MRSA in breeding pigs in Germany in 2015

Methicillin resistant Staphylococcus aureus has been known to be prevalent in the pig production for nearly 15 years now (Meemken et al., 2010). In 2008 a survey carried out in the EU determined a high prevalence of MRSA in herds of breeding pigs also in Germany (EFSA, 2010). Likewise, MRSA were identified in Germany in herds of fattening pigs (Alt et al., 2011), pigs at slaughter (Tenhagen et al., 2009), on carcasses (Beneke et al., 2011) and in meat from pigs at retail (BVL, 2013). The current investigation was carried out to determine the current prevalence of MRSA in herds of breeding pigs, analyse patterns in the type of MRSA isolated from pigs and determine differences between the MRSA observed in units only housing sows and those housing weaned piglets. In previous studies it could be shown, that the prevalence of MRSA is higher in weaned piglets than in sows but little is known about differences in the types of MRSA that can be isolated in the different units

Prevalence of mcr-1 in E. coli from Livestock and Food in Germany, 2010–2015

Since the first description of a plasmid-mediated colistin resistance gene (mcr-1) in November 2015 multiple reports of mcr-1 positive isolates indicate a worldwide spread of this newly discovered resistance gene in Enterobacteriaceae. Although the occurrence of mcr-1 positive isolates of livestock, food, environment and human origin is well documented only few systematic studies on the prevalence of mcr-1 are available yet. Here, comprehensive data on the prevalence of mcr-1 in German livestock and food isolates are presented. Over 10.600 E. coli isolates from the national monitoring on zoonotic agents from the years 2010–2015 were screened for phenotypic colistin resistance (MIC value >2 mg/l). Of those, 505 resistant isolates were screened with a newly developed TaqMan-based real-time PCR for the presence of the mcr-1 gene. In total 402 isolates (79.8% of colistin resistant isolates) harboured the mcr-1 gene. The prevalence was depending on the food production chain. The highest prevalence was detected in the turkey food chain (10.7%), followed by broilers (5.6%). A low prevalence was determined in pigs, veal calves and laying hens. The mcr-1 was not detected in beef cattle, beef and dairy products in all years investigated. In conclusion, TaqMan based real-time PCR provides a fast and accurate tool for detection of mcr-1 gene. The overall detection rate of 3.8% for mcr-1 among all E. coli isolates tested is due to high prevalence of mcr-1 in poultry production chains. More epidemiological studies of other European countries are urgently needed to assess German prevalence data

Identification of a blaVIM-1-Carrying IncA/C2 Multiresistance Plasmid in an Escherichia coli Isolate Recovered from the German Food Chain

Within the German national monitoring of zoonotic agents, antimicrobial resistance determination also targets carbapenemase-producing (CP) Escherichia coli by selective isolation from food and livestock. In this monitoring in 2019, the CP E. coli 19-AB01133 was recovered from pork shoulder. The isolate was assigned to the phylogenetic group B1 and exhibited the multi-locus sequence-type ST5869. Molecular investigations, including whole genome sequencing, of 19-AB01133 revealed that the isolate carried the resistance genes blaVIM-1, blaSHV-5 and blaCMY-13 on a self-transmissible IncA/C2 plasmid. The plasmid was closely related to the previously described VIM-1-encoding plasmid S15FP06257_p from E. coli of pork origin in Belgium. Our results indicate an occasional spread of the blaVIM-1 gene in Enterobacteriaceae of the European pig population. Moreover, the blaVIM-1 located on an IncA/C2 plasmid supports the presumption of a new, probably human source of carbapenemase-producing Enterobacteriaceae (CPE) entering the livestock and food chain sector

ChromID® CARBA Agar Fails to Detect Carbapenem-Resistant Enterobacteriaceae With Slightly Reduced Susceptibility to Carbapenems

After first detections of carbapenemase-producing Enterobacteriaceae (CPE) in animals, the European Union Reference Laboratory for Antimicrobial Resistance has provided a protocol for the isolation of carbapenemase-producingEscherichia(E.)colifrom cecum content and meat. Up to now, only few isolates were recovered using this procedure. In our experience, the choice of the selective agar is important for the efficacy of the method. Currently, the use of the prevailing method fails to detect CPE that exhibit a low resistance against carbapenems. Thus, this study aims to evaluate the suitability of selective media with antibiotic supplements and commercial ChromID(R)CARBA agar for a reliable CPE detection. For comparative investigations, detection of freeze-dried carbapenemase-resistant bacteria was studied on different batches of the ChromID(R)CARBA agar as well as on MacConkey agar supplemented with 1 mg/L cefotaxime and 0.125 mg/L meropenem (McC+CTX+MEM). The suitability of the different media was assessed within a time of 25 weeks, starting at least six weeks before expiration of the media. Carbapenem-resistant isolates exhibiting a serine-based hydrolytic resistance mechanism (e.g.,bla(KPC)genes) were consistently detected over 25 weeks on the different media. In contrast, carbapenemase producers with only slightly reduced susceptibility and exhibiting a zinc-catalyzed activity (e.g.,bla(VIM),bla(NDM), andbla(IMP)) could only be cultivated on long-time expired ChromID(R)CARBA, but within the whole test period on McC+CTX+MEM. Thus, ChromID(R)CARBA agar appears to be not suitable for the detection of CPE with slightly increased minimum inhibitory concentrations (MIC) against carbapenems, which have been detected in German livestock and thus, are of main interest in the national monitoring programs. Our data are in concordance with the results of eleven state laboratories that had participated in this study with their ChromID(R)CARBA batches routinely used for the German CPE monitoring. Based on the determined CPE detection rate, we recommend the use of McC+CTX+MEM for monitoring purposes. This study indicates that the use of ChromID(R)CARBA agar might lead to an underestimation of the current CPE occurrence in food and livestock samples

Biocide Susceptibility and Antimicrobial Resistance of Escherichia coli Isolated from Swine Feces, Pork Meat and Humans in Germany

Phenotypic susceptibility testing of Escherichia (E.) coli is an essential tool to gain a better understanding of the potential impact of biocide selection pressure on antimicrobial resistance. We, therefore, determined the biocide and antimicrobial susceptibility of 216 extended-spectrum β-lactamase-producing (ESBL) and 177 non-ESBL E. coli isolated from swine feces, pork meat, voluntary donors and inpatients and evaluated associations between their susceptibilities. Minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) of benzalkonium chloride, chlorhexidine digluconate (CHG), chlorocresol (PCMC), glutaraldehyde (GDA), isopropanol (IPA), octenidine dihydrochloride and sodium hypochlorite (NaOCl) showed unimodal distributions, indicating the absence of bacterial adaptation to biocides due to the acquisition of resistance mechanisms. Although MIC95 and MBC95 did not vary more than one doubling dilution step between isolates of porcine and human origin, significant differences in MIC and/or MBC distributions were identified for GDA, CHG, IPA, PCMC and NaOCl. Comparing non-ESBL and ESBL E. coli, significantly different MIC and/or MBC distributions were found for PCMC, CHG and GDA. Antimicrobial susceptibility testing revealed the highest frequency of resistant E. coli in the subpopulation isolated from inpatients. We observed significant but weakly positive correlations between biocide MICs and/or MBCs and antimicrobial MICs. In summary, our data indicate a rather moderate effect of biocide use on the susceptibility of E. coli to biocides and antimicrobials

Isolation Procedure for CP E. coli from Caeca Samples under Review towards an Increased Sensitivity

Due to the increasing reports of carbapenemase-producing Enterobacteriaceae (CPE) from livestock in recent years, the European Reference Laboratory for Antimicrobial Resistances (EURL-AR) provided a protocol for their recovery from caecum and meat samples. This procedure exhibited limitations for the detection of CPE with low carbapenem MIC values. Therefore, it was modified by a second, selective enrichment in lysogeny broth with cefotaxime (CTX 1 mg/L) and with meropenem (MEM 0.125 mg/L) at 37 °C under microaerophilic conditions. By Real-time PCR, these enrichments are pre-screened for the most common carbapenemase genes. Another adaptation was the use of in-house prepared MacConkey agar with MEM and MEM+CTX instead of commercial selective agar. According to the EURL-method, we achieved 100% sensitivity and specificity using the in-house media instead of commercial agar, which decreased the sensitivity to ~75%. Comparing the method with and without the second enrichment, no substantial influence on sensitivity and specificity was detected. Nevertheless, this enrichment has simplified the CPE-isolation regarding the accompanying microbiota and the separation of putative colonies. In conclusion, the sensitivity of the method can be increased with slight modifications

Azithromycin resistance in Escherichia coli and Salmonella from food-producing animals and meat in Europe.

OBJECTIVES To characterize the genetic basis of azithromycin resistance in Escherichia coli and Salmonella collected within the EU harmonized antimicrobial resistance (AMR) surveillance programme in 2014-18 and the Danish AMR surveillance programme in 2016-19. METHODS WGS data of 1007 E. coli [165 azithromycin resistant (MIC > 16 mg/L)] and 269 Salmonella [29 azithromycin resistant (MIC > 16 mg/L)] were screened for acquired macrolide resistance genes and mutations in rplDV, 23S rRNA and acrB genes using ResFinder v4.0, AMRFinder Plus and custom scripts. Genotype-phenotype concordance was determined for all isolates. Transferability of mef(C)-mph(G)-carrying plasmids was assessed by conjugation experiments. RESULTS mph(A), mph(B), mef(B), erm(B) and mef(C)-mph(G) were detected in E. coli and Salmonella, whereas erm(C), erm(42), ere(A) and mph(E)-msr(E) were detected in E. coli only. The presence of macrolide resistance genes, alone or in combination, was concordant with the azithromycin-resistant phenotype in 69% of isolates. Distinct mph(A) operon structures were observed in azithromycin-susceptible (n = 50) and -resistant (n = 136) isolates. mef(C)-mph(G) were detected in porcine and bovine E. coli and in porcine Salmonella enterica serovar Derby and Salmonella enterica 1,4, [5],12:i:-, flanked downstream by ISCR2 or TnAs1 and associated with IncIγ and IncFII plasmids. CONCLUSIONS Diverse azithromycin resistance genes were detected in E. coli and Salmonella from food-producing animals and meat in Europe. Azithromycin resistance genes mef(C)-mph(G) and erm(42) appear to be emerging primarily in porcine E. coli isolates. The identification of distinct mph(A) operon structures in susceptible and resistant isolates increases the predictive power of WGS-based methods for in silico detection of azithromycin resistance in Enterobacterales

Resistance situation in veterinary pathogenic bacteria of the Enterobacteriaceae familiy with special concideration of the fluoroquinolones

1 Einleitung - 5 - 2 Vorstellung der Publikationen - 12 - 3 Diskussion - 19 - 3.1 Resistenzraten - 19 - 3.1.1 Resistenzen in aus Schweinen isolierten Enterobacteriaceae - 21 - 3.1.1.1 Infektionen des Urogenitaltrakts (inklusive Mastitis-Metritis- Agalaktie-Syndrom) - 21 - 3.1.2 Resistenzen in aus Pferden isolierten Enterobacteriaceae - 23 - 3.1.2.1 Infektionen des Genitaltrakts - 23 - 3.1.3 Resistenzen in aus Hunden und Katzen isolierten Enterobacteriaceae - 25 - 3.1.3.1 Infektionen des Respirationstrakts - 25 - 3.1.3.2 Infektionen des Urogenitaltraks - 26 - 3.1.3.3 Infektionen von Haut/Ohr/Maul - 27 - 3.1.3.4 Infektionen des Gastrointestinaltrakts - 29 - 3.1.4 Fluorchinolon- Resistenz - 29 - 3.2 Aktivitätsvergleich der Fluorchinolone - 32 - 4 Schlussfolgerung - 34 - 5 Zusammenfassung - 36 - 6 Summary - 37 - 7 Literatur - 38 - 8 Anhang - 44 -Die Entwicklung und Ausbreitung von Antibiotikaresistenzen in Bakterien stellt ein immer größer werdendes Problem in Human- und Veterinärmedizin dar. Eine Abschätzung der Resistenzlage, auch in Tieren welche engen Kontakt zum Halter haben, ist aus mehrerlei Hinsicht von Bedeutung. Sie ermöglicht sowohl eine Behandlung der Tiere im Krankheitsfall, als auch die Abschätzung einer eventuellen Gefährdung für den Menschen. Die vorliegenden Studien haben im Rahmen des BfT-GermVet gezeigt, dass die Resistenzraten in aus erkrankten Hobbytieren isolierten E. coli im allgemeinen niedriger sind als die vergleichbarer Isolate aus Menschen. Für Enterobacteriacea der Genera Proteus und Klebsiella sind allgemein höhere Resistenzraten bekannt. Hier zeigten sich in der vorliegenden Studie dennoch niedrigere Werte als in der Humanmedizin, mit der Ausnahme der Resistenzraten gegenüber Fluorchinolonen. Gegenüber diesem Wirkstoff wiesen Klebsiella spp. und Proteus spp. mit bis zu 29 % resistenter Isolate höhere Raten auf als in der Humanmedizin berichtet wurden, E. coli war jedoch deutlich sensibler. Die große Bedeutung der Fluorchinolone gab auch Anlass zum Aktivitätsvergleich verschiedener Fluorchinolone in vitro, um Fehldiagnosen in der Resistenztestung gegenüber diesen Wirkstoffen zu minimieren. Dieser Vergleich zeigte zum Teil deutliche Unterschiede, wobei zusätzlich Variationen zwischen den verschiedenen Bakterienspezies beobachtet werden konnten. Eine Festlegung einer Stellvertretersubstanz für Fluorchinolone ist also abzulehnen. Die hier durchgeführten Studien sind nur eine erste Erhebung der Resistenzsituation in pathogenen Bakterien von Hobbytieren in Deutschland. Um Veränderungen in der Antibiotikaempfindlichkeit dieser Bakterien sehen zu können, möglichst noch bevor sie sich in den Resistenzraten niederschlagen, ist jedoch eine weitere regelmäßige Untersuchung notwendig. Durch die Übernahme in das GERM-Vet Progamm ist diese Beobachtung für einige der untersuchten Kategorien gewährleistet, so daß in Zukunft auch die Entwicklung der Antibiotikaresistenz in Bakterien von Hunden, Katzen und Pferden in Deutschland exakt verfolgt werden kann.Development and spread of bacteria resistant to antimicrobial agents is a major concern to human and veterinary health. Estimation of the resistance situation especially in animals with close contact to their owners is thereby of great importance. On one hand it allows a propper therapy of diseased animals; on the other hand it also gives us the opportunity to estimate a possible hazzard to human health. The present part of the BfT-GermVet study shows that percentages of resistant bacteria from animals are generally lower than those of comparable isolates from humans. Enterobacteriaceae from the genera Proteus and Klebsiella are known to contribute to high percentages of resitant isolates. The present study though shows lower percentages than those known from human medicine with exception of those for Fluoroquinolones. Here percentages of Klebsiella spp. and Proteus spp. with up to 29 % were notably higher than those from humans, while those for E. coli-isolates were notably lower. The important role of Fluoroquinolones also generated the idea to compare the in vitro activity of different Fluoroquinolones in order to minimize false diagnoses in the detection of antimicrobial resistance against these agents. This comparison shows clear differences between the agents, with additional variations between the different bacterial species. The determination of a single Fluoroquinolone as a representative for the whole family is thereby not acceptable. The present studies are only cornerstones in the monitoring of antimicrobial resistance in pathogenic bacteria from companion animals in Germany. Further investigations of these bacteria are needed to show changes in their antimicrobial susceptibility before they have to be classified as resistant. A first step already taken is the inclusion in the GERM-Vet Program. This ensures detailed observations of pathogenic bacteria from dogs, cats and horses in Germany

Plasmid-Coded Linezolid Resistance in Methicillin-Resistant Staphylococcus aureus from Food and Livestock in Germany

Resistance of methicillin-resistant Staphylococcus aureus (MRSA) from food and livestock to last resort antibiotics such as linezolid is highly concerning, since treatment options for infections in humans might be diminished. Known mechanisms of linezolid resistance include point mutations in the 23S rRNA gene and in the ribosomal proteins L3, L4 and L22 as well as an acquisition of the cfr, optrA or poxtA gene. The objective of our study was to characterize antimicrobial resistance (AMR) determinants and phylogenetic relationships among linezolid-resistant (LR-) MRSA from food and livestock. In total, from more than 4000 incoming isolates in the years 2012 to 2021, only two strains from 2015 originating from pig samples exhibited linezolid resistance in the antimicrobial susceptibility testing with MICs of ≥8 mg/L. These LR-MRSA were characterized in detail by whole-genome sequencing and phylogenetic analyses using cgMLST. The LR-MRSA strains showed resistances to ten and eight different antibiotics, respectively. Both strains harbored plasmid-coded cfr genes mediating the linezolid resistance. The cfr genes showed identical sequences in both strains. In addition to the cfr gene, genes for phenicol and clindamycin resistance were detected on the respective plasmids, opening the possibility for a co-selection. The LR-MRSA differed distantly in the phylogenetic analyses and also to other MRSA from pig samples in the year 2015. In conclusion, the occurrence of LR-MRSA in food and livestock seems to be very rare in Germany. However, carriage of plasmids with linezolid resistance determinants could lead to further linezolid-resistant strains by horizontal gene transfer

MRSA in breeding pigs in Germany in 2015

Methicillin resistant Staphylococcus aureus has been known to be prevalent in the pig production for nearly 15 years now (Meemken et al., 2010). In 2008 a survey carried out in the EU determined a high prevalence of MRSA in herds of breeding pigs also in Germany (EFSA, 2010). Likewise, MRSA were identified in Germany in herds of fattening pigs (Alt et al., 2011), pigs at slaughter (Tenhagen et al., 2009), on carcasses (Beneke et al., 2011) and in meat from pigs at retail (BVL, 2013). The current investigation was carried out to determine the current prevalence of MRSA in herds of breeding pigs, analyse patterns in the type of MRSA isolated from pigs and determine differences between the MRSA observed in units only housing sows and those housing weaned piglets. In previous studies it could be shown, that the prevalence of MRSA is higher in weaned piglets than in sows but little is known about differences in the types of MRSA that can be isolated in the different units.</p