Genome Comparison of Erythromycin Resistant Campylobacter from Turkeys Identifies Hosts and Pathways for Horizontal Spread of erm(B) Genes.

Pathogens in the genus Campylobacter are the most common cause of food-borne bacterial gastro-enteritis. Campylobacteriosis, caused principally by Campylobacter jejuni and Campylobacter coli, is transmitted to humans by food of animal origin, especially poultry. As for many pathogens, antimicrobial resistance in Campylobacter is increasing at an alarming rate. Erythromycin prescription is the treatment of choice for clinical cases requiring antimicrobial therapy but this is compromised by mobility of the erythromycin resistance gene erm(B) between strains. Here, we evaluate resistance to six antimicrobials in 170 Campylobacter isolates (133 C. coli and 37 C. jejuni) from turkeys. Erythromycin resistant isolates (n = 85; 81 C. coli and 4 C. jejuni) were screened for the presence of the erm(B) gene, that has not previously been identified in isolates from turkeys. The genomes of two positive C. coli isolates were sequenced and in both isolates the erm(B) gene clustered with resistance determinants against aminoglycosides plus tetracycline, including aad9, aadE, aph(2″)-IIIa, aph(3')-IIIa, and tet(O) genes. Comparative genomic analysis identified identical erm(B) sequences among Campylobacter from turkeys, Streptococcus suis from pigs and Enterococcus faecium and Clostridium difficile from humans. This is consistent with multiple horizontal transfer events among different bacterial species colonizing turkeys. This example highlights the potential for dissemination of antimicrobial resistance across bacterial species boundaries which may compromise their effectiveness in antimicrobial therapy.

Molecular characterization and antimicrobial resistance of STEC strains isolated from healthy cattle in 2011 and 2013 in Spain.

Prevention of Shiga toxin-producing Escherichia coli (STEC) foodborne outbreaks is hampered by its complex epidemiology. We assessed the distribution of virulence genes (VGs), main serogroups/serotypes for public health [haemolytic uraemic syndrome (HUS)-related], antimicrobial resistance (AMR) profiles and pulsed-field gel electrophoresis (PFGE) patterns in a collection of STEC isolates obtained from cattle hide (n = 149) and faecal (n = 406) samples collected during a national survey conducted in Spain in 2011 and 2013. Isolates were cultured using McConkey and CT-SMAC agar after enrichment, and confirmed as STEC by PCR. STEC prevalence in hides (15·4%) was higher than in faeces (10·7%) and O157:H7 was more frequent in the former (2·7% vs. 0·99%). Non-O157 HUS-related serogroups were present albeit at low frequencies. The non-O157 isolates were more heterogeneous than O157:H7 in their VG patterns, with 25/64 presenting VGs from both STEC and enterotoxigenic pathotypes (hybrid isolates). Of the STEC isolates, 62·5% were resistant at least to one antimicrobial, and no differences in AMR between O157:H7 and non-O157 were detected. All isolates had different profiles by PFGE and did not form a cluster. Overall, our results demonstrated that STEC in the cattle reservoir is still a matter of concern for human health due to the presence of HUS-related serogroups, the occurrence of certain VGs, AMR and the additional risks that hybrid isolates may pose, and thus warrants further investigation.

Prevalence of Type VI Secretion System in Spanish Campylobacter jejuni Isolates.

Infections from Campylobacter jejuni pose a serious public health problem and are now considered the leading cause of foodborne bacterial gastroenteritis throughout the world. Sequencing of C. jejuni genomes has previously allowed a number of loci to be identified, which encode virulence factors that aid survival and pathogenicity. Recently, a Type VI secretion system (T6SS) consisting of 13 conserved genes was described in C. jejuni strains and recognised to promote pathogenicity and adaptation to the environment. In this study, we determined the presence of this T6SS in 63 Spanish C. jejuni isolates from the food chain and urban effluents using whole-genome sequencing. Our findings demonstrated that nine (14%) strains harboured the 13 ORFs found in prototype strain C. jejuni 108. Further studies will be necessary to determine the prevalence and importance of T6SS-positive C. jejuni strains.

Campylobacter shared between free-ranging cattle and sympatric wild ungulates in a natural environment (NE Spain).

Campylobacter infections are a public health concern and an increasingly common cause of food-borne zoonoses in the European Union. However, little is known about their spill-over from free-ranging livestock to sympatric wild ungulates, especially in regards to uncommon Campylobacter species. In this study, we aim to determine the prevalence of C. coli, C. jejuni and other C. spp. in game ungulates (wild boar Sus scrofa and Iberian ibex Capra pyrenaica) and free-ranging sympatric cattle in a National Game Reserve in NE Spain. Furthermore, we explore the extent to which Campylobacter species are shared among these co-habiting hosts. Faecal samples from Iberian ibex (n = 181) were negative for C. spp. By direct plating, two wild boars out of 150 were positive for C. coli (1.3%, 95% CI 0.16-4.73), and one was positive for C. jejuni (0.67%, 95% CI 0.02-3.66). The latter was predominant in cattle: 5.45% (n = 55, 95% CI 1.14-5.12), while C. coli was not isolated from this host. C. lanienae was the most frequent species in wild boar at 10% (95% CI 5.7-15.96), and one cow cohabiting with positive wild boars in the same canyon also carried C. lanienae. Four enrichment protocols (using Bolton or Preston broth combined with either mCCDA or CFA) were added for 172 samples (57 from wild boars, 55 cattle and 60 Iberian ibexes) to increase the number of isolates obtained allowing the detection of statistically significant differences. The prevalence of C. lanienae was statistically significantly higher in wild boar than in cattle (P < 0.01), but the prevalence of C. jejuni was higher in the latter (P = 0.045). These results suggest that wild boar and cattle carry their own predominant Campylobacter species, while Iberian ibex do not seem to play an important role in the epidemiology of Campylobacter. However, there is a potential spill-over of C. spp., and thus, further research is needed to elucidate the factors determining inter-species transmission.

Lack of evidence of spill-over of Salmonella enterica between cattle and sympatric Iberian ibex (Capra pyrenaica) from a protected area in Catalonia, NE Spain.

Salmonella enterica is a zoonotic agent of worldwide importance found in a wide range of wild hosts. However, its prevalence in many popular game species has never been assessed. Iberian ibex (Capra pyrenaica) is the main game caprinae of the Iberian Peninsula and around two thousand individuals are hunted every year for trophy or for home consumption. In this work, 313 Iberian ibexes from the Ports de Tortosa i Beseit National Game Reserve (NE Spain) were tested for Salmonella enterica in faeces, and anti microbial susceptibility was determined. The exact location of shooting or capture was recorded with a GPS device to study the links of Salmonella infection with cattle presence and human proximity. Additionally, samples were taken from cattle grazing inside this reserve (n = 73). Only three Iberian ibexes (0.96%, 95% CI 0.2-2.8) were positive to Salmonella (serotype Enteritidis, Bardo and 35:r:z35), while prevalence was moderate in cattle: 21.92% (95% CI 13.10-33.14, serotype Meleagridis, Anatum, Kedougou and Othmarschen). All isolates were susceptible to the anti microbial agents tested. Moreover, a case of fatal septicaemic salmonellosis in an 11-year-old male Iberian ibex is described where Salmonella enterica serotype Enteritidis was isolated from the lung, liver and spleen samples. The low prevalence of Salmonella in Iberian ibex and the lack of shared serotypes suggest no association to cattle. Despite this, game meat aimed for human consumption should be examined, and it is strongly recommended that hunters and game keepers manipulate animals and carcasses under maximal hygienic conditions to avoid environmental contamination and human contagion.

Antimicrobial resistance in indicator Escherichia coli isolates from free-ranging livestock and sympatric wild ungulates in a natural environment (Northeastern Spain).

Antimicrobial resistance was assessed in indicator Escherichia coli isolates from free-ranging livestock and sympatric wild boar (Sus scrofa) and Iberian ibex (Capra pyrenaica) in a National Game Reserve in northeastern Spain. The frequency of antimicrobial resistance was low (0% to 7.9%). However, resistance to an extended-spectrum cephalosporin and fluoroquinolones was detected.

The effect of different isolation protocols on detection and molecular characterization of Campylobacter from poultry.

We determined whether different methods to isolate Campylobacter (including the ISO standard 10272:2006-1) affected the genotypes detectable from poultry, at three points during slaughter: caecal content, neck skin and meat. Carcasses from 28 independent flocks were thus sampled (subset A). In addition, ten neck skin samples from four flocks, ten caecal samples from ten different flocks and ten unrelated meat samples obtained from local supermarkets were collected (subset B). Campylobacter was isolated using eight different protocols: with and without enrichment using Bolton broth, Preston broth or Campyfood broth (CFB), followed by culture on either modified Charcoal Cefoperazone Deoxycholate Agar (mCCDA) or Campyfood agar (CFA). All obtained isolates were genotyped for flaA-SVR, and over half of the isolates were also typed by MLST. The strain richness, as a measure of number of detected fla-genotypes, obtained from subset A neck skin and caecal samples was higher than that of meat samples. In half of the cases, within a flock, at least one identical fla-genotype was obtained at all three slaughter stages, suggestive of autologous contamination of carcasses. Enrichment reduced the observed richness of isolates, while CFA plates increased richness compared to mCCDA plates, irrespective of inclusion of an enrichment step. Because the isolation protocol used influences both the yield and the fla-genotype richness obtained from poultry, this variable should be taken into account when different studies are being compared.

Cattle drive Salmonella infection in the wildlife-livestock interface.

The genus Salmonella is found throughout the world and is a potential pathogen for most vertebrates. It is also the most common cause of food-borne illness in humans, and wildlife is an emerging source of food-borne disease in humans due to the consumption of game meat. Wild boar is one of the most abundant European game species and these wild swine are known to be carriers of zoonotic and food-borne pathogens such as Salmonella. Isolation of the pathogen, serotyping and molecular biology are necessary for elucidating epidemiological connections in multi-host populations. Although disease management at population level can be addressed using a number of different strategies, such management is difficult in free-living wildlife populations due to the lack of experience with the wildlife-livestock interface. Herein, we provide the results of a 4-year Salmonella survey in sympatric populations of wild boar and cattle in the Ports de Tortosa i Beseit National Game Reserve (NE Spain). We also evaluated the effects of two management strategies, cattle removal and increased wild boar harvesting (i.e. by hunting and trapping), on the prevalence of the Salmonella serovar community. The serovars Meleagridis and Anatum were found to be shared by cattle and wild boar, a finding that was confirmed by 100% DNA similarity patterns using pulse field gel electrophoresis. Cattle removal was more efficient than the culling of wild boar as a means of reducing the prevalence of shared serotypes, which underlines the role of cattle as a reservoir of Salmonella for wild boar. To our knowledge, this is the first attempt to manage Salmonella in the wild, and the results have implications for management.

Evaluation of four protocols for the detection and isolation of thermophilic Campylobacter from different matrices.

AIMS: To identify the optimal method for detection of thermophilic Campylobacter at various stages in the food chain, three culture-dependent (direct plating, Bolton and Preston enrichment) and one molecular method (qPCR) were compared for three matrices: poultry faeces (n = 38), neck skin (n = 38) and packed fresh meat (n = 38). METHODS AND RESULTS: Direct plating was compared to enrichment with either Bolton broth (ISO 10272:2006-1) or Preston broth, followed by culture on two selective agars: modified charcoal cefoperazone desoxycholate agar (mCCDA) and Campyfood agar (CFA). Direct plating on CFA provided the highest number of positive samples for faeces and neck skin samples. Enrichment of meat samples in Preston followed by plating on mCCDA gave significantly higher number of positives than the recommended ISO method. Real-time qPCR yielded the highest number of positive samples. CONCLUSION: Direct plating on CFA is optimal for Campylobacter isolation from highly contaminated samples such as faeces or neck skin. When enrichment is required for less-contaminated samples such as poultry meat, Preston broth is the best choice. The maximum of detectable cells predicted by qPCR is a sensitive and powerful evaluation tool. SIGNIFICANCE AND IMPACT OF THE STUDY: The recommended ISO protocol had the least sensitivity, and application of this method could result in underreporting. We detected a high prevalence of Campylobacter on packed meat to be distributed, which suggests this is still a significant risk for consumers.

Detection of methicillin-resistant Staphylococcus aureus in Iberian pigs.

AIMS: Iberian pigs are bred in Spain for the production of high-value dry-cured products, whose export volumes are increasing. Animals are typically reared outdoors, although indoor farming is becoming popular. We compared carriage of methicillin-resistant Staphylococcus aureus (MRSA) in Iberian pigs, raised indoors and outdoors, with intensively farmed Standard White pigs. METHODS AND RESULTS: From June 2007 to February 2008, 106 skin swabs were taken from Iberian pigs and 157 samples from SWP at slaughterhouses in Spain. We found that Iberian pigs carried MRSA, although with a significantly lower prevalence (30/106; 28%) than SWP (130/157; 83%). A higher prevalence of indoor Iberian pigs compared with animals reared under outdoor conditions was not significant; however, all but one positive indoor Iberian pig samples were detected from one slaughterhouse. Overall, 16 different spa types were identified, with t011 predominating in all three animal populations. A subset of isolates was characterized by MLST. Most of these belonged to ST398. MRSA isolates from Iberian pigs presented a higher susceptibility to antibiotics than those isolated from SWP. CONCLUSIONS: Despite limited contact with humans, pigs raised outdoors are colonized by an MRSA population that genetically overlaps with that of intensively farmed pigs, although antimicrobial resistance is lower. SIGNIFICANCE AND IMPACT OF THE STUDY: To our knowledge, this is the first detection of MRSA in food animals raised in free-range conditions.

Multiresistant Salmonella enterica serovar 4,[5],12:i:- in Europe: a new pandemic strain?

A marked increase in the prevalence of S. enterica serovar 4,[5],12:i:- with resistance to ampicillin, streptomycin, sulphonamides and tetracyclines (R-type ASSuT) has been noted in food-borne infections and in pigs/pig meat in several European countries in the last ten years. One hundred and sixteen strains of S. enterica serovar 4,[5],12:i:- from humans, pigs and pig meat isolated in England and Wales, France, Germany, Italy, Poland, Spain and the Netherlands were further subtyped by phage typing, pulsed-field gel electrophoresis and multilocus variable number tandem repeat analysis to investigate the genetic relationship among strains. PCR was performed to identify the fljB flagellar gene and the genes encoding resistance to ampicillin, streptomycin, sulphonamides and tetracyclines. Class 1 and 2 integrase genes were also sought. Results indicate that genetically related serovar 4,[5],12:i:- strains of definitive phage types DT193 and DT120 with ampicillin, streptomycin, sulphonamide and tetracycline resistance encoded by blaTEM, strA-strB, sul2 and tet(B) have emerged in several European countries, with pigs the likely reservoir of infection. Control measures are urgently needed to reduce spread of infection to humans via the food chain and thereby prevent the possible pandemic spread of serovar 4,[5],12:i:- of R-type ASSuT as occurred with S. Typhimurium DT104 during the 1990s.

Surveillance of antimicrobial resistance in Escherichia coli strains isolated from pigs at Spanish slaughterhouses.

Antimicrobial resistance can make the efficient treatment of bacterial infections in humans and animals more difficult. Antimicrobial use in food animals may be one of the factors contributing to resistance. The Spanish surveillance network VAV has established a baseline of antimicrobial resistance in Escherichia coli strains from healthy pigs. Minimum inhibitory concentration and patterns of resistance to antimicrobials used in animals and humans were determined for 205 faecal strains isolated in a sampling frame of four slaughterhouses in Spain from 220 pigs in 1998. Higher levels of resistance were seen against antimicrobial agents authorised for use in food animals especially tetracycline, sulphonamides, trimethoprim and amoxycillin. All isolates were susceptible to antimicrobials employed mainly in humans such as ceftazidime, cefotaxime, imipenem, aztreonam and amikacin.

Antibiotic resistance monitoring: the Spanish programme. The VAV Network. Red de Vigilancia de Resistencias Antibióticas en Bacterias de Origen Veterinario.

Antimicrobial resistance is a problem in modern public health and antimicrobial use and especially misuse, the most important selecting force for bacterial antibiotic resistance. As this resistance must be monitored we have designed the Spanish network 'Red de Vigilancia de Resistencias Antibióticas en Bacterias de Origen Veterinario'. This network covers the three critical points of veterinary responsibility, bacteria from sick animals, bacteria from healthy animals and bacteria from food animals. Key bacteria, antimicrobials and animal species have been defined for each of these groups along with laboratory methods for testing antimicrobial susceptibility and for data analysis and reporting. Surveillance of sick animals was first implemented using Escherichia coli as the sentinel bacterium. Surveillance of E. coli and Enterococcus faecium from healthy pigs was implemented in 1998. In July 1999, data collection on Salmonella spp. was initiated in poultry slaughterhouses. Additionally, the prevalence of vancomycin resistant E. faecium was also monitored. This network has specific topics of interest related to methods of determining resistance, analysis and reporting of data, methods of use for veterinary practitioners and collaboration with public health authorities.