants, and Agricultural Wastes, 50:6, 417-421, DOI: 10.1080/03601234.2015.1011959 To link to this article: http://dx.doi.org/10.1080/03601234.2015.1011959
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Journal of Environmental Science and Health, Part B (2015) 50, 417–421 Copyright © Taylor & Francis Group, LLC ISSN: 0360-1234 (Print); 1532-4109 (Online) DOI: 10.1080/03601234.2015.1011959
Resistance and virulence factors of Escherichia coli isolated from chicken SILVIE PAVLICKOVA1, MAGDA DOLEZALOVA1 and IVAN HOLKO1,2 1
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2
Faculty of Technology, Department of Environmental Protection Engineering, Tomas Bata University in Zlin, Zlin, Czech Republic Vetservis s.r.o., Nitra, Slovak Republic
Chicken meat has become an important part of the human diet and besides contamination by pathogenic Escherichia coli there is a risk of antibiotic resistance spreading via the food chain. The purpose of this study was to examine the prevalence of resistance against eight antibiotics and the presence of 14 virulence factors among 75 Escherichia coli strains isolated from chicken meat in the Czech Republic after classification into phylogenetic groups by the multiplex PCR method. More than half of strains belonged to A phylogroup, next frequently represented was B1 phylogroup, which suggests the commensal strains. The other strains were classified into phylogroups B2 and D, which had more virulence factors. Almost half of all E. coli strains were resistant to at least one of eighttested antibiotics. A multidrug resistance was observed in 13% of strains. The most prevalent virulence genes were iucD, iss and tsh. None of genes encoding toxins was detected. Most of E. coli strains isolated from chicken meat can be considered as nonpathogenic on the basis of analysis of virulence factors, antibiotic resistance and phylogroups assignment. It can provide a useful tool for prediction of a potential risk from food contaminated by E. coli. Keywords: Escherichia coli, antibiotic resistance, phylogenetic groups, virulence factors.
Introduction Poultry meat represents a substantial part of the human diet. The average of annual consumption of poultry has increased steadily. However, chicken meat shows high level of Escherichia coli contamination, and these E. coli might be more resistant to antibiotics than E. coli obtained from other types of meat.[1] Therefore, the quality of poultry purchased in retail markets is a concern for suppliers, consumers and public health worldwide.[2] In 2006, the European Union has gradually banned the use of all growth-promoting antimicrobials.[3] However, because of high production of meat for human consumption, antibiotics are still used for the treatment, prevention and control of bacterial infections in animal husbandry.[2,4] Resistant strains of E. coli are isolated especially from poultry and pigs.[2,5–7] Incidence of resistant isolates in cattle is lower.[3,7] Resistant strains of E. coli were also found in eggs, milk and milk products.[8,9]
Address corresponding to Silvie Pavlickova, Faculty of Technology, Department of Environmental Protection Engineering, Tomas Bata University in Zlin, Nam. T. G. M. 275, Zlin 762 72, Czech Republic; E-mail: [email protected] Received October 14, 2014.
Resistant bacteria present in the intestines of slaughtered animals can contaminate the carcasses during slaughtering, and thereby transfer genes of resistance into the human intestinal flora directly or via the food chain. Thereafter, these resistant bacteria are indirect threat to human health.[10] E. coli have been categorized into intestinal pathogenic, extraintestinal pathogenic (ExPEC), and commensal E. coli. While intestinal pathotypes are well defined, ExPEC exhibit considerable genome diversity and have a broad range of virulence factors encoded on mobile DNA elements.[11] The iss (increased serum survival) gene and its protein product (ISS) of avian pathogenic E. coli (APEC) are important characteristics of resistance to the complement system. The iss gene is probably located in a conserved portion of some plasmids.[12] The broad group of E. coli known as enterohemorrhagic E. coli (EHEC), including E. coli O157:H7, contain the eae gene for attachment and effacing. The Einv gene is responsible for encoding enteroinvasive mechanisms. The Eagg gene is known for its encoding enteroaggregative mechanisms.[13] Genes encoding temperature-sensitive hemagglutinin (tsh), aerobactin (iucD), and salmochelin (iroN) systems, ColV operon (cvi/cva), serum resistance protein (iss) and transfer protein (traT) are known for their frequent or exclusive localization on large transmissible R plasmids or ColV plasmids in APEC.[12] These genes are clearly associated with APEC strains.[11]
418 The aim of this study was to characterize E. coli from chicken meat and thus assess the potential risk to human health.
Pavlickova et al. strains were assigned to the phylogenetic groups or subgroups.[16,17]
Virulence factors and colicins
Materials and methods
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Strains and cultivation media A total of 75 E. coli strains from retail chicken meat were isolated at Faculty of Technology, Tomas Bata University in Zlin, the Czech Republic. Ten grams of skin from breast and legs were aseptically removed from each chicken carcass. These skin samples were homogenized for 30 min in 90 mL saline solution, seeded on Endo agar (Oxoid Ltd., United Kingdom) plates and incubated at 37 C/24 h. Suspected violet colonies were isolated and identified to the species level by using commercial identification microsystem ENTEROtest24 (ErbaLachema Brno, Czech Republic). This test is designed for routine, definitive identification of important strains of family Enterobacteriaceae. The kit contains 24 biochemical tests (dehydrated substrates for biochemical reactions, e.g, IND, ONP), which are placed in microplate. Obtained data were evaluated by software TNW Lite 6.5 (ErbaLachema Brno, Czech Republic). The strains were stored at ¡80 C in glycerol. Strains were cultivated on Nutrient Agar (beef extract 10 g, peptone 10 g, agar 15 g, NaCl 5 g, distilled water 1,000 mL, pH 7,2) or on Mueller Hinton Agar.
Antimicrobial resistance In all tested strains, antimicrobial susceptibility testing to eight antibiotics was performed by disc diffusion method according to EUCAST methodology.[14] Antibiotic discs (Oxoid Ltd.): amoxicillin/clavulanic acid (AMC 30 mg), ampicillin (AMP 10 mg), cefotaxime (CTX 5 mg), cefuroxime (CXM 30 mg), ciprofloxacin (CIP 5 mg), chloramphenicol (C 30 mg), imipenem (IPM 10 mg), and sulfamethoxazole/trimethoprim (SXT 25 mg) were used. The diameters of the inhibition zones were evaluated (susceptible, intermediate, resistant) according to EUCAST breakpoints.
Phylogenetic groups A previously described multiplex PCR method (chuA, yjaA, TspE4.C2) was used for determination of phylogenetic groups.[15] A few colonies of each strain were solved in 100 mL of 1_c PCR buffer (ThermoPol Buffer, NEB, USA) and heated up to 95 C for 20 min. Supernatant after centrifugation (10,000 g L¡1 min¡1) was taken as DNA template for all PCR testing. The amplification products were visualized by 1% agarose gel electrophoresis and
All strains were tested for presence of 14 virulence factors (eaeA, Einv, Eagg, iss, iucD, neuC, papC, tsh, vat, ST1, ST2, CNF1, CNF2, and LT1) and for colicins B (cba) and colicin M (cma) by single PCR as was described previously.[11,18–20]
Statistical analysis The results of antibiotic resistance and virulence factors were evaluated by Pearson’s correlation coefficient between the measured variables. The analysis was performed using statistical software STATISTICA CZ.[21]
Results and discussion The goal of this work was to evaluate the possibility of antibiotic resistance spreading and incidence of virulence factors in retail chicken meat. A total of 75 E. coli isolates from chicken meat were examined for occurrence of antibiotic resistance, presence of virulence factors and were also assigned into phylogenetic groups and subgroups (Table 1). In this study, more than half of strains belonged to A phylogroup (42% A0, 16% A1), slightly less frequently was represented B1 phylogroup (25%), and just 17% fell within phylogroups B2 and D together. In this study, most of the strains isolated from chicken meat are not ExPEC because that usually belongs to groups B2 and D.[17] In healthy food-producing animals, a predominant distribution of group B1 was reported in E. coli from cattle, while group A was predominantly prevalent in pigs and chickens,[17] what is in accordance with this study. In comparison with human isolates, most of the ExPEC strains (NMEC) were assigned to B2, but also half of all isolates from healthy humans were surprisingly assigned to B2. It suggests that these strains might harbor pathogenic potential.[22] Table 1. Characterization of E. coli strains isolated from chicken meat (%). Phylogenetic A B1 B2 D
Groupsa
Antibiotic Resistanceb
Virulence Factorsb
58 25 13 4
44 63 60 0
54 90 90 100
percentage of all tested strains (n D 75). percentage in each phylogroup (nA D 43, nB1 D 19, nB2 D 10, nD D 3).
a
b
419
Resistance and virulence factors Table 2. Occurrence of virulence factors in phylogroups A, B1, B2 and D. A
eaeA iss iucD neuC papC Tsh Vat
B1
B2
D
n*
%
n*
%
n*
%
n*
%
0 9 16 1 2 11 0
0 12 21 1 3 15 0
0 12 5 0 1 7 1
0 16 7 0 1 9 1
2 1 4 0 0 6 0
3 1 5 0 0 8 0
0 2 1 0 1 1 1
0 3 1 0 1 1 1
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*Number of positive virulence factors.
None of the strains isolated from retail meat demonstrated any occurrence of these virulence factors and toxins: Einv, Eagg, ST1, ST2, CNF1, CNF2, and LT1. Almost half of strains from A phylogroup had no virulence factors and carried a little antibiotic resistance (aminopenicillins). The most prevalent virulence genes in E. coli from chicken meat in this work were iucD, iss and tsh (Table 2). One strain unexpectedly carried the neuC gene, which is related with neonatal meningitis. Five strains encoded both tested colicins together (B and M). In this study, incidence of 14 tested virulence factors are more frequent among E. coli of phylogroups B2 and D (16%) in opposite to strains of phylogroups A and B1 (10%) (Table 3). Similar finding was shown in another study.[23] The analysis of phylogenetic groups, together with virulence factor detection, may provide a useful tool to predict potential risk associated with E. coli strains.[24] The most prevalent virulence genes (iucD, iss, and tsh) are associated with APEC.[11] Moreover, some strains can be considered as APEC, because they probably harbor plasmid pAPEC-O1-ColBM or its part. A 174-kb ColBM Table 3. Occurrence of antibiotic resistance in phylogroups A, B1, B2 and D. A
a
AMC AMP C CIP CTX CXM IPM SXT
B1
B2
D
n*
%
n*
%
n*
%
n*
%
9/3 13/1 4/0 8/4 0/2 0/2 0/2 3/0
12/4 17/1 5/0 11/5 0/3 0/3 0/3 4/0
9/0 12/0 3/0 2/5 0/4 0/0 0/1 4/0
12/0 16/0 4/0 3/7 0/5 0/0 0/1 5/0
2/1 2/1 1/0 2/0 0/2 0/2 0/0 0/0
3/1 3/1 1/0 3/0 0/3 0/3 0/0 0/0
0/0 0/0 0/0 0/0 0/0 0/1 0/0 0/0
0/0 0/0 0/0 0/0 0/0 0/1 0/0 0/0
*Number of resistant/intermediate E. coli strains. a AMC-amoxicillin/clavulanic acid; AMP-ampicillin; C-chloramphenicol; CIP-ciprofloxacin; CTX-cefotaxime; CXM-cefuroxime; IPM-imipenem; SXT-sulphametoxazole/trimethoprim.
plasmid was originally identified in UPEC strains, where it was found to encode the colicins B and M. pAPEC-O1ColBM was found to contain several genes that have previously been associated with APEC virulence—etsABC, sitABCD, iucABCD, iutA, iss, iroBCDEN, cvaA, cvaB, eitABCD and tsh.[12] Four strains (120, 121, 273 and 285) were proved to encode genes for colicin B and M, and concurrently two of three virulence factors (iucD, iss and tsh), which are closely related with this plasmid. The distribution of phylogenetic groups in this E. coli collection can serve as bacterial source tracking tool. For example, the strains 273 and 285 (both B23) from this study can be considered as human feces contamination in chicken retail meat, because all strains from B23 were previously observed as of human origin.[17] As this strains were isolated from retail chickens, it is more likely that they are APEC. However, human ExPEC strains appeared to be closely related to APEC strains.[25] Surprisingly, one strain was positive for neuC gene (A0), which is highly prevalent among NMEC (92%), less among APEC (30%) strains, which are assigned especially in B2 and D phylogroup.[11] Antimicrobial susceptibility of 75 E. coli strains isolated from chicken meat was determined against 8 antibiotics (Table 3). From all tested strains almost 49% of all E. coli strains were resistant to at least one of eight tested antibiotics and 13% of isolates had multidrug resistance. The highest resistance was observed to aminopenicillins: ampicillin (38%) and amoxicillin/clavulanic acid (33%). The most frequently used groups of antibiotics in poultry are penicillins, tetracyclines, sulfonamides, potentiated sulfonamides and quinolones. Aminopenicillins (AMC and AMP) are also considered as drugs of first choice in the treatment of poultry.[26] In comparison with other European countries, E. coli strains isolated from food animals has shown a higher prevalence of resistance to AMP (35– 75%),[2,7] and AMC (23–45%).[2,27] It might be due to the fact, that these antibiotics have been commonly used for prevention or treatment of diseases in poultry production. Moreover, E. coli strains from retail meat were also found resistant to antibiotics such as trimethoprim/sulphamethoxazole, third-generation cephalosporins and fluoroquinolones.[28] In our study 16% of strains were resistant to CIP and 10% strains showed resistance to SXT. Other studies have shown a higher resistance to CIP, especially in chickens.[29] This might reflect a higher use of quinolones in chickens compared with pigs or other animals.[7] In all tested strains were observed 11% resistance to chloramphenicol. Similarly, the higher resistance to chloramphenicol was reported in chicken (21%)[29] and other animals (16%).[27] None of all strains were resistant to cefotaxime and cefuroxime. The low incidence of antibiotic resistance of these antibiotics (0–3.8%) were confirmed by other studies.[2,7] Until recently, carbapenems were the choice for the therapy of multidrug resistant gram-negative bacterial infections. Currently, carbapenemases producing strains from
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420 Enterobacteriaceae represent a serious threat to human health.[30] Although Antimicrobial resistance interactive database of European Centre for Disease Prevention and Control (ECDC) has reported only 0.0–0.2% of resistant E. coli strains, the amount of intermediate strains has increased to 1% in 2011 in the Czech Republic.[31] In this study, was found 4% of intermediate strains to imipenem. Therefore, should be increased attention in usage of these antibiotics, and thus prevent the emergence of resistance. Resistance to three or more antibiotics is taken as multidrug resistance.[32] From all tested strains 13% of isolates had multidrug resistance. Recently, it was reported that multidrug resistance is widespread in E. coli of poultry origin and is associated with conjugative plasmid.[33] The multi-resistant strains belonged to group A and B1. This is in accordance with study of Johnson et al.,[33] where isolates from phylogroup A and B1 tended to have a higher prevalence of resistance. Nevertheless, these groups were proved as groups with lower prevalence of virulence factors. Association between antibiotic resistance (AMP) and presence of virulence factor (gene iss) was also demonstrated in another study.[33] In this work, statistically significant correlation (P < 0.05) was proved between the presence of antibiotic resistance and virulence factors.
Conclusion Escherichia coli from chicken meat in the Czech Republic were found to be frequently resistant to five antibiotics, and 13% of strains were found to be multiresistant. More than half of tested strains belonged to A0 and A1phylogroup with lower incidence of virulence factors, what can determine commensal strains. The other strains were classified into B2 and D phylogroups, which contains more virulence factors. Most of E. coli strains isolated from chicken meat can be considered as nonpathogenic on the basis of phylogenetic analysis, presence of virulence factors and antibiotic resistance. The analysis of these factors together can provide a useful tool for prediction of a potential risk associated with E. coli from food.
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Funding This work was supported by Internal Grant of Tomas Bata University in Zlin (No. IGA/FT/2014/005).
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Journal of Environmental Science and Health, Part B: Pesticides, Food Contaminants, and Agricultural Wastes Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/lesb20
Resistance and virulence factors of Escherichia coli isolated from chicken a
a
Silvie Pavlickova , Magda Dolezalova & Ivan Holko
ab
a
Faculty of Technology, Department of Environmental Protection Engineering, Tomas Bata University in Zlin, Zlin, Czech Republic b
Vetservis s.r.o., Nitra, Slovak Republic Published online: 06 Apr 2015.
Click for updates To cite this article: Silvie Pavlickova, Magda Dolezalova & Ivan Holko (2015) Resistance and virulence factors of Escherichia coli isolated from chicken, Journal of Environmental Science and Health, Part B: Pesticides, Food Contaminants, and Agricultural Wastes, 50:6, 417-421, DOI: 10.1080/03601234.2015.1011959 To link to this article: http://dx.doi.org/10.1080/03601234.2015.1011959
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Journal of Environmental Science and Health, Part B (2015) 50, 417–421 Copyright © Taylor & Francis Group, LLC ISSN: 0360-1234 (Print); 1532-4109 (Online) DOI: 10.1080/03601234.2015.1011959
Resistance and virulence factors of Escherichia coli isolated from chicken SILVIE PAVLICKOVA1, MAGDA DOLEZALOVA1 and IVAN HOLKO1,2 1
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2
Faculty of Technology, Department of Environmental Protection Engineering, Tomas Bata University in Zlin, Zlin, Czech Republic Vetservis s.r.o., Nitra, Slovak Republic
Chicken meat has become an important part of the human diet and besides contamination by pathogenic Escherichia coli there is a risk of antibiotic resistance spreading via the food chain. The purpose of this study was to examine the prevalence of resistance against eight antibiotics and the presence of 14 virulence factors among 75 Escherichia coli strains isolated from chicken meat in the Czech Republic after classification into phylogenetic groups by the multiplex PCR method. More than half of strains belonged to A phylogroup, next frequently represented was B1 phylogroup, which suggests the commensal strains. The other strains were classified into phylogroups B2 and D, which had more virulence factors. Almost half of all E. coli strains were resistant to at least one of eighttested antibiotics. A multidrug resistance was observed in 13% of strains. The most prevalent virulence genes were iucD, iss and tsh. None of genes encoding toxins was detected. Most of E. coli strains isolated from chicken meat can be considered as nonpathogenic on the basis of analysis of virulence factors, antibiotic resistance and phylogroups assignment. It can provide a useful tool for prediction of a potential risk from food contaminated by E. coli. Keywords: Escherichia coli, antibiotic resistance, phylogenetic groups, virulence factors.
Introduction Poultry meat represents a substantial part of the human diet. The average of annual consumption of poultry has increased steadily. However, chicken meat shows high level of Escherichia coli contamination, and these E. coli might be more resistant to antibiotics than E. coli obtained from other types of meat.[1] Therefore, the quality of poultry purchased in retail markets is a concern for suppliers, consumers and public health worldwide.[2] In 2006, the European Union has gradually banned the use of all growth-promoting antimicrobials.[3] However, because of high production of meat for human consumption, antibiotics are still used for the treatment, prevention and control of bacterial infections in animal husbandry.[2,4] Resistant strains of E. coli are isolated especially from poultry and pigs.[2,5–7] Incidence of resistant isolates in cattle is lower.[3,7] Resistant strains of E. coli were also found in eggs, milk and milk products.[8,9]
Address corresponding to Silvie Pavlickova, Faculty of Technology, Department of Environmental Protection Engineering, Tomas Bata University in Zlin, Nam. T. G. M. 275, Zlin 762 72, Czech Republic; E-mail: [email protected] Received October 14, 2014.
Resistant bacteria present in the intestines of slaughtered animals can contaminate the carcasses during slaughtering, and thereby transfer genes of resistance into the human intestinal flora directly or via the food chain. Thereafter, these resistant bacteria are indirect threat to human health.[10] E. coli have been categorized into intestinal pathogenic, extraintestinal pathogenic (ExPEC), and commensal E. coli. While intestinal pathotypes are well defined, ExPEC exhibit considerable genome diversity and have a broad range of virulence factors encoded on mobile DNA elements.[11] The iss (increased serum survival) gene and its protein product (ISS) of avian pathogenic E. coli (APEC) are important characteristics of resistance to the complement system. The iss gene is probably located in a conserved portion of some plasmids.[12] The broad group of E. coli known as enterohemorrhagic E. coli (EHEC), including E. coli O157:H7, contain the eae gene for attachment and effacing. The Einv gene is responsible for encoding enteroinvasive mechanisms. The Eagg gene is known for its encoding enteroaggregative mechanisms.[13] Genes encoding temperature-sensitive hemagglutinin (tsh), aerobactin (iucD), and salmochelin (iroN) systems, ColV operon (cvi/cva), serum resistance protein (iss) and transfer protein (traT) are known for their frequent or exclusive localization on large transmissible R plasmids or ColV plasmids in APEC.[12] These genes are clearly associated with APEC strains.[11]
418 The aim of this study was to characterize E. coli from chicken meat and thus assess the potential risk to human health.
Pavlickova et al. strains were assigned to the phylogenetic groups or subgroups.[16,17]
Virulence factors and colicins
Materials and methods
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Strains and cultivation media A total of 75 E. coli strains from retail chicken meat were isolated at Faculty of Technology, Tomas Bata University in Zlin, the Czech Republic. Ten grams of skin from breast and legs were aseptically removed from each chicken carcass. These skin samples were homogenized for 30 min in 90 mL saline solution, seeded on Endo agar (Oxoid Ltd., United Kingdom) plates and incubated at 37 C/24 h. Suspected violet colonies were isolated and identified to the species level by using commercial identification microsystem ENTEROtest24 (ErbaLachema Brno, Czech Republic). This test is designed for routine, definitive identification of important strains of family Enterobacteriaceae. The kit contains 24 biochemical tests (dehydrated substrates for biochemical reactions, e.g, IND, ONP), which are placed in microplate. Obtained data were evaluated by software TNW Lite 6.5 (ErbaLachema Brno, Czech Republic). The strains were stored at ¡80 C in glycerol. Strains were cultivated on Nutrient Agar (beef extract 10 g, peptone 10 g, agar 15 g, NaCl 5 g, distilled water 1,000 mL, pH 7,2) or on Mueller Hinton Agar.
Antimicrobial resistance In all tested strains, antimicrobial susceptibility testing to eight antibiotics was performed by disc diffusion method according to EUCAST methodology.[14] Antibiotic discs (Oxoid Ltd.): amoxicillin/clavulanic acid (AMC 30 mg), ampicillin (AMP 10 mg), cefotaxime (CTX 5 mg), cefuroxime (CXM 30 mg), ciprofloxacin (CIP 5 mg), chloramphenicol (C 30 mg), imipenem (IPM 10 mg), and sulfamethoxazole/trimethoprim (SXT 25 mg) were used. The diameters of the inhibition zones were evaluated (susceptible, intermediate, resistant) according to EUCAST breakpoints.
Phylogenetic groups A previously described multiplex PCR method (chuA, yjaA, TspE4.C2) was used for determination of phylogenetic groups.[15] A few colonies of each strain were solved in 100 mL of 1_c PCR buffer (ThermoPol Buffer, NEB, USA) and heated up to 95 C for 20 min. Supernatant after centrifugation (10,000 g L¡1 min¡1) was taken as DNA template for all PCR testing. The amplification products were visualized by 1% agarose gel electrophoresis and
All strains were tested for presence of 14 virulence factors (eaeA, Einv, Eagg, iss, iucD, neuC, papC, tsh, vat, ST1, ST2, CNF1, CNF2, and LT1) and for colicins B (cba) and colicin M (cma) by single PCR as was described previously.[11,18–20]
Statistical analysis The results of antibiotic resistance and virulence factors were evaluated by Pearson’s correlation coefficient between the measured variables. The analysis was performed using statistical software STATISTICA CZ.[21]
Results and discussion The goal of this work was to evaluate the possibility of antibiotic resistance spreading and incidence of virulence factors in retail chicken meat. A total of 75 E. coli isolates from chicken meat were examined for occurrence of antibiotic resistance, presence of virulence factors and were also assigned into phylogenetic groups and subgroups (Table 1). In this study, more than half of strains belonged to A phylogroup (42% A0, 16% A1), slightly less frequently was represented B1 phylogroup (25%), and just 17% fell within phylogroups B2 and D together. In this study, most of the strains isolated from chicken meat are not ExPEC because that usually belongs to groups B2 and D.[17] In healthy food-producing animals, a predominant distribution of group B1 was reported in E. coli from cattle, while group A was predominantly prevalent in pigs and chickens,[17] what is in accordance with this study. In comparison with human isolates, most of the ExPEC strains (NMEC) were assigned to B2, but also half of all isolates from healthy humans were surprisingly assigned to B2. It suggests that these strains might harbor pathogenic potential.[22] Table 1. Characterization of E. coli strains isolated from chicken meat (%). Phylogenetic A B1 B2 D
Groupsa
Antibiotic Resistanceb
Virulence Factorsb
58 25 13 4
44 63 60 0
54 90 90 100
percentage of all tested strains (n D 75). percentage in each phylogroup (nA D 43, nB1 D 19, nB2 D 10, nD D 3).
a
b
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Resistance and virulence factors Table 2. Occurrence of virulence factors in phylogroups A, B1, B2 and D. A
eaeA iss iucD neuC papC Tsh Vat
B1
B2
D
n*
%
n*
%
n*
%
n*
%
0 9 16 1 2 11 0
0 12 21 1 3 15 0
0 12 5 0 1 7 1
0 16 7 0 1 9 1
2 1 4 0 0 6 0
3 1 5 0 0 8 0
0 2 1 0 1 1 1
0 3 1 0 1 1 1
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*Number of positive virulence factors.
None of the strains isolated from retail meat demonstrated any occurrence of these virulence factors and toxins: Einv, Eagg, ST1, ST2, CNF1, CNF2, and LT1. Almost half of strains from A phylogroup had no virulence factors and carried a little antibiotic resistance (aminopenicillins). The most prevalent virulence genes in E. coli from chicken meat in this work were iucD, iss and tsh (Table 2). One strain unexpectedly carried the neuC gene, which is related with neonatal meningitis. Five strains encoded both tested colicins together (B and M). In this study, incidence of 14 tested virulence factors are more frequent among E. coli of phylogroups B2 and D (16%) in opposite to strains of phylogroups A and B1 (10%) (Table 3). Similar finding was shown in another study.[23] The analysis of phylogenetic groups, together with virulence factor detection, may provide a useful tool to predict potential risk associated with E. coli strains.[24] The most prevalent virulence genes (iucD, iss, and tsh) are associated with APEC.[11] Moreover, some strains can be considered as APEC, because they probably harbor plasmid pAPEC-O1-ColBM or its part. A 174-kb ColBM Table 3. Occurrence of antibiotic resistance in phylogroups A, B1, B2 and D. A
a
AMC AMP C CIP CTX CXM IPM SXT
B1
B2
D
n*
%
n*
%
n*
%
n*
%
9/3 13/1 4/0 8/4 0/2 0/2 0/2 3/0
12/4 17/1 5/0 11/5 0/3 0/3 0/3 4/0
9/0 12/0 3/0 2/5 0/4 0/0 0/1 4/0
12/0 16/0 4/0 3/7 0/5 0/0 0/1 5/0
2/1 2/1 1/0 2/0 0/2 0/2 0/0 0/0
3/1 3/1 1/0 3/0 0/3 0/3 0/0 0/0
0/0 0/0 0/0 0/0 0/0 0/1 0/0 0/0
0/0 0/0 0/0 0/0 0/0 0/1 0/0 0/0
*Number of resistant/intermediate E. coli strains. a AMC-amoxicillin/clavulanic acid; AMP-ampicillin; C-chloramphenicol; CIP-ciprofloxacin; CTX-cefotaxime; CXM-cefuroxime; IPM-imipenem; SXT-sulphametoxazole/trimethoprim.
plasmid was originally identified in UPEC strains, where it was found to encode the colicins B and M. pAPEC-O1ColBM was found to contain several genes that have previously been associated with APEC virulence—etsABC, sitABCD, iucABCD, iutA, iss, iroBCDEN, cvaA, cvaB, eitABCD and tsh.[12] Four strains (120, 121, 273 and 285) were proved to encode genes for colicin B and M, and concurrently two of three virulence factors (iucD, iss and tsh), which are closely related with this plasmid. The distribution of phylogenetic groups in this E. coli collection can serve as bacterial source tracking tool. For example, the strains 273 and 285 (both B23) from this study can be considered as human feces contamination in chicken retail meat, because all strains from B23 were previously observed as of human origin.[17] As this strains were isolated from retail chickens, it is more likely that they are APEC. However, human ExPEC strains appeared to be closely related to APEC strains.[25] Surprisingly, one strain was positive for neuC gene (A0), which is highly prevalent among NMEC (92%), less among APEC (30%) strains, which are assigned especially in B2 and D phylogroup.[11] Antimicrobial susceptibility of 75 E. coli strains isolated from chicken meat was determined against 8 antibiotics (Table 3). From all tested strains almost 49% of all E. coli strains were resistant to at least one of eight tested antibiotics and 13% of isolates had multidrug resistance. The highest resistance was observed to aminopenicillins: ampicillin (38%) and amoxicillin/clavulanic acid (33%). The most frequently used groups of antibiotics in poultry are penicillins, tetracyclines, sulfonamides, potentiated sulfonamides and quinolones. Aminopenicillins (AMC and AMP) are also considered as drugs of first choice in the treatment of poultry.[26] In comparison with other European countries, E. coli strains isolated from food animals has shown a higher prevalence of resistance to AMP (35– 75%),[2,7] and AMC (23–45%).[2,27] It might be due to the fact, that these antibiotics have been commonly used for prevention or treatment of diseases in poultry production. Moreover, E. coli strains from retail meat were also found resistant to antibiotics such as trimethoprim/sulphamethoxazole, third-generation cephalosporins and fluoroquinolones.[28] In our study 16% of strains were resistant to CIP and 10% strains showed resistance to SXT. Other studies have shown a higher resistance to CIP, especially in chickens.[29] This might reflect a higher use of quinolones in chickens compared with pigs or other animals.[7] In all tested strains were observed 11% resistance to chloramphenicol. Similarly, the higher resistance to chloramphenicol was reported in chicken (21%)[29] and other animals (16%).[27] None of all strains were resistant to cefotaxime and cefuroxime. The low incidence of antibiotic resistance of these antibiotics (0–3.8%) were confirmed by other studies.[2,7] Until recently, carbapenems were the choice for the therapy of multidrug resistant gram-negative bacterial infections. Currently, carbapenemases producing strains from
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420 Enterobacteriaceae represent a serious threat to human health.[30] Although Antimicrobial resistance interactive database of European Centre for Disease Prevention and Control (ECDC) has reported only 0.0–0.2% of resistant E. coli strains, the amount of intermediate strains has increased to 1% in 2011 in the Czech Republic.[31] In this study, was found 4% of intermediate strains to imipenem. Therefore, should be increased attention in usage of these antibiotics, and thus prevent the emergence of resistance. Resistance to three or more antibiotics is taken as multidrug resistance.[32] From all tested strains 13% of isolates had multidrug resistance. Recently, it was reported that multidrug resistance is widespread in E. coli of poultry origin and is associated with conjugative plasmid.[33] The multi-resistant strains belonged to group A and B1. This is in accordance with study of Johnson et al.,[33] where isolates from phylogroup A and B1 tended to have a higher prevalence of resistance. Nevertheless, these groups were proved as groups with lower prevalence of virulence factors. Association between antibiotic resistance (AMP) and presence of virulence factor (gene iss) was also demonstrated in another study.[33] In this work, statistically significant correlation (P < 0.05) was proved between the presence of antibiotic resistance and virulence factors.
Conclusion Escherichia coli from chicken meat in the Czech Republic were found to be frequently resistant to five antibiotics, and 13% of strains were found to be multiresistant. More than half of tested strains belonged to A0 and A1phylogroup with lower incidence of virulence factors, what can determine commensal strains. The other strains were classified into B2 and D phylogroups, which contains more virulence factors. Most of E. coli strains isolated from chicken meat can be considered as nonpathogenic on the basis of phylogenetic analysis, presence of virulence factors and antibiotic resistance. The analysis of these factors together can provide a useful tool for prediction of a potential risk associated with E. coli from food.
Pavlickova et al.
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Funding This work was supported by Internal Grant of Tomas Bata University in Zlin (No. IGA/FT/2014/005).
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