Comparative Genomic Analysis of Antimicrobial-Resistant Escherichia coli from South American Camelids in Central Germany

South American camelids (SAC) are increasingly kept in Europe in close contact with humans and other livestock species and can potentially contribute to transmission chains of epizootic, zoonotic and antimicrobial-resistant (AMR) agents from and to livestock and humans. Consequently, SAC were included as livestock species in the new European Animal Health Law. However, the knowledge on bacteria exhibiting AMR in SAC is too scarce to draft appropriate monitoring and preventive programs. During a survey of SAC holdings in central Germany, 39 Escherichia coli strains were isolated from composite fecal samples by selecting for cephalosporin or fluoroquinolone resistance and were here subjected to whole-genome sequencing. The data were bioinformatically analyzed for strain phylogeny, detection of pathovars, AMR genes and plasmids. Most (33/39) strains belonged to phylogroups A and B1. Still, the isolates were highly diverse, as evidenced by 28 multi-locus sequence types. More than half of the isolates (23/39) were genotypically classified as multidrug resistant. Genes mediating resistance to trimethoprim/sulfonamides (22/39), aminoglycosides (20/39) and tetracyclines (18/39) were frequent. The most common extended-spectrum-β-lactamase gene was bla CTX-M-1 (16/39). One strain was classified as enteropathogenic E. coli . The positive results indicate the need to include AMR bacteria in yet-to-be-established animal disease surveillance protocols for SAC

Clostridioides difficile in South American Camelids in Germany: First Insights into Molecular and Genetic Characteristics and Antimicrobial Resistance

Little is known about zoonotic pathogens and their antimicrobial resistance in South American camelids (SAC) in Germany including Clostridioides (C.) difficile. The aim of this study was to investigate prevalence, molecular characteristics and antimicrobial resistance of C. difficile in SAC. Composite SAC faecal samples were collected in 43 husbandries in Central Germany and cultured for C. difficile. Toxinotyping and ribotyping was done by PCR. Whole genome sequencing was performed with Illumina® Miseq™. The genomes were screened for antimicrobial resistance determinants. Genetic relatedness of the isolates was investigated using core genome multi locus sequence typing (cgMLST) and single nucleotide polymorphism analysis. Antimicrobial susceptibility testing was done using the Etest® method. Eight C. difficile isolates were recovered from seven farms. The isolates belonged to different PCR ribotypes. All isolates were toxinogenic. cgMLST revealed a cluster containing isolates recovered from different farms. Seven isolates showed similar resistance gene patterns. Different phenotypic resistance patterns were found. Agreement between phenotypic and genotypic resistance was identified only in some cases. Consequently, SAC may act as a reservoir for C. difficile. Thus, SAC may pose a risk regarding zoonotic transmission of toxinogenic, potentially human-pathogenic and resistant C. difficile isolates

Portable Differential Detection of CTX-M ESBL Gene Variants, blaCTX-M-1 and blaCTX-M-15, from Escherichia coli Isolates and Animal Fecal Samples Using Loop-Primer Endonuclease Cleavage Loop-Mediated Isothermal Amplification

Cefotaximase-Munich (CTX-M) extended-spectrum beta-lactamase (ESBL) enzymes produced by Enterobacteriaceae confer resistance to clinically relevant third-generation cephalosporins. CTX-M group 1 variants, CTX-M-1 and CTX-M-15, are the leading ESBL-producing Enterobacteriaceae associated with animal and human infection, respectively, and are an increasing antimicrobial resistance (AMR) global health concern. The blaCTX-M-1 and blaCTX-M-15 genes encoding these variants have an approximate nucleotide sequence similarity of 98.7%, making effective differential diagnostic monitoring difficult. Loop-primer endonuclease cleavage loop-mediated isothermal amplification (LEC-LAMP) enables rapid real-time multiplex pathogen detection with single-base specificity and portable on-site testing. We have developed an internally controlled multiplex CTX-M-1/15 LEC-LAMP assay for the differential detection of blaCTX-M-1 and blaCTX-M-15. Assay analytical specificity was established using a panel of human, animal, and environmental Escherichia coli isolates positive for blaCTX-M-1 (n = 18), blaCTX-M-15 (n = 35), and other closely related blaCTX-Ms (n = 38) from Ireland, Germany, and Portugal, with analytical sensitivity determined using probit regression analysis. Animal fecal sample testing using the CTX-M-1/15 LEC-LAMP assay in combination with a rapid DNA extraction protocol was carried out on porcine fecal samples previously confirmed to be PCR-positive for E. coli blaCTX-M. Portable instrumentation was used to further analyze each fecal sample and demonstrate the on-site testing capabilities of the LEC-LAMP assay with the rapid DNA extraction protocol. The CTX-M-1/15 LEC-LAMP assay demonstrated complete analytical specificity for the differential detection of both variants with sensitive low-level detection of 8.5 and 9.8 copies per reaction for blaCTX-M-1 and blaCTX-M-15, respectively, and E. coli blaCTX-M-1 was identified in all blaCTX-M positive porcine fecal samples tested.This research was funded by the European Union Horizon 2020 Research and Innovation Program under grant agreement No. 773830: One Health European Joint Program, JRP13-AMRSH5-WORLDCOM project.info:eu-repo/semantics/publishedVersio

Santos Ovejero del Agua. Catedrático de la Facultad de Veterinaria y su relación con la de Biología de León

Santos Ovejero del Agua fue un personaje de una personalidad extraordinaria, sin cuya presencia sería difícil de entender la vida social, académica, universitaria y empresarial de la ciudad y provincia de León desde el final de la guerra civil hasta el final de los años 60. Nacido, criado y fallecido en León (1906-1983), suma un espíritu inquieto, bien formado, gran personalidad, de perfil internacional, que ostentó responsabilidades importantes en la vida pública universitaria de la ciudad y fue catedrático y decano de la Facultad de Veterinaria, un visionario de sus posibilidades de proyección social, que proporcionó las primeras claves para la ampliación de estudios universitarios en lo que después sería Facultad de Biología y pionero en la actividad industrial biotecnológica de la Sanidad Animal

Übertrag von Salmonellen über Schlachtschweine im Transport und in den Wartebuchten

This study was carried out during two sampling periods (5 days each) in March 2015 and September and October 2015, in a slaughterhouse in northern Germany. The aim of this study was to determine the transfer of Salmonella from trucks into lairage. Specially, the presence of Salmonella on trucks delivering pigs for slaughter, the role of the drivers in the Salmonella transfer and the role of the animals in the diversity of Salmonella strains. Samples were taken from transport (trucks and boots of the drivers), from lairage: pigs (rectum and skin swabs), lairage environment (before and after the occupation the pigs) and finally carcasses in the chilling room. In total, 859 samples were collected to get the information, if Salmonella could pass into the chilling room, and also to determine the diversity of the serovars in the lairage facilities. From 859 samples, 52 (6%) were Salmonella positive. 4 from the first sampling period, where pigs of category I were sampled, and 48 from the second sampling, where pigs of category I and II were available. Two serotypes were found: S. Derby with a proportion of 57.7% (30/52) and S. Typhimurium of 42.3% (22/52). With the PGFE technique, 6 different identical Salmonella patterns were found. In total, 48.1% (25/52) of the positive samples were recovered from pigs’ skin and rectum swabs. The lairage environment after occupation of the pigs, provided 21.1% (11/52) of the total positive samples, and transport (boots and floor truck) 15.4% (8/52). Pens before occupation and the lairage facilities before disinfection had the same proportion, 7.7% (4/52) of the positive samples. Neither samples after disinfection of the lairage facilities nor the carcasses at the chilling room, were Salmonella positive. All positive samples were found in the dirty area. Salmonella found in previous positions did not reach the end of the processing chain in the abattoir. Salmonella seems to get eliminated during the harvesting technology. In total, 30 shipments were sampled. The transport samples consisted of truck´s floor and boots of the drivers. In total, 56 transport samples were taken and 8 of them were positive: one sample came from a truck floor. The rest of the transport positive samples (7) were found on the boots of drives. Two identical PFGE patterns were found, one in the first period and the other in the second period. In both of them, boots contaminated the lairage environment and also pigs from other shipments. In conclusion, the drivers´ boots were an important part of the contamination cycle and a vector for Salmonella contamination in the lairage area, the truck apparently does not play a major role. Lairage environment was more frequently contaminated after (11/52) than before occupation (4/52). Salmonella positive samples were found before disinfection (4/52) of the whole lairage facilities. Disinfection eliminated Salmonella, no positive sample was found after this procedure. The pigs were also an important part of the Salmonella contamination of the lairage. Positive pigs carried Salmonella into lairage, contaminating not only the environment of the lairage but also animals from other suppliers. So, questions asked in this study can be answered as follows: • Does the truck transfer Salmonella into the abattoir (are the trucks´ surfaces testing positive)? In this study, truck surfaces did not play a major role in transfer of Salmonella into the lairage. • How far could Salmonella pass from the truck into the harvesting technology under the given circumstances? Salmonella passed from the truck into the lairage and stayed there. Salmonella did not reach the chilling room. • Is the driver a part of this contamination cycle? In this study, the drivers´ boots were an important vector in the Salmonella contamination cycle. • Is the lairage Salmonella positive? Lairage was positive before and after the occupation of the sampled pigs, but no positive samples could be found after the disinfection of the whole facilities. • Does the animal influence the diversity of Salmonella serovars in the chain? Two serovars, which are widely distributed in the pig chain, were found. The pigs influenced the diversity of the different patterns.Diese Studie wurde über zwei Perioden von je 5 Tagen im März 2015 und September und Oktober desselben Jahres, in einem Schlachtbetrieb in Norddeutschland durchgeführt. Es wurden die Nachweishäufigkeit von Salmonellen auf Transportern, in und auf Tieren im Wartebereich sowie ein mögliches Festsetzen dieser Flora als inhouse- Flora untersucht. Es wurden Proben während der Transportphase (Transporter und Stiefel der Fahrer) gezogen. Im Bereich der Wartebuchten wurden Schweine beprobt (Rektal- und Hautproben) und Proben aus dem Umfeld genommen (vor und nach der Belegung von Buchten durch die Tiere). Zuletzt wurden Proben von zugehörigen Karkassen in den Kühlräumen genommen. Insgesamt wurden 859 Proben gewonnen, um der Frage des Transfers bis in die Kühlung nachzugehen. Von 859 Proben waren 52 (6 %) Salmonella- positiv, 4 aus der ersten Untersuchungsperiode, in der nur Tiere aus Beständen der Salmonellen-Kategorie I geschlachtet wurden. Während der zweite Untersuchungsperiode wurden Schweinen aus Beständen der Kat. I und II geschlachtet. Hier waren 48 Proben Salmonella-positiv. Es wurden zwei Serotypen nachgewiesen: S. Derby in einem Prozentsatz von 57,7 (30/52) und S. Typhimurium in einer Anzahl von 42,3 % (22/52). Mittels der PFGE-Methode wurden 6 unterschiedliche Bandentypen identifiziert. 48,1 % (25/52) der positiven Proben wurden von Haut- und Rektal- Tupfern isoliert. Aus dem Umfeld der Wartebuchten nach der Belegung durch die Schweine kamen 21,1 % (11/52) der Gesamtzahl der positiven Proben und vom Transport (Stiefel und Transportböden) 15,4 % (8/52). Die Wartebuchten vor der Belegung und vor der Desinfektion lieferten beide 7,7 % (4/52) der positiven Proben. Weder Proben, die nach der Desinfektion gezogen wurden, noch die beprobten Karkassen in der Kühlung waren positiv. Alle positiven Proben wurden im sog. schwarzen Bereich gefunden. Es konnte kein positiver Nachweis in der Kühlung erbracht werden. Dies könnte an der Technologie des Fleischgewinnungsprozesses liegen. Insgesamt wurden 30 Lieferungen geprüft. Die Proben aus dem Transport stammen von den Böden der Transporter und den Stiefeln der Fahrer. Insgesamt wurden 56 Transportproben gezogen, von denen sich 8 als positiv herausstellten. Eine Probe kam von einem Transporterboden, die übrigen (7) stammen von den Stiefeln der Fahrer. Es wurden zwei identische PFGE- Bandentypen gefunden, eine in der ersten und die andere während der zweiten Untersuchungsperiode. Beide signalisieren eine Kontamination des Wartebuchtenumfeldes und der Schweine anderer Lieferungen durch die Stiefel. In Konsequenz müssen die Stiefel der Fahrer als ein wichtiger Teil des Kontaminationszyklus und als Vektor der Kontamination durch Salmonellen in den Wartestallungen angesehen werden. Offenbar spielten die Transporter in dieser Untersuchung keine wesentliche Rolle. Das Wartebuchtenumfeld war häufiger kontaminiert nach der Belegung (11/52) als vor der Belegung (4/25). Salmonella- positive Proben fanden sich vor der Desinfektion (4/52) des gesamten Areals, nach der Desinfektion dagegen wurden positive Proben nicht mehr gefunden. Die einkommenden Schlachtschweine waren ebenfalls ein wichtiger Teil der Kontamination mit Salmonellen im Wartebereich. Positive Schweine können Salmonella in den Wartebereich einbringen, wobei sie nicht nur das Umfeld kontaminieren, sondern auch eine Kontamination von Tieren anderer Herkünfte führen bewirken können. Somit können die anfangs gestellten Fragen wie folgt beantwortet werden: • Ist der Transporter einen Überträger für Salmonella und erfolgt ein Eintrag in den Schlachtbetrieb (sind die Transporter-Oberflächen positiv)? Die Oberflächen der Transporter spielten bei der Übertragung von Salmonellen in den Wartebereich hinein keine größere Rolle. • Wie weit können Salmonellen aus den Transportern in die Schlachtbetriebstechnologie eingetragen werden? Die Erreger wurden aus dem Transporter in den Wartebereich eingetragen, gelangten nicht allerding in die Kühlung. • Sind die Fahrer am Kontaminationszyklus beteiligt? Die Stiefel der Fahrer waren ein wichtiger Vektor im Salmonella- Kontaminationszyklus. • Ist der Wartebereich Salmonella-positiv? Der Wartebereich war vor und nach der Belegung durch die beprobten Schlachtschweine positiv, allerdings nicht nach einer Desinfektion des Bereiches. • Beeinflussen die Tiere die Diversität der Salmonella-Serovare in der Kette? Es wurden zwei Serotypen gefunden, die in der Schweine-Produktionskette weit verbreitet sind

Genomic characterization of multidrug-resistant Salmonella serovars Derby and Rissen from the pig value chain in Vietnam

Nontyphoidal Salmonella (NTS) is the most reported cause of bacterial foodborne zoonoses in Vietnam, and contaminated pork is one of the main sources of human infection. In recent years, the prevalence of NTS carrying multiple antimicrobial resistance genes (ARGs) have been increased. The genomic characterization along the pig value chain and the identification of ARGs and plasmids have the potential to improve food safety by understanding the dissemination of ARGs from the farm to the table. We report an analysis of 13 S. Derby and 10 S. Rissen isolates, collected in 2013 at different stages in Vietnamese slaughterhouses and markets. VITEK 2 Compact System was used to characterize the phenotypical antimicrobial resistance of the isolates. In addition, whole-genome sequencing (WGS) was used to detect ARGs and plasmids conferring multidrug resistance. Whole genome single nucleotide polymorphism typing was used to determine the genetic diversity of the strains and the spread of ARGs along the pig value chain. Altogether, 86.9% (20/23) of the samples were resistant to at least one antibiotic. Resistance to ampicillin was most frequently detected (73.9%), followed by piperacillin and moxifloxacin (both 69.6%). At least one ARG was found in all strains, and 69.6% (16/23) were multidrug-resistant (MDR). The observed phenotype and genotype of antimicrobial resistance were not always concordant. Plasmid replicons were found in almost all strains [95.6% (22/23)], and the phylogenetic analysis detected nine clusters (S. Derby, n = 5; S. Rissen, n = 4). ARGs and plasmid content were almost identical within clusters. We found six MDR IncHI1s with identical plasmid sequence type in strains of different genetic clusters at the slaughterhouse and the market. In conclusion, high rates of multidrug resistance were observed in Salmonella strains from Vietnam in 2013. Genomic analysis revealed many resistance genes and plasmids, which have the potential to spread along the pig value chain from the slaughterhouse to the market. This study pointed out that bioinformatics analyses of WGS data are essential to detect, trace back, and control the MDR strains along the pig value chain. Further studies are necessary to assess the more recent MDR Salmonella strains spreading in Vietnam

<i>Clostridioides difficile</i> in South American Camelids in Germany: First Insights into Molecular and Genetic Characteristics and Antimicrobial Resistance

Little is known about zoonotic pathogens and their antimicrobial resistance in South American camelids (SAC) in Germany including Clostridioides (C.) difficile. The aim of this study was to investigate prevalence, molecular characteristics and antimicrobial resistance of C. difficile in SAC. Composite SAC faecal samples were collected in 43 husbandries in Central Germany and cultured for C. difficile. Toxinotyping and ribotyping was done by PCR. Whole genome sequencing was performed with Illumina® Miseq™. The genomes were screened for antimicrobial resistance determinants. Genetic relatedness of the isolates was investigated using core genome multi locus sequence typing (cgMLST) and single nucleotide polymorphism analysis. Antimicrobial susceptibility testing was done using the Etest® method. Eight C. difficile isolates were recovered from seven farms. The isolates belonged to different PCR ribotypes. All isolates were toxinogenic. cgMLST revealed a cluster containing isolates recovered from different farms. Seven isolates showed similar resistance gene patterns. Different phenotypic resistance patterns were found. Agreement between phenotypic and genotypic resistance was identified only in some cases. Consequently, SAC may act as a reservoir for C. difficile. Thus, SAC may pose a risk regarding zoonotic transmission of toxinogenic, potentially human-pathogenic and resistant C. difficile isolates