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Antimicrobial resistance and class 1 and 2 integrons in Escherichia coli from meat turkeys in Northern Italy a
b
a
c
a
d
d
A. Piccirillo , D. Giovanardi , G. Dotto , G. Grilli , C. Montesissa , C. Boldrin , C. Salata a
& M. Giacomelli a
Department of Comparative Biomedicine and Food Science, University of Padua, Padua, Italy b
Laboratorio Tre Valli, Verona, Italy
c
Department of Veterinary Science and Public Health, University of Milan, Milano, Italy
d
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Microbiology and Virology Unit, Padua University Hospital, Padua, Italy Accepted author version posted online: 11 Jul 2014.Published online: 14 Aug 2014.
To cite this article: A. Piccirillo, D. Giovanardi, G. Dotto, G. Grilli, C. Montesissa, C. Boldrin, C. Salata & M. Giacomelli (2014) Antimicrobial resistance and class 1 and 2 integrons in Escherichia coli from meat turkeys in Northern Italy, Avian Pathology, 43:5, 396-405, DOI: 10.1080/03079457.2014.943690 To link to this article: http://dx.doi.org/10.1080/03079457.2014.943690
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Avian Pathology, 2014 Vol. 43, No. 5, 396–405, http://dx.doi.org/10.1080/03079457.2014.943690
ORIGINAL ARTICLE
Antimicrobial resistance and class 1 and 2 integrons in Escherichia coli from meat turkeys in Northern Italy A. Piccirillo1*, D. Giovanardi2, G. Dotto1, G. Grilli3, C. Montesissa1, C. Boldrin4, C. Salata4, and M. Giacomelli1 Department of Comparative Biomedicine and Food Science, University of Padua, Padua, Italy, 2Laboratorio Tre Valli, Verona, Italy, 3Department of Veterinary Science and Public Health, University of Milan, Milano, Italy, and 4Microbiology and Virology Unit, Padua University Hospital, Padua, Italy
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1
This study is aimed at determining the antimicrobial resistance (AMR) and the presence of class 1 and 2 integrons in 48 avian pathogenic Escherichia coli (APEC) strains isolated from meat turkeys during three sequential production cycles. Thirty avian faecal E. coli (AFEC) strains from the first cycle were also analysed. Strains were tested for AMR against 25 antimicrobials by disk diffusion test and were screened for the presence of integrons and associated gene cassettes by polymerase chain reaction followed by sequencing. Genetic relatedness of isolates was established by pulsed-field gel electrophoresis. High levels of resistance were detected to tetracyclines, penicillins and sulphonamides in APEC and AFEC. Resistance to aminoglycosides, fluoroquinolones, cephalosporins and phenicols was variable, based on the antimicrobial drug and the isolate (APEC vs. AFEC). Full susceptibility to colistin was detected. Multidrug resistance of up to seven antimicrobial classes was exhibited by APEC (93.8%) and AFEC (100%). Nearly 44% of strains tested positive for class 1 and/or class 2 integrons containing the dfrA, aadA and sat2 genes, alone or in combination, coding for streptomycin/spectinomycin, trimethoprim and streptothricin resistance, respectively. The estX and orfF genes of unknown function were also detected. A significant association was found between the presence of integrons and the resistance to aminoglycosides and potentiated sulphonamides. The results of this study showed that AMR, multidrug resistance and class 1 and 2 integrons are widespread among pathogenic and commensal E. coli from Italian turkeys. More attention should be addressed to limit the use of antimicrobials in turkeys and the AMR of turkey E. coli.
Introduction Avian colibacillosis is a localized or systemic disease of poultry caused by avian pathogenic Escherichia coli (APEC) and one of the major causes of economic losses in the poultry industry worldwide (Barnes et al., 2008). E. coli is a common inhabitant of the intestinal microflora of poultry at abundances of up to 106 colony-forming units per gram of faeces. In healthy chickens, 10 to 15% of faecal coliforms may belong to potentially pathogenic serogroups, carrying virulence and antimicrobial resistance genes (Barnes et al., 2008). Serogroups most frequently associated with disease in poultry are O1, O2, O35, O36 and O78 (Dho-Moulin & Fairbrother, 1999). Prevention and control of colibacillosis in poultry are addressed at reducing or eliminating the introduction of APEC into the poultry house, protecting the birds from predisposing factors associated with infection, immunizing the birds by use of inactivated and live vaccines and treating birds with antimicrobial agents (Barnes et al., 2008; Gyles, 2008). Since management and vaccination procedures are not always effective, antimicrobial therapy represents the most common intervention used to reduce losses from avian colibacillosis. However, APEC strains are increasingly
becoming resistant to a wide range of antimicrobial drugs (Singer & Hofacre, 2006; Gyles, 2008), indicating that the protection against avian colibacillosis in poultry could be impaired in the near future. The spread of antimicrobial resistance among bacterial populations via horizontal gene transfer represents a serious threat both in veterinary and human medicine (FAO/WHO/ OIE, 2008). Recently, integrons have been shown to play a significant role in the acquisition and the dissemination of antimicrobial resistance among bacteria, especially when associated with mobile DNA elements, such as transposons and plasmids (Mazel, 2006). Integrons constitute a sitespecific recombination system capable of capturing and expressing exogenous open reading frames in the form of gene cassettes, encoding for resistance against antimicrobials and disinfectants (Hall, 2012). According to the sequence homology of the integrase genes, encoding the enzymes mediating the integration and the excision of gene cassettes, several distinct classes of integrons have been described to date (Partridge et al., 2009; Hall, 2012). Class 1 integrons are the most frequently detected and the best characterized, mainly among the members of the family of Enterobacteriaceae; class 2 integrons have been observed
*To whom correspondence should be addressed: Tel: +39 049 8272968. Fax: +39 049 8272973. E-mail: [email protected] (Received 29 November 2013; accepted 16 June 2014) © 2014 Houghton Trust Ltd
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Antimicrobial resistance and integrons in turkey E. coli
only in a few Gram-negative microorganisms (Partridge et al., 2009). Related to the important role played by integrons in the horizontal transfer of antimicrobial resistance genes, over the past years many studies have been conducted to detect their presence in commensal and pathogenic E. coli from humans and animals (Partridge et al., 2009; Hall, 2012). Nevertheless, little is known about the distribution of integrons in E. coli isolated from commercial meat turkeys. Only a few studies have been carried throughout the world and most of them are focused on E. coli isolated from turkey meat (Johnson et al., 2005; Khaitsa et al., 2008; Cook et al., 2009; Soufi et al., 2009, 2011). To our knowledge, studies targeted at revealing the distribution and the gene cassette content of class 1 and 2 integrons in E. coli from intensive turkey flocks are not available in the literature. Therefore, the aims of this study were to determine the antimicrobial resistance profiles, the distribution of class 1 and class 2 integrons and the integronassociated gene cassettes in APEC strains isolated from diseased turkeys in Italy. Analyses were also performed in a number of avian faecal E. coli (AFEC) strains isolated from healthy birds. All strains were characterized by pulsed-field gel electrophoresis (PFGE) in order to determine the genetic relationship among them.
Materials and Methods Sample collection. Between June 2009 and July 2010, dead male turkeys (≤14 weeks old) from three sequential production cycles in a single commercial farm were submitted to post-mortem examination. Total mortality of flocks ranged from 6.5% in the first production cycle to 8.0% in the second cycle and 7.0% in the third cycle. A downtime of 21 days among the different production cycles was implemented in the farm, during which the turkey house was cleaned and disinfected, and litter was removed. Pathological lesions were clearly indicative of colisepticaemia with pericarditis, pneumonia and airsacculitis, or arthritis. Samples from all carcasses, viscera (i.e. brain, pericardial sac, liver, lungs) and joints were collected for microbiological examination. Further, cloacal swabs from healthy turkeys of the first production cycle were collected in order to isolate faecal E. coli. No information on therapeutic antimicrobial usage in the flocks was recorded. E. coli isolation, identification and serogrouping. Swabs from viscera, joints and cloaca were individually cultured onto 5% sheep blood agar (OXOID, Basingstoke, UK) and Eosin-Methylene Blue agar (OXOID), and were incubated aerobically at 37°C for 18 to 24 h. One presumptive E. coli colony from each sample was subcultured on tryptic soy agar (OXOID) and then biochemically identified using the commercial kit RapID E20 (bioMérieux Vitek, Hazelwood, MO, USA). Serogroups were determined according to the technique proposed by Blanco et al. (1992), identifying 37 different somatic antigens. Pulsed-field gel electrophoresis. All E. coli strains were subjected to PFGE typing according to the protocol of Ribot et al. (2006), modified slightly. Electrophoresis separation of DNAs was performed in the CHEF-DRIII PFGE System (Biorad, Hercules, California, USA) and images captured using Gel Doc EZ Imager (Biorad). E. coli ATCC 25922 was used as reference strain. Interpretation of PFGE patterns was performed with GelCompar II version 6.6.11 (Applied Maths, St-Martens-Latem, Belgium) and similarity was determined using the Dice similarity coefficient. Isolates were considered to be related if their Dice similarity index was ≥80% and according to the criteria defined by Tenover (≤6 bands of difference) (Tenover et al., 1995). Antimicrobial susceptibility testing. All E. coli strains were tested for antimicrobial susceptibility by the disk diffusion method, according to CLSI (2008) standards, against 25 antimicrobial drugs belonging to eight classes: aminoglycosides (aminosidine, 60 µg; apramycin, 15 µg;
397
gentamicin, 10 µg; spectinomycin, 10 µg; streptomycin, 10 µg; kanamycin, 30 µg), cephalosporins (cephalotin, 30 µg; ceftiofur, 30 µg; cefotaxime, 30 µg; ceftazidime, 30 µg; cefepime, 30 µg), fluoroquinolones (nalidixic acid, 30 µg; enrofloxacin, 5 µg; ciprofloxacin, 5 µg; norfloxacin, 10 µg), penicillins (ampicillin, 10 µg; amoxicillin + clavulanic acid, 30 µg), phenicols (chloramphenicol 30 µg; florfenicol, 30 µg), potentiated sulphonamides (triple sulphonamides, 300 µg; trimethoprim + sulfamethoxazole, 25 µg), tetracyclines (tetracycline, 30 µg; oxytetracycline, 30 µg; doxycycline, 30 µg) and polymyxins (colistin, 10 µg). All antimicrobial-containing disks were obtained from OXOID. E. coli ATCC 25922 was used as quality control strain. The level of antimicrobial resistance was defined as the percentage of resistant isolates as a proportion of the isolates tested; multidrug resistance (MDR) was defined as resistance to ≥3 different antimicrobial classes where resistance was detected against at least one antimicrobial agent. Penicillins and cephalosporins were considered separate classes. Detection of class 1 and class 2 integrons and characterization of gene cassettes. Total DNA was extracted by boiling for 20 min of a single colony emulsified in 100 µl sterile RNase/DNase free water (Sigma-Aldrich, Milan, Italy). All E. coli isolates were screened for the presence of class 1 and class 2 integrons by real-time polymerase chain reaction (PCR) assay using previously described primer sets (Ekkapobyotin et al., 2008) and the protocol designed by Piccirillo et al. (2013). Real-time PCR amplifications were performed in a LightCycler®480 Real-Time PCR System (Roche Diagnostics, Basel, Switzerland). All class 1 and class 2 integron-positive strains were examined by sequencing to determine the gene cassette content using the primers and the protocols suggested by Lévesque et al. (1995) and White et al. (2001). Both strands were sequenced using the BigDye Terminator Cycle Sequencing Kit v3.1 in the ABI PRISM 3100 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA), according to the manufacturer’s instructions. Editing of chromatograms and assembly of nucleotide sequences were performed by the software ChromasPro v. 1.42 (Technelysium Pty Ltd, Tewantin, Australia). Gene cassettes were identified by the BLASTN software available online (http://blast.ncbi.nlm.nih.gov/). Statistical analysis. To test for significance in the difference of resistance between APEC and AFEC, among the three production cycles and among the serogroups, as well as in the association between resistance and presence of integrons, the intermediate isolates were grouped together with the susceptible isolates and the Fisher’s exact test was performed using the General Linear Model (GLM) procedure (SAS Institute Inc., Cary, NC, USA). P ≤ 0.05 was considered statistically significant.
Results E. coli isolation, identification and serogrouping. From 37 carcasses, 48 APEC strains were isolated: 10 from the first production cycle, 28 from the second cycle and 10 from the third cycle (Table 1). Most strains were isolated from the brain (22/48 isolates), followed by the pericardium (7/48 isolates), lungs (7/48 isolates), tibiotarsal (6/48 isolates) and coxofemoral (4/48 isolates) joints, liver (1/48 isolate) and spleen (1/48 isolate). Twenty-eight isolates belonged to the O78 serogroup, 14 isolates to the O2 serogroup, one isolate to the O111 serogroup, and five isolates were untypeable with standard O antisera. Thirty AFEC isolates from faecal samples from healthy birds of the first production cycle were also obtained (Table 2). Pulsed-field gel electrophoresis analysis. All APEC and AFEC isolates except for one and three, respectively, were successfully analysed by PFGE. Twenty-eight different PFGE types were identified: 11 for APEC strains and 17 for AFEC strains (Figure 1). Among APEC isolates, three PFGE types were detected in the first production cycle, seven types in the second cycle and two types in the third cycle. One PFGE type (i.e. XVI) was shared among strains of the first and the second production cycles. Higher strain
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A. Piccirillo et al. Table 1.
Antimicrobial resistance, class 1 and 2 integrons and gene cassette content of APEC isolates.
Integrons Production cycle 1
Isolate identificationa 19038-1 17421-1 20317-1 14964-1 19700-1 13432-1 18041-1 18456-1 20318-1 14359-1
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2
27620-1 27028-1 3302-4 29610-1 1037-2 1038-2 1038-4 2216-1 3302-1 3302-4 29610-1 1038-3 29610-2 29611-1 410-1 1037-1 1038-1 2497-1 2759-1 3302-1 3302-4 28234-1 28234-1 1037-1 1037-1 1037-2 3302-1
3
27619-2 12685-1 12685-2
Origin Lung Brain Pericardial sac Pericardial sac Brain Brain Liver Brain Tibiotarsal joint Pericardial sac Brain Brain Coxofemoral joint Pericardial sac Tibiotarsal joint Brain Lung Lung Coxofemoral joint Tibiotarsal joint Brain Brain Brain Brain Brain Tibiotarsal joint Lung Lung Coxofemoral joint Tibiotarsal joint Tibiotarsal joint Pericardial sac Spleen Brain Pericardial sac Brain Coxofemoral joint Brain Pericardial sac Brain
PFGE type
Serogroup
MDR patternb
Class 1
Class 2
O111 O2 O78
NTc V XVI
ChTe ChSulTe ChPenTe
– – –
– – –
O78
XVI
CefChPenTe
–
–
O78 O78 NT O78 O78
XVI XVI IX XVI XVI
ChPenFenTe AmChPenFenTe CefChPenSulTe AmChPenSulTe AmChPenSulTe
– – – – –
– – – – –
O78
XVI
AmCefChPenFenTe
–
–
O2 O2 O78
XVIII XVIII XXVI
Pen CefPen ChPenTe
– – –
– – –
O78
XVI
AmChPenTe
–
–
O78
XVII
AmChPenTe
–
–
O78 O78 NT O78
XXVI XXVI XXIV XXVI
AmChPenTe AmChPenTe AmPenSulTe CefChPenTe
– – aadA1 –
O78
XXVI
CefChPenTe
–
O78 O78 O78 O78 O78 O78
XVI XXVI XVI XVI XVII XVII
AmCefChPenTe AmCefChPenTe AmChPenSulTe AmChPenSulTe AmChPenSulTe AmChPenSulTe
– – – – estX, aadA1 –
– – – – – –
NT O78 O78
XXVIII XXVI XXVI
AmChPenSulTe AmChPenSulTe AmChPenSulTe
dfrA1, aadA1 – –
– – –
O78
XXVI
AmChPenSulTe
–
–
O78
XXVI
AmChPenSulTe
–
–
O2
XIX
AmChPenFenSulTe
dfrA1, aadA1
–
O2 O78 O78
XIX XVII XVII
AmChPenFenSulTe AmCefChPenSulTe AmCefChPenSulTe
dfrA1, aadA1 – –
– – –
O78 O78
XVII XXVI
AmCefChPenSulTe AmCefChPenSulTe
– –
– –
O2 O2
XVIII VI
AmCefChPenFenSulTe dfrA1, aadA1 AmChPenSulTe –
O2
VI
AmChPenSulTe
–
– – sat2, aadA1 – –
– dfrA1, sat2, aadA1 dfrA1, sat2, aadA1
Antimicrobial resistance and integrons in turkey E. coli
399
Table 1 (Continued)
Integrons
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Production cycle
Isolate identificationa
Origin
PFGE type
Serogroup
MDR patternb
Class 1
Class 2 dfrA1, sat2, aadA1 dfrA1, sat2, aadA1 dfrA1, sat2, aadA1 dfrA1, sat2, aadA1 dfrA1, sat2, aadA1 dfrA1, sat2, aadA1
13282-1
Brain
O2
VI
AmChPenSulTe
–
14486-1
Brain
O2
VI
AmChPenSulTe
–
15189-1
Brain
O2
VI
AmChPenSulTe
–
15189-2
Brain
O2
VI
AmChPenSulTe
–
15189-3
Brain
O2
VI
AmChPenSulTe
–
14486-2
Brain
O2
III
AmCefChPenSulTe
–
14486-1 14486-2
Lung Lung
NT NT
III III
AmCefChPenSulTe AmCefChPenSulTe
– –
a
Strains with the same identification number were isolated from the same carcass (1037-1, 1037-2, 3302-1, 3302-4, 14486-1, 14486-2, 28234-1, 29610-1). bMDR = multidrug resistance; Am = aminoglycosides; Cef = cephalosporins; Ch = quinolones; Pen = penicillins; Fen = phenicols; Sul = potentiated sulphonamides; Te = tetracyclines. cNT, not typeable.
diversity was detected in AFEC compared with APEC (Simpson’s index of diversity = 0.94 and 0.86, respectively), with no overlapping among their PFGE types. Among APEC and AFEC isolates, four and 13 PFGE types were represented by single strains, respectively. Antimicrobial resistance of APEC and AFEC isolates. Overall, except for a few drugs (i.e. nalidixic acid, ciprofloxacin, and florfenicol), AFEC showed higher percentages of resistance than APEC (Figure 2). Most isolates, both APEC and AFEC, were resistant to tetracyclines (95.8% for all the drugs in APEC; 96.7% for doxycycline to 100% for tetracycline and oxytetracycline in AFEC), as well as to penicillins (95.8% for all the drugs in APEC; from 90% for amoxicillin + clavulanic acid to 100% for ampicillin in AFEC). A high percentage of AFEC was also resistant to potentiated sulphonamides (80% for trimethoprim + sulfamethoxazole and 90% for triple sulphonamides), whereas APEC were less resistant to both antimicrobials (47.9% and 58.3%, respectively). Conversely, APEC exhibited higher frequencies of resistance to fluoroquinolones than AFEC (from 6.2% for enrofloxacin and ciprofloxacin to 93.7% for nalidixic acid and from 3.3% ciprofloxacin to 46.7% for nalidixic acid, respectively). The percentage of resistance to aminoglycosides ranged from 0% for apramycin to 75% for streptomycin among APEC and from 6.7% for apramycin to 86.7% for streptomycin among AFEC. Resistance to cephalosporins was higher in AFEC (from 3.3% for ceftazidime to 43.3% for cephalotin) than APEC (from 0% for 3 cephalosporins to 31.2% for cephalotin). Low levels of resistance were detected to phenicols, both in APEC (8.3% for chloramphenicol and 4.2% for florfenicol) and in AFEC (36.7% for chloramphenicol and 0% for florfenicol). All isolates were fully susceptible to colistin. The resistance rates to triple sulphonamides, trimethoprim combined with sulfamethoxazole, gentamicin, kanamycin, aminosidine, ceftiofur, cefotaxime, and chloramphenicol of AFEC were significantly (P ≤ 0.05) higher compared with APEC; resistance to nalidixic acid was significantly (P ≤ 0.05) higher in APEC
than in AFEC. The prevalence of resistance of APEC to triple sulphonamides, trimethoprim combined with sulfamethoxazole, ampicillin, amoxicillin combined with clavulanic acid, streptomycin, and spectinomycin increased significantly (P ≤ 0.05) over the three production cycles; the prevalence of resistance to florfenicol decreased significantly (P ≤ 0.05) over the same interval. No significant differences (P > 0.05) in resistance rates were observed among serogroups of APEC. The majority (96.1%) of E. coli isolated from diseased and healthy birds exhibited resistance to three or more antimicrobial classes (Tables 1 and 2). Only three APEC isolates showed resistance to one and two classes of antimicrobials, and none was fully susceptible. In particular, among the 48 APEC isolates, three (6.2%), nine (18.7%), 22 (45.8%), 10 (20.8%) and one (2.1%) isolates showed MDR to three, four, five, six and seven antimicrobial classes, respectively. Out of the 30 AFEC, three (10%), eight (26.7%), six (20%), eight (26.7%) and five (16.7%) isolates were resistant to three, four, five, six and seven antimicrobial classes, respectively. MDR patterns comprised 19 different combinations, with the most common including five classes (i.e. aminoglycosides, fluoroquinolones, penicillins, sulphonamides and tetracyclines) among APEC (18/ 48 isolates) and four classes (i.e. aminoglycosides, penicillins, sulphonamides and tetracyclines) among AFEC (7/30 isolates). Presence of class 1 and 2 integrons and gene cassettes content in APEC and AFEC isolates. Real-time PCR screening for intI1 and intI2 genes identified 34 (43.6%) strains carrying class 1 and/or class 2 integrons (Tables 1 and 2). Out of the 48 APEC isolates, the intI1 gene was detected in six (12.5%) isolates and the intI2 gene in nine (18.7%) isolates. Among the 30 AFEC isolates, 19/30 (63.3%) strains were positive for class 1 integrons and only two (6.7%) for class 2 integrons. Two strains (one APEC and one AFEC) carried both classes of integrons. The prevalence of class 1 integrons was significantly higher (P ≤ 0.001) in AFEC as compared with APEC, while that
400
A. Piccirillo et al. Table 2.
Antimicrobial resistance, class 1 and 2 integrons and gene cassette content of AFEC isolates.
Integrons Production cycle
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1
Isolate identificationa 18042-5 17401-2 16756-3 17401-1 17401-4 16756-4 15446-3 16185-1 16185-2 16185-3 14966-3 17401-3 17401-5 16756-1 16756-2 16185-4 14966-5 18042-1 18042-2 18042-3 16756-5 15446-1 15446-2 14966-1 14966-4 18042-4 15446-4 15446-5 16185-5 14966-2
PFGE type IV XXIII IV XXIc NT NT XXVII II XII II XI XXII XXV X IV VIII XIII VII X X II XIV II I NT X XV I II XX
MDR patternb
Class 1
Class 2
AmPenTe PenSulTe AmPenTe AmPenSulTe AmPenSulTe AmPenSulTe AmPenSulTe AmPenSulTe AmPenSulTe AmPenSulTe AmChPenTe AmCefPenSulTe AmChPenSulTe AmPenFenSulTe AmPenFenSulTe AmCefPenSulTe CefChPenSulTe AmChPenFenSulTe AmCefPenFenSulTe AmCefPenFenSulTe AmCefChPenSulTe AmCefChPenSulTe AmCefChPenSulTe AmChPenFenSulTe AmCefChPenSulTe AmCefChPenFenSulTe AmCefChPenFenSulTe AmCefChPenFenSulTe AmCefChPenFenSulTe AmCefChPenFenSulTe
– – – – dfrA1, aadA1 – – dfrA12, orfF, aadA2 dfrA1, aadA1 dfrA12, orfF, aadA2 – dfrA1, aadA1 dfrA1, aadA1 dfrA1, aadA1 dfrA1, aadA1 dfrA1, aadA1 – dfrA1, aadA1 dfrA1, aadA1 dfrA1, aadA1 dfrA12, orfF – dfrA12, aadA2 – – dfrA1, aadA1 dfrA12, orfF, aadA2 sat2 dfrA12, orfF, aadA2 dfrA7
– – – – – – – – – – – – – – – – – – – – – – – sat2, aadA1 – – – sat2, aadA1 – –
a
Strains with the same identification number were isolated from the same carcass (1037-1, 1037-2, 3302-1, 3302-4, 14486-1, 14486-2, 28234-1, 29610-1). Faecal samples were obtained from individual birds. bMDR = multidrug resistance; Am = aminoglycosides; Cef = cephalosporins; Ch = quinolones; Pen = penicillins; Fen = phenicols; Sul = potentiated sulphonamides; Te = tetracyclines. cNT, not typeable.
of class 2 integrons was not significantly (P > 0.05) different between APEC and AFEC. Prevalence of integrons was quite different among APEC of the various production cycles and serogroups. Indeed, none of the isolates of the first production cycle carried integrons, whereas isolates from the second and third production cycles were positive (6/28 and 8/10 isolates, respectively). Only class 1 integrons were identified in isolates from the second cycle, except for one strain carrying both classes of integrons; isolates from the third cycle carried only class 2 integrons. Furthermore, most of the intI1-positive and intI2positive strains belonged to the O2 serogroup (11/14), 2/14 strains were untypeable and only one strain belonged to the O78 serogroup. As shown in Tables 1 and 2, 10 different gene cassettes including aadA (variants aadA1 and aadA2), dfrA (variants dfrA1, dfrA7, dfrA12), sat2, orfF and estX genes, alone or in combination, were identified among APEC and AFEC. The aadA genes encode for the aminoglycoside adenylyltransferases (streptomycin/spectinomycin resistance), the dfrA genes for the dihydrofolate reductases (trimethoprim resistance), the sat2 gene for the streptothricin acetyltransferases, the estX gene for a putative esterase, and the orfF gene for a protein of unknown function. The most common cassette array was dfrA1-aadA1 carried by 11 intI1-positive AFEC and four APEC strains, followed by the dfrA1-sat2-aadA1
array carried by eight intI2-positive APEC strains. Isolates carrying both classes of integrons harboured the aadA1 and sat2-aadA1 genes (APEC) and the sat2 and sat2-aadA1 genes (AFEC) in class 1 and 2 integrons, respectively. Diversity of gene cassette arrays was higher in AFEC than APEC (seven vs. five different cassette arrays, respectively). In APEC isolates from the second production cycle, the most prevalent array was dfrA1-aadA1, whereas in intI2positive APEC isolates from the third production cycle only one array (dfrA1-sat2-aadA1) was identified. The only intI1-positive O78 serogroup carried an unusual cassette array, the estX-aadA1. Association between presence of integrons and antimicrobial resistance in E. coli isolates. For a clear assessment of the relation between antimicrobial resistance and integrons, resistance determinants not associated with integrons were excluded from the analysis. The differences between intI1-positive and intI1-negative isolates in resistance to gentamycin, streptomycin, aminosidine, triple sulphonamides and trimethoprim combined with sulfamethoxazole were significantly (P ≤ 0.05) higher in intI1-positive compared with intI1-negative isolates. The differences in intI2-positive and intI2-negative isolates in resistance to spectinomycin, triple sulphonamides and trimethoprim combined with sulfamethoxazole were
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Antimicrobial resistance and integrons in turkey E. coli
401
Figure 1. Dendrogram of the 28 different PFGE types identified among APEC and AFEC isolates. The scale bar on the top indicates similarity based on the Dice coefficient and PFGE types refer to patterns greater than 80% similarity.
significantly (P ≤ 0.05) higher in intI2-positive compared with intI2-negative isolates. All intI1-positive and intI2positive E. coli isolates were MDR, while intI1-negative and intI2-negative isolates were 93.2%.
Discussion Currently available studies showed that high resistance and multidrug resistance are common among avian pathogenic E. coli from turkeys (Salmon & Watts, 2000; Cormican et al., 2001; van den Bogaard et al., 2001; Altekruse et al., 2002; EFSA, 2010; Randall et al., 2011, 2013; Gosling et al., 2012). In the current study, a very high level of resistance to several antimicrobial drugs was detected and multidrug resistance to three or more antimicrobial classes was widespread both among APEC and AFEC. Moreover, a high percentage of isolates was found positive for class 1 and 2 integrons. Although some data on the presence and characterization of integrons in turkey E. coli are available in the literature (Bass et al., 1999; Singh et al., 2005; Nógrády et al., 2006), none of these studies was targeted to ascertain the distribution of these genetic elements in turkey flocks. Therefore, with this study we provide the first data on the presence and the gene cassette content of class 1 and 2 integrons in E. coli isolated from commercial meat turkey flocks. The highest levels of resistance were detected against tetracyclines, penicillins and sulphonamides, as widely documented among pathogenic and commensal E. coli of turkey origin (Salmon & Watts, 2000; Cormican et al., 2001; van den Bogaard et al., 2001; Altekruse et al., 2002;
Lister, 2005; EFSA, 2010; Randall et al., 2011, 2013; Gosling et al., 2012). The high levels of resistance to these classes may be attributed to a long-term selection pressure, since these antimicrobials are among the oldest and the most extensively used in the poultry industry all over the world (Singer & Hofacre, 2006), including Italy. In contrast, the very high resistance rates to streptomycin and nalidixic acid were unexpected since these drugs have not been used for a long time in our country. Similarly to our findings, a widespread resistance to these antimicrobials has been documented in turkey APEC and AFEC (Cormican et al., 2001; Giraud et al., 2001; Altekruse et al., 2002; Bryan et al., 2004; EFSA, 2010; Randall et al., 2011, 2013; Gosling et al., 2012). However, it has been reported that the resistance to specific antimicrobials, such as streptomycin, may be prevalent despite its discontinuance as a therapeutic agent (Bass et al., 1999) and the development of resistance to nalidixic acid may not be necessarily linked with treatment (Hofacre et al., 2000). In accordance with previous data (Salmon & Watts, 2000; Cormican et al., 2001; Lister, 2005; EFSA, 2010; Randall et al., 2011, 2013; Gosling et al., 2012; Kempf et al., 2013a), a prevailing susceptibility against several members of the aminoglycoside family (streptomycin excepted) was found in E. coli from turkeys, mainly among APEC. This finding was expected since these antimicrobials are scarcely employed in the turkey industry in Italy. Similarly, low levels of resistance to fluoroquinolones (except for nalidixic acid) were found. Partially contrasting results regarding fluoroquinolone resistance can be found in the literature (Salmon & Watts, 2000; Cormican et al., 2001; Giraud
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A. Piccirillo et al.
Figure 2. Percentage of APEC (2a) and AFEC (2b) isolates susceptible (S), intermediate (I) and resistant (R) to antimicrobials. APR = apramycin; GM = gentamicin; STR = streptomycin; K = kanamycin; AM = aminosidine; SPT = spectinomycin; CF = cephalotin; CFT = ceftiofur; CTX = cefotaxime; CAZ = ceftazidime; FEP = cefepime; NA = nalidixic acid; ENR = enrofloxacin; CIP = ciprofloxacin; NOR = norfloxacin; AMP = ampicillin; AMC = amoxicillin+clavulanic acid; CAF = chloramphenicol; FFC = florfenicol; S3 = triple sulphonamides; SXT = trimethoprim+sulfamethoxazole; TE = tetracycline; DO = doxycycline; OT = oxytetracycline; CT = colistin.
et al., 2001; van den Bogaard et al., 2001; Altekruse et al., 2002; Lister, 2005; Taylor et al., 2008; EFSA, 2010; Randall et al., 2011, 2013; Gosling et al., 2012). It has been recognized that fluoroquinolone resistance can be related to the usage pattern of these drugs in a given country, which is in turn influenced by the market dynamics, mainly the cost of commercial products (Bywater, 2005). In Italy, the parsimonious use of costly products, together with the policy of the integrated company, may have limited the development of fluoroquinolone resistance in E. coli examined in the present study. A low susceptibility to cephalotin and ceftiofur was detected in both APEC and AFEC. Resistance to thirdgeneration and fourth-generation cephalosporins of clinical and faecal turkey E. coli has been previously reported (Salmon & Watts, 2000; Altekruse et al., 2002; Randall et al., 2011, 2013; Gosling et al., 2012). Dutil et al. (2010) demonstrated that ceftiofur resistance was related to the use of this drug in poultry hatcheries. Although cephalosporins are not approved for use in poultry, the administration of this drug in 1-day-old chicks at the hatchery cannot be excluded. One should emphasize that the predominant
susceptibility to third-generation and fourth-generation cephalosporins, as well as to fluoroquinolones, is of great value from a public health point of view given the importance of these antimicrobials as critically important antibiotics for human medicine (WHO, 2012). The moderate levels of resistance to chloramphenicol and florfenicol detected in our study are unclear, since the first drug is not approved for use in food-producing animals in Europe (Commission Regulation No. 37/2010 of 22 December 2009) and the second is not authorized for use in poultry in Italy. However, this finding has been previously documented in other studies (Salmon & Watts, 2000; Cormican et al., 2001; Altekruse et al., 2002; EFSA, 2010; Randall et al., 2011, 2013; Gosling et al., 2012). Finally, the full susceptibility to colistin observed in E. coli is valuable both for poultry and human concerns, since this agent is widely used in poultry production and represents a last-resort treatment in human medicine (Livermore, 2004). In accordance with our results, colistin resistance seems to be extremely rare (
Avian Pathology Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/cavp20
Antimicrobial resistance and class 1 and 2 integrons in Escherichia coli from meat turkeys in Northern Italy a
b
a
c
a
d
d
A. Piccirillo , D. Giovanardi , G. Dotto , G. Grilli , C. Montesissa , C. Boldrin , C. Salata a
& M. Giacomelli a
Department of Comparative Biomedicine and Food Science, University of Padua, Padua, Italy b
Laboratorio Tre Valli, Verona, Italy
c
Department of Veterinary Science and Public Health, University of Milan, Milano, Italy
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Microbiology and Virology Unit, Padua University Hospital, Padua, Italy Accepted author version posted online: 11 Jul 2014.Published online: 14 Aug 2014.
To cite this article: A. Piccirillo, D. Giovanardi, G. Dotto, G. Grilli, C. Montesissa, C. Boldrin, C. Salata & M. Giacomelli (2014) Antimicrobial resistance and class 1 and 2 integrons in Escherichia coli from meat turkeys in Northern Italy, Avian Pathology, 43:5, 396-405, DOI: 10.1080/03079457.2014.943690 To link to this article: http://dx.doi.org/10.1080/03079457.2014.943690
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Avian Pathology, 2014 Vol. 43, No. 5, 396–405, http://dx.doi.org/10.1080/03079457.2014.943690
ORIGINAL ARTICLE
Antimicrobial resistance and class 1 and 2 integrons in Escherichia coli from meat turkeys in Northern Italy A. Piccirillo1*, D. Giovanardi2, G. Dotto1, G. Grilli3, C. Montesissa1, C. Boldrin4, C. Salata4, and M. Giacomelli1 Department of Comparative Biomedicine and Food Science, University of Padua, Padua, Italy, 2Laboratorio Tre Valli, Verona, Italy, 3Department of Veterinary Science and Public Health, University of Milan, Milano, Italy, and 4Microbiology and Virology Unit, Padua University Hospital, Padua, Italy
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1
This study is aimed at determining the antimicrobial resistance (AMR) and the presence of class 1 and 2 integrons in 48 avian pathogenic Escherichia coli (APEC) strains isolated from meat turkeys during three sequential production cycles. Thirty avian faecal E. coli (AFEC) strains from the first cycle were also analysed. Strains were tested for AMR against 25 antimicrobials by disk diffusion test and were screened for the presence of integrons and associated gene cassettes by polymerase chain reaction followed by sequencing. Genetic relatedness of isolates was established by pulsed-field gel electrophoresis. High levels of resistance were detected to tetracyclines, penicillins and sulphonamides in APEC and AFEC. Resistance to aminoglycosides, fluoroquinolones, cephalosporins and phenicols was variable, based on the antimicrobial drug and the isolate (APEC vs. AFEC). Full susceptibility to colistin was detected. Multidrug resistance of up to seven antimicrobial classes was exhibited by APEC (93.8%) and AFEC (100%). Nearly 44% of strains tested positive for class 1 and/or class 2 integrons containing the dfrA, aadA and sat2 genes, alone or in combination, coding for streptomycin/spectinomycin, trimethoprim and streptothricin resistance, respectively. The estX and orfF genes of unknown function were also detected. A significant association was found between the presence of integrons and the resistance to aminoglycosides and potentiated sulphonamides. The results of this study showed that AMR, multidrug resistance and class 1 and 2 integrons are widespread among pathogenic and commensal E. coli from Italian turkeys. More attention should be addressed to limit the use of antimicrobials in turkeys and the AMR of turkey E. coli.
Introduction Avian colibacillosis is a localized or systemic disease of poultry caused by avian pathogenic Escherichia coli (APEC) and one of the major causes of economic losses in the poultry industry worldwide (Barnes et al., 2008). E. coli is a common inhabitant of the intestinal microflora of poultry at abundances of up to 106 colony-forming units per gram of faeces. In healthy chickens, 10 to 15% of faecal coliforms may belong to potentially pathogenic serogroups, carrying virulence and antimicrobial resistance genes (Barnes et al., 2008). Serogroups most frequently associated with disease in poultry are O1, O2, O35, O36 and O78 (Dho-Moulin & Fairbrother, 1999). Prevention and control of colibacillosis in poultry are addressed at reducing or eliminating the introduction of APEC into the poultry house, protecting the birds from predisposing factors associated with infection, immunizing the birds by use of inactivated and live vaccines and treating birds with antimicrobial agents (Barnes et al., 2008; Gyles, 2008). Since management and vaccination procedures are not always effective, antimicrobial therapy represents the most common intervention used to reduce losses from avian colibacillosis. However, APEC strains are increasingly
becoming resistant to a wide range of antimicrobial drugs (Singer & Hofacre, 2006; Gyles, 2008), indicating that the protection against avian colibacillosis in poultry could be impaired in the near future. The spread of antimicrobial resistance among bacterial populations via horizontal gene transfer represents a serious threat both in veterinary and human medicine (FAO/WHO/ OIE, 2008). Recently, integrons have been shown to play a significant role in the acquisition and the dissemination of antimicrobial resistance among bacteria, especially when associated with mobile DNA elements, such as transposons and plasmids (Mazel, 2006). Integrons constitute a sitespecific recombination system capable of capturing and expressing exogenous open reading frames in the form of gene cassettes, encoding for resistance against antimicrobials and disinfectants (Hall, 2012). According to the sequence homology of the integrase genes, encoding the enzymes mediating the integration and the excision of gene cassettes, several distinct classes of integrons have been described to date (Partridge et al., 2009; Hall, 2012). Class 1 integrons are the most frequently detected and the best characterized, mainly among the members of the family of Enterobacteriaceae; class 2 integrons have been observed
*To whom correspondence should be addressed: Tel: +39 049 8272968. Fax: +39 049 8272973. E-mail: [email protected] (Received 29 November 2013; accepted 16 June 2014) © 2014 Houghton Trust Ltd
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Antimicrobial resistance and integrons in turkey E. coli
only in a few Gram-negative microorganisms (Partridge et al., 2009). Related to the important role played by integrons in the horizontal transfer of antimicrobial resistance genes, over the past years many studies have been conducted to detect their presence in commensal and pathogenic E. coli from humans and animals (Partridge et al., 2009; Hall, 2012). Nevertheless, little is known about the distribution of integrons in E. coli isolated from commercial meat turkeys. Only a few studies have been carried throughout the world and most of them are focused on E. coli isolated from turkey meat (Johnson et al., 2005; Khaitsa et al., 2008; Cook et al., 2009; Soufi et al., 2009, 2011). To our knowledge, studies targeted at revealing the distribution and the gene cassette content of class 1 and 2 integrons in E. coli from intensive turkey flocks are not available in the literature. Therefore, the aims of this study were to determine the antimicrobial resistance profiles, the distribution of class 1 and class 2 integrons and the integronassociated gene cassettes in APEC strains isolated from diseased turkeys in Italy. Analyses were also performed in a number of avian faecal E. coli (AFEC) strains isolated from healthy birds. All strains were characterized by pulsed-field gel electrophoresis (PFGE) in order to determine the genetic relationship among them.
Materials and Methods Sample collection. Between June 2009 and July 2010, dead male turkeys (≤14 weeks old) from three sequential production cycles in a single commercial farm were submitted to post-mortem examination. Total mortality of flocks ranged from 6.5% in the first production cycle to 8.0% in the second cycle and 7.0% in the third cycle. A downtime of 21 days among the different production cycles was implemented in the farm, during which the turkey house was cleaned and disinfected, and litter was removed. Pathological lesions were clearly indicative of colisepticaemia with pericarditis, pneumonia and airsacculitis, or arthritis. Samples from all carcasses, viscera (i.e. brain, pericardial sac, liver, lungs) and joints were collected for microbiological examination. Further, cloacal swabs from healthy turkeys of the first production cycle were collected in order to isolate faecal E. coli. No information on therapeutic antimicrobial usage in the flocks was recorded. E. coli isolation, identification and serogrouping. Swabs from viscera, joints and cloaca were individually cultured onto 5% sheep blood agar (OXOID, Basingstoke, UK) and Eosin-Methylene Blue agar (OXOID), and were incubated aerobically at 37°C for 18 to 24 h. One presumptive E. coli colony from each sample was subcultured on tryptic soy agar (OXOID) and then biochemically identified using the commercial kit RapID E20 (bioMérieux Vitek, Hazelwood, MO, USA). Serogroups were determined according to the technique proposed by Blanco et al. (1992), identifying 37 different somatic antigens. Pulsed-field gel electrophoresis. All E. coli strains were subjected to PFGE typing according to the protocol of Ribot et al. (2006), modified slightly. Electrophoresis separation of DNAs was performed in the CHEF-DRIII PFGE System (Biorad, Hercules, California, USA) and images captured using Gel Doc EZ Imager (Biorad). E. coli ATCC 25922 was used as reference strain. Interpretation of PFGE patterns was performed with GelCompar II version 6.6.11 (Applied Maths, St-Martens-Latem, Belgium) and similarity was determined using the Dice similarity coefficient. Isolates were considered to be related if their Dice similarity index was ≥80% and according to the criteria defined by Tenover (≤6 bands of difference) (Tenover et al., 1995). Antimicrobial susceptibility testing. All E. coli strains were tested for antimicrobial susceptibility by the disk diffusion method, according to CLSI (2008) standards, against 25 antimicrobial drugs belonging to eight classes: aminoglycosides (aminosidine, 60 µg; apramycin, 15 µg;
397
gentamicin, 10 µg; spectinomycin, 10 µg; streptomycin, 10 µg; kanamycin, 30 µg), cephalosporins (cephalotin, 30 µg; ceftiofur, 30 µg; cefotaxime, 30 µg; ceftazidime, 30 µg; cefepime, 30 µg), fluoroquinolones (nalidixic acid, 30 µg; enrofloxacin, 5 µg; ciprofloxacin, 5 µg; norfloxacin, 10 µg), penicillins (ampicillin, 10 µg; amoxicillin + clavulanic acid, 30 µg), phenicols (chloramphenicol 30 µg; florfenicol, 30 µg), potentiated sulphonamides (triple sulphonamides, 300 µg; trimethoprim + sulfamethoxazole, 25 µg), tetracyclines (tetracycline, 30 µg; oxytetracycline, 30 µg; doxycycline, 30 µg) and polymyxins (colistin, 10 µg). All antimicrobial-containing disks were obtained from OXOID. E. coli ATCC 25922 was used as quality control strain. The level of antimicrobial resistance was defined as the percentage of resistant isolates as a proportion of the isolates tested; multidrug resistance (MDR) was defined as resistance to ≥3 different antimicrobial classes where resistance was detected against at least one antimicrobial agent. Penicillins and cephalosporins were considered separate classes. Detection of class 1 and class 2 integrons and characterization of gene cassettes. Total DNA was extracted by boiling for 20 min of a single colony emulsified in 100 µl sterile RNase/DNase free water (Sigma-Aldrich, Milan, Italy). All E. coli isolates were screened for the presence of class 1 and class 2 integrons by real-time polymerase chain reaction (PCR) assay using previously described primer sets (Ekkapobyotin et al., 2008) and the protocol designed by Piccirillo et al. (2013). Real-time PCR amplifications were performed in a LightCycler®480 Real-Time PCR System (Roche Diagnostics, Basel, Switzerland). All class 1 and class 2 integron-positive strains were examined by sequencing to determine the gene cassette content using the primers and the protocols suggested by Lévesque et al. (1995) and White et al. (2001). Both strands were sequenced using the BigDye Terminator Cycle Sequencing Kit v3.1 in the ABI PRISM 3100 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA), according to the manufacturer’s instructions. Editing of chromatograms and assembly of nucleotide sequences were performed by the software ChromasPro v. 1.42 (Technelysium Pty Ltd, Tewantin, Australia). Gene cassettes were identified by the BLASTN software available online (http://blast.ncbi.nlm.nih.gov/). Statistical analysis. To test for significance in the difference of resistance between APEC and AFEC, among the three production cycles and among the serogroups, as well as in the association between resistance and presence of integrons, the intermediate isolates were grouped together with the susceptible isolates and the Fisher’s exact test was performed using the General Linear Model (GLM) procedure (SAS Institute Inc., Cary, NC, USA). P ≤ 0.05 was considered statistically significant.
Results E. coli isolation, identification and serogrouping. From 37 carcasses, 48 APEC strains were isolated: 10 from the first production cycle, 28 from the second cycle and 10 from the third cycle (Table 1). Most strains were isolated from the brain (22/48 isolates), followed by the pericardium (7/48 isolates), lungs (7/48 isolates), tibiotarsal (6/48 isolates) and coxofemoral (4/48 isolates) joints, liver (1/48 isolate) and spleen (1/48 isolate). Twenty-eight isolates belonged to the O78 serogroup, 14 isolates to the O2 serogroup, one isolate to the O111 serogroup, and five isolates were untypeable with standard O antisera. Thirty AFEC isolates from faecal samples from healthy birds of the first production cycle were also obtained (Table 2). Pulsed-field gel electrophoresis analysis. All APEC and AFEC isolates except for one and three, respectively, were successfully analysed by PFGE. Twenty-eight different PFGE types were identified: 11 for APEC strains and 17 for AFEC strains (Figure 1). Among APEC isolates, three PFGE types were detected in the first production cycle, seven types in the second cycle and two types in the third cycle. One PFGE type (i.e. XVI) was shared among strains of the first and the second production cycles. Higher strain
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A. Piccirillo et al. Table 1.
Antimicrobial resistance, class 1 and 2 integrons and gene cassette content of APEC isolates.
Integrons Production cycle 1
Isolate identificationa 19038-1 17421-1 20317-1 14964-1 19700-1 13432-1 18041-1 18456-1 20318-1 14359-1
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2
27620-1 27028-1 3302-4 29610-1 1037-2 1038-2 1038-4 2216-1 3302-1 3302-4 29610-1 1038-3 29610-2 29611-1 410-1 1037-1 1038-1 2497-1 2759-1 3302-1 3302-4 28234-1 28234-1 1037-1 1037-1 1037-2 3302-1
3
27619-2 12685-1 12685-2
Origin Lung Brain Pericardial sac Pericardial sac Brain Brain Liver Brain Tibiotarsal joint Pericardial sac Brain Brain Coxofemoral joint Pericardial sac Tibiotarsal joint Brain Lung Lung Coxofemoral joint Tibiotarsal joint Brain Brain Brain Brain Brain Tibiotarsal joint Lung Lung Coxofemoral joint Tibiotarsal joint Tibiotarsal joint Pericardial sac Spleen Brain Pericardial sac Brain Coxofemoral joint Brain Pericardial sac Brain
PFGE type
Serogroup
MDR patternb
Class 1
Class 2
O111 O2 O78
NTc V XVI
ChTe ChSulTe ChPenTe
– – –
– – –
O78
XVI
CefChPenTe
–
–
O78 O78 NT O78 O78
XVI XVI IX XVI XVI
ChPenFenTe AmChPenFenTe CefChPenSulTe AmChPenSulTe AmChPenSulTe
– – – – –
– – – – –
O78
XVI
AmCefChPenFenTe
–
–
O2 O2 O78
XVIII XVIII XXVI
Pen CefPen ChPenTe
– – –
– – –
O78
XVI
AmChPenTe
–
–
O78
XVII
AmChPenTe
–
–
O78 O78 NT O78
XXVI XXVI XXIV XXVI
AmChPenTe AmChPenTe AmPenSulTe CefChPenTe
– – aadA1 –
O78
XXVI
CefChPenTe
–
O78 O78 O78 O78 O78 O78
XVI XXVI XVI XVI XVII XVII
AmCefChPenTe AmCefChPenTe AmChPenSulTe AmChPenSulTe AmChPenSulTe AmChPenSulTe
– – – – estX, aadA1 –
– – – – – –
NT O78 O78
XXVIII XXVI XXVI
AmChPenSulTe AmChPenSulTe AmChPenSulTe
dfrA1, aadA1 – –
– – –
O78
XXVI
AmChPenSulTe
–
–
O78
XXVI
AmChPenSulTe
–
–
O2
XIX
AmChPenFenSulTe
dfrA1, aadA1
–
O2 O78 O78
XIX XVII XVII
AmChPenFenSulTe AmCefChPenSulTe AmCefChPenSulTe
dfrA1, aadA1 – –
– – –
O78 O78
XVII XXVI
AmCefChPenSulTe AmCefChPenSulTe
– –
– –
O2 O2
XVIII VI
AmCefChPenFenSulTe dfrA1, aadA1 AmChPenSulTe –
O2
VI
AmChPenSulTe
–
– – sat2, aadA1 – –
– dfrA1, sat2, aadA1 dfrA1, sat2, aadA1
Antimicrobial resistance and integrons in turkey E. coli
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Table 1 (Continued)
Integrons
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Production cycle
Isolate identificationa
Origin
PFGE type
Serogroup
MDR patternb
Class 1
Class 2 dfrA1, sat2, aadA1 dfrA1, sat2, aadA1 dfrA1, sat2, aadA1 dfrA1, sat2, aadA1 dfrA1, sat2, aadA1 dfrA1, sat2, aadA1
13282-1
Brain
O2
VI
AmChPenSulTe
–
14486-1
Brain
O2
VI
AmChPenSulTe
–
15189-1
Brain
O2
VI
AmChPenSulTe
–
15189-2
Brain
O2
VI
AmChPenSulTe
–
15189-3
Brain
O2
VI
AmChPenSulTe
–
14486-2
Brain
O2
III
AmCefChPenSulTe
–
14486-1 14486-2
Lung Lung
NT NT
III III
AmCefChPenSulTe AmCefChPenSulTe
– –
a
Strains with the same identification number were isolated from the same carcass (1037-1, 1037-2, 3302-1, 3302-4, 14486-1, 14486-2, 28234-1, 29610-1). bMDR = multidrug resistance; Am = aminoglycosides; Cef = cephalosporins; Ch = quinolones; Pen = penicillins; Fen = phenicols; Sul = potentiated sulphonamides; Te = tetracyclines. cNT, not typeable.
diversity was detected in AFEC compared with APEC (Simpson’s index of diversity = 0.94 and 0.86, respectively), with no overlapping among their PFGE types. Among APEC and AFEC isolates, four and 13 PFGE types were represented by single strains, respectively. Antimicrobial resistance of APEC and AFEC isolates. Overall, except for a few drugs (i.e. nalidixic acid, ciprofloxacin, and florfenicol), AFEC showed higher percentages of resistance than APEC (Figure 2). Most isolates, both APEC and AFEC, were resistant to tetracyclines (95.8% for all the drugs in APEC; 96.7% for doxycycline to 100% for tetracycline and oxytetracycline in AFEC), as well as to penicillins (95.8% for all the drugs in APEC; from 90% for amoxicillin + clavulanic acid to 100% for ampicillin in AFEC). A high percentage of AFEC was also resistant to potentiated sulphonamides (80% for trimethoprim + sulfamethoxazole and 90% for triple sulphonamides), whereas APEC were less resistant to both antimicrobials (47.9% and 58.3%, respectively). Conversely, APEC exhibited higher frequencies of resistance to fluoroquinolones than AFEC (from 6.2% for enrofloxacin and ciprofloxacin to 93.7% for nalidixic acid and from 3.3% ciprofloxacin to 46.7% for nalidixic acid, respectively). The percentage of resistance to aminoglycosides ranged from 0% for apramycin to 75% for streptomycin among APEC and from 6.7% for apramycin to 86.7% for streptomycin among AFEC. Resistance to cephalosporins was higher in AFEC (from 3.3% for ceftazidime to 43.3% for cephalotin) than APEC (from 0% for 3 cephalosporins to 31.2% for cephalotin). Low levels of resistance were detected to phenicols, both in APEC (8.3% for chloramphenicol and 4.2% for florfenicol) and in AFEC (36.7% for chloramphenicol and 0% for florfenicol). All isolates were fully susceptible to colistin. The resistance rates to triple sulphonamides, trimethoprim combined with sulfamethoxazole, gentamicin, kanamycin, aminosidine, ceftiofur, cefotaxime, and chloramphenicol of AFEC were significantly (P ≤ 0.05) higher compared with APEC; resistance to nalidixic acid was significantly (P ≤ 0.05) higher in APEC
than in AFEC. The prevalence of resistance of APEC to triple sulphonamides, trimethoprim combined with sulfamethoxazole, ampicillin, amoxicillin combined with clavulanic acid, streptomycin, and spectinomycin increased significantly (P ≤ 0.05) over the three production cycles; the prevalence of resistance to florfenicol decreased significantly (P ≤ 0.05) over the same interval. No significant differences (P > 0.05) in resistance rates were observed among serogroups of APEC. The majority (96.1%) of E. coli isolated from diseased and healthy birds exhibited resistance to three or more antimicrobial classes (Tables 1 and 2). Only three APEC isolates showed resistance to one and two classes of antimicrobials, and none was fully susceptible. In particular, among the 48 APEC isolates, three (6.2%), nine (18.7%), 22 (45.8%), 10 (20.8%) and one (2.1%) isolates showed MDR to three, four, five, six and seven antimicrobial classes, respectively. Out of the 30 AFEC, three (10%), eight (26.7%), six (20%), eight (26.7%) and five (16.7%) isolates were resistant to three, four, five, six and seven antimicrobial classes, respectively. MDR patterns comprised 19 different combinations, with the most common including five classes (i.e. aminoglycosides, fluoroquinolones, penicillins, sulphonamides and tetracyclines) among APEC (18/ 48 isolates) and four classes (i.e. aminoglycosides, penicillins, sulphonamides and tetracyclines) among AFEC (7/30 isolates). Presence of class 1 and 2 integrons and gene cassettes content in APEC and AFEC isolates. Real-time PCR screening for intI1 and intI2 genes identified 34 (43.6%) strains carrying class 1 and/or class 2 integrons (Tables 1 and 2). Out of the 48 APEC isolates, the intI1 gene was detected in six (12.5%) isolates and the intI2 gene in nine (18.7%) isolates. Among the 30 AFEC isolates, 19/30 (63.3%) strains were positive for class 1 integrons and only two (6.7%) for class 2 integrons. Two strains (one APEC and one AFEC) carried both classes of integrons. The prevalence of class 1 integrons was significantly higher (P ≤ 0.001) in AFEC as compared with APEC, while that
400
A. Piccirillo et al. Table 2.
Antimicrobial resistance, class 1 and 2 integrons and gene cassette content of AFEC isolates.
Integrons Production cycle
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1
Isolate identificationa 18042-5 17401-2 16756-3 17401-1 17401-4 16756-4 15446-3 16185-1 16185-2 16185-3 14966-3 17401-3 17401-5 16756-1 16756-2 16185-4 14966-5 18042-1 18042-2 18042-3 16756-5 15446-1 15446-2 14966-1 14966-4 18042-4 15446-4 15446-5 16185-5 14966-2
PFGE type IV XXIII IV XXIc NT NT XXVII II XII II XI XXII XXV X IV VIII XIII VII X X II XIV II I NT X XV I II XX
MDR patternb
Class 1
Class 2
AmPenTe PenSulTe AmPenTe AmPenSulTe AmPenSulTe AmPenSulTe AmPenSulTe AmPenSulTe AmPenSulTe AmPenSulTe AmChPenTe AmCefPenSulTe AmChPenSulTe AmPenFenSulTe AmPenFenSulTe AmCefPenSulTe CefChPenSulTe AmChPenFenSulTe AmCefPenFenSulTe AmCefPenFenSulTe AmCefChPenSulTe AmCefChPenSulTe AmCefChPenSulTe AmChPenFenSulTe AmCefChPenSulTe AmCefChPenFenSulTe AmCefChPenFenSulTe AmCefChPenFenSulTe AmCefChPenFenSulTe AmCefChPenFenSulTe
– – – – dfrA1, aadA1 – – dfrA12, orfF, aadA2 dfrA1, aadA1 dfrA12, orfF, aadA2 – dfrA1, aadA1 dfrA1, aadA1 dfrA1, aadA1 dfrA1, aadA1 dfrA1, aadA1 – dfrA1, aadA1 dfrA1, aadA1 dfrA1, aadA1 dfrA12, orfF – dfrA12, aadA2 – – dfrA1, aadA1 dfrA12, orfF, aadA2 sat2 dfrA12, orfF, aadA2 dfrA7
– – – – – – – – – – – – – – – – – – – – – – – sat2, aadA1 – – – sat2, aadA1 – –
a
Strains with the same identification number were isolated from the same carcass (1037-1, 1037-2, 3302-1, 3302-4, 14486-1, 14486-2, 28234-1, 29610-1). Faecal samples were obtained from individual birds. bMDR = multidrug resistance; Am = aminoglycosides; Cef = cephalosporins; Ch = quinolones; Pen = penicillins; Fen = phenicols; Sul = potentiated sulphonamides; Te = tetracyclines. cNT, not typeable.
of class 2 integrons was not significantly (P > 0.05) different between APEC and AFEC. Prevalence of integrons was quite different among APEC of the various production cycles and serogroups. Indeed, none of the isolates of the first production cycle carried integrons, whereas isolates from the second and third production cycles were positive (6/28 and 8/10 isolates, respectively). Only class 1 integrons were identified in isolates from the second cycle, except for one strain carrying both classes of integrons; isolates from the third cycle carried only class 2 integrons. Furthermore, most of the intI1-positive and intI2positive strains belonged to the O2 serogroup (11/14), 2/14 strains were untypeable and only one strain belonged to the O78 serogroup. As shown in Tables 1 and 2, 10 different gene cassettes including aadA (variants aadA1 and aadA2), dfrA (variants dfrA1, dfrA7, dfrA12), sat2, orfF and estX genes, alone or in combination, were identified among APEC and AFEC. The aadA genes encode for the aminoglycoside adenylyltransferases (streptomycin/spectinomycin resistance), the dfrA genes for the dihydrofolate reductases (trimethoprim resistance), the sat2 gene for the streptothricin acetyltransferases, the estX gene for a putative esterase, and the orfF gene for a protein of unknown function. The most common cassette array was dfrA1-aadA1 carried by 11 intI1-positive AFEC and four APEC strains, followed by the dfrA1-sat2-aadA1
array carried by eight intI2-positive APEC strains. Isolates carrying both classes of integrons harboured the aadA1 and sat2-aadA1 genes (APEC) and the sat2 and sat2-aadA1 genes (AFEC) in class 1 and 2 integrons, respectively. Diversity of gene cassette arrays was higher in AFEC than APEC (seven vs. five different cassette arrays, respectively). In APEC isolates from the second production cycle, the most prevalent array was dfrA1-aadA1, whereas in intI2positive APEC isolates from the third production cycle only one array (dfrA1-sat2-aadA1) was identified. The only intI1-positive O78 serogroup carried an unusual cassette array, the estX-aadA1. Association between presence of integrons and antimicrobial resistance in E. coli isolates. For a clear assessment of the relation between antimicrobial resistance and integrons, resistance determinants not associated with integrons were excluded from the analysis. The differences between intI1-positive and intI1-negative isolates in resistance to gentamycin, streptomycin, aminosidine, triple sulphonamides and trimethoprim combined with sulfamethoxazole were significantly (P ≤ 0.05) higher in intI1-positive compared with intI1-negative isolates. The differences in intI2-positive and intI2-negative isolates in resistance to spectinomycin, triple sulphonamides and trimethoprim combined with sulfamethoxazole were
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Antimicrobial resistance and integrons in turkey E. coli
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Figure 1. Dendrogram of the 28 different PFGE types identified among APEC and AFEC isolates. The scale bar on the top indicates similarity based on the Dice coefficient and PFGE types refer to patterns greater than 80% similarity.
significantly (P ≤ 0.05) higher in intI2-positive compared with intI2-negative isolates. All intI1-positive and intI2positive E. coli isolates were MDR, while intI1-negative and intI2-negative isolates were 93.2%.
Discussion Currently available studies showed that high resistance and multidrug resistance are common among avian pathogenic E. coli from turkeys (Salmon & Watts, 2000; Cormican et al., 2001; van den Bogaard et al., 2001; Altekruse et al., 2002; EFSA, 2010; Randall et al., 2011, 2013; Gosling et al., 2012). In the current study, a very high level of resistance to several antimicrobial drugs was detected and multidrug resistance to three or more antimicrobial classes was widespread both among APEC and AFEC. Moreover, a high percentage of isolates was found positive for class 1 and 2 integrons. Although some data on the presence and characterization of integrons in turkey E. coli are available in the literature (Bass et al., 1999; Singh et al., 2005; Nógrády et al., 2006), none of these studies was targeted to ascertain the distribution of these genetic elements in turkey flocks. Therefore, with this study we provide the first data on the presence and the gene cassette content of class 1 and 2 integrons in E. coli isolated from commercial meat turkey flocks. The highest levels of resistance were detected against tetracyclines, penicillins and sulphonamides, as widely documented among pathogenic and commensal E. coli of turkey origin (Salmon & Watts, 2000; Cormican et al., 2001; van den Bogaard et al., 2001; Altekruse et al., 2002;
Lister, 2005; EFSA, 2010; Randall et al., 2011, 2013; Gosling et al., 2012). The high levels of resistance to these classes may be attributed to a long-term selection pressure, since these antimicrobials are among the oldest and the most extensively used in the poultry industry all over the world (Singer & Hofacre, 2006), including Italy. In contrast, the very high resistance rates to streptomycin and nalidixic acid were unexpected since these drugs have not been used for a long time in our country. Similarly to our findings, a widespread resistance to these antimicrobials has been documented in turkey APEC and AFEC (Cormican et al., 2001; Giraud et al., 2001; Altekruse et al., 2002; Bryan et al., 2004; EFSA, 2010; Randall et al., 2011, 2013; Gosling et al., 2012). However, it has been reported that the resistance to specific antimicrobials, such as streptomycin, may be prevalent despite its discontinuance as a therapeutic agent (Bass et al., 1999) and the development of resistance to nalidixic acid may not be necessarily linked with treatment (Hofacre et al., 2000). In accordance with previous data (Salmon & Watts, 2000; Cormican et al., 2001; Lister, 2005; EFSA, 2010; Randall et al., 2011, 2013; Gosling et al., 2012; Kempf et al., 2013a), a prevailing susceptibility against several members of the aminoglycoside family (streptomycin excepted) was found in E. coli from turkeys, mainly among APEC. This finding was expected since these antimicrobials are scarcely employed in the turkey industry in Italy. Similarly, low levels of resistance to fluoroquinolones (except for nalidixic acid) were found. Partially contrasting results regarding fluoroquinolone resistance can be found in the literature (Salmon & Watts, 2000; Cormican et al., 2001; Giraud
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A. Piccirillo et al.
Figure 2. Percentage of APEC (2a) and AFEC (2b) isolates susceptible (S), intermediate (I) and resistant (R) to antimicrobials. APR = apramycin; GM = gentamicin; STR = streptomycin; K = kanamycin; AM = aminosidine; SPT = spectinomycin; CF = cephalotin; CFT = ceftiofur; CTX = cefotaxime; CAZ = ceftazidime; FEP = cefepime; NA = nalidixic acid; ENR = enrofloxacin; CIP = ciprofloxacin; NOR = norfloxacin; AMP = ampicillin; AMC = amoxicillin+clavulanic acid; CAF = chloramphenicol; FFC = florfenicol; S3 = triple sulphonamides; SXT = trimethoprim+sulfamethoxazole; TE = tetracycline; DO = doxycycline; OT = oxytetracycline; CT = colistin.
et al., 2001; van den Bogaard et al., 2001; Altekruse et al., 2002; Lister, 2005; Taylor et al., 2008; EFSA, 2010; Randall et al., 2011, 2013; Gosling et al., 2012). It has been recognized that fluoroquinolone resistance can be related to the usage pattern of these drugs in a given country, which is in turn influenced by the market dynamics, mainly the cost of commercial products (Bywater, 2005). In Italy, the parsimonious use of costly products, together with the policy of the integrated company, may have limited the development of fluoroquinolone resistance in E. coli examined in the present study. A low susceptibility to cephalotin and ceftiofur was detected in both APEC and AFEC. Resistance to thirdgeneration and fourth-generation cephalosporins of clinical and faecal turkey E. coli has been previously reported (Salmon & Watts, 2000; Altekruse et al., 2002; Randall et al., 2011, 2013; Gosling et al., 2012). Dutil et al. (2010) demonstrated that ceftiofur resistance was related to the use of this drug in poultry hatcheries. Although cephalosporins are not approved for use in poultry, the administration of this drug in 1-day-old chicks at the hatchery cannot be excluded. One should emphasize that the predominant
susceptibility to third-generation and fourth-generation cephalosporins, as well as to fluoroquinolones, is of great value from a public health point of view given the importance of these antimicrobials as critically important antibiotics for human medicine (WHO, 2012). The moderate levels of resistance to chloramphenicol and florfenicol detected in our study are unclear, since the first drug is not approved for use in food-producing animals in Europe (Commission Regulation No. 37/2010 of 22 December 2009) and the second is not authorized for use in poultry in Italy. However, this finding has been previously documented in other studies (Salmon & Watts, 2000; Cormican et al., 2001; Altekruse et al., 2002; EFSA, 2010; Randall et al., 2011, 2013; Gosling et al., 2012). Finally, the full susceptibility to colistin observed in E. coli is valuable both for poultry and human concerns, since this agent is widely used in poultry production and represents a last-resort treatment in human medicine (Livermore, 2004). In accordance with our results, colistin resistance seems to be extremely rare (
