Methicillin-Resistant Staphylococcus aureus from Municipal Abattoirs in Nigeria: Showing Highly Similar Clones and Possible Transmission from Slaughter Animals to Humans.

In recent years, methicillin-resistant Staphylococcus aureus (MRSA) has gained interest in veterinary medicine due to its zoonotic potential. Currently, little information is available on the genotypic and virulence characteristics of MRSA isolates detected in Nigerian abattoirs. To better understand the epidemiology of MRSA associated with the abattoir food chain environment in Nigeria, a total of 18 isolates (humans: n = 5, slaughter animals: n = 5, and environment: n = 8), previously spa typed, were recovered and characterized by Staphylococcus cassette chromosome mec (SCCmec) typing, and phenotypic and genotypic antimicrobial susceptibility testing. In addition, 10 of the 18 MRSA strains with a new spa type (t16571) were subjected to multilocus sequence typing. The similarity of strains was analyzed based on the results of the DNA microarray analysis. The 18 MRSA strains harbored two distinct SCCmec types (IVa and V) and belonged to four clonal clusters (CC1, CC7, CC88, and CC152). All MRSA of the new spa type t16571 (n = 10) harbored the SCCmec type IVa. Seven of the MRSA t16571 strains belonged to ST88, while three other strains were assigned to ST3614. The 18 MRSA isolates were categorized into six virulence profiles, and the detection rate for the Panton-Valentine Leukocidin gene was high (33.3%). The antimicrobial susceptibility profiles of the 18 MRSA varied widely between strains, but phenotypic resistance corresponded to relevant resistance genes harbored. The detection of highly similar MRSA strains in slaughter animals, abattoir workers, and the environment underlines the need to use adequate measures at Nigerian abattoirs to prevent further spread and transmission of MRSA to humans or food.

Contamination of home-grown and retail vegetables with Clostridioidesdifficile.

The opportunistic intestinal pathogen Clostridioides difficile is the number one cause of nosocomial diarrhea in humans. In this study, C. difficile was isolated from 26.7% of potatoes and 1.9% of salads from German retail. The majority of strains possessed toxinogenic PCR-ribotypes that are associated with human clinical cases pointing towards a potential risk to human health.

Microarray-based detection of resistance genes in coagulase-negative staphylococci isolated from cattle and buffalo with mastitis in Egypt.

The present study aimed to provide a detailed characterization of coagulase-negative staphylococci (CoNS) isolated from cows and buffaloes with mastitis. The study included seventy-five CoNS isolates (60 came from cattle and 15 from buffaloes) originating from 68 individual quarters of 67 dairy cows (53 cattle and 14 buffaloes). The animals belonged to five different small holding dairy herds (n = 140 cows) that show clinical or subclinical mastitis. CoNS isolates were phenotypically characterized using MALDI-TOF-MS and were further genotypically characterized by microarray-based assays. Furthermore, the antimicrobial susceptibility of CoNS strains which carried the mecA gene was examined by broth microdilution. The occurrence of CoNS in the respective five herds was 10.5%, 14.7%, 14.8%, 12.8%, and 9.9%, with an average of 12.4%. Six different CoNS species were identified: S. sciuri (n = 37; 30 from cattle and 7 from buffaloes), S. chromogenes (n = 14; 8 from cattle and 6 from buffaloes), S. haemolyticus (n = 10; nine from cattle and one buffalo), S. xylosus (n = 10; nine from cattle and one buffalo), S. hyicus (n = 2), S. warneri (n = 1), and unidentified CoNS (n = 1). Twenty percent (20%) of CoNS isolates (17.3% of cattle origin) carried at least one antimicrobial resistance gene, while 4% of the isolate including two isolates of S. haemolyticus and one S. warneri of cattle origin carried the mecA gene and were phenotypically identified as methicillin-resistant strains. The genes detected were blaZ (16%), followed by tet(K) (8%), aacA-aphD (4%), aphA3 (2.6%), msr(A) (2.6%), [far1 (2.6%), and fusC (2.6%)], sat (2.6%), and cat (1.3%) conferring resistance to penicillin, tetracycline, gentamicin, neomycin/kanamycin, erythromycin, fusidic acid, streptothricin, and chloramphenicol, respectively. The majority of investigated CoNS strains displayed considerably low prevalence of resistance genes, while resistance to more than three antibiotics was found in S. haemolyticus and S. warneri. Implementing effective preventive measures is, therefore, important for limiting the transmission of CoNS, rather than using antibiotics to control mastitis in bovines.

High toxinogenic potential of Staphylococcus aureus from wild ungulates in Brandenburg, Germany with a low level of antibiotic resistance.

Introduction: Data regarding the occurrence and virulence of Staphylococcus (S.) aureus in wild living animals is rare. However, S. aureus may carry a multitude of virulence factors and express resistance to several antimicrobial substances. Handling game meat may thus lead to serious infections or food poisoning. The aim of this study was to provide insights into the occurrence and characteristics of S. aureus in wild ungulates from Brandenburg, Germany. Methods: Nasal swabs of externally healthy-looking wild boars, roe, fallow and red deer were collected in hunts during season 2021/2022 and analyzed for S. aureus by selective enrichment. Species were determined using matrix assisted laser desorption ionization mass spectrometry and tested for phenotypic antimicrobial resistance. Whole-genome sequencing was conducted for genotyping, determination of virulence associated genes and analysis of phylogenetic relationships. Results: S. aureus were recovered from approximately 8% of nasal swabs. However, the strains were only obtained from the sampled wild ruminants. S. aureus isolates were associated with sequence types (ST) 1, ST30, ST133, ST425, ST582 and ST6238. Isolates of ST1 clustered closely together in the phylogenetic analysis. Genes encoding staphylococcal enterotoxin (SE) or SE-like (SEl) were found in 14/17 isolates. In particular, a seh gene was present in 12/17 isolates. Moreover, two isolates harbored a multiplicity of genes encoding SE or SEl. In addition, the toxic shock syndrome toxin encoding tst gene was detected in one isolate. This isolate was resistant to penicillin and cefoxitin and accordingly harbored the blaZ gene. Discussion: Wild ungulates intended for human consumption may carry potentially virulent S. aureus. In one case, the close phylogenetic relationship of S. aureus isolates indicates a possible intraspecific spread within a common territory. However, for others, the origin or the spread pattern can only be inferred. Handling of animals or their carcasses might contribute to staphylococcal infections in humans. Moreover, food poisoning due to SE producing strains may occur, if recommended hygiene practices are not applied during processing of game meat.

Genomic study of European Clostridioides difficile ribotype 002/sequence type 8.

Clostridioides difficile has significant clinical importance as a leading cause of healthcare-associated infections, with symptoms ranging from mild diarrhoea to severe colitis, and possible life-threatening complications. C. difficile ribotype (RT) 002, mainly associated with MLST sequence type (ST) 8, is one of the most common RTs found in humans. This study aimed at investigating the genetic characteristics of 537 C. difficile genomes of ST8/RT002. To this end, we sequenced 298 C. difficile strains representing a new European genome collection, with strains from Germany, Denmark, France and Portugal. These sequences were analysed against a global dataset consisting of 1,437 ST8 genomes available through Enterobase. Our results showed close genetic relatedness among the studied ST8 genomes, a diverse array of antimicrobial resistance (AMR) genes and the presence of multiple mobile elements. Notably, the pangenome analysis revealed an open genomic structure. ST8 shows relatively low overall variation. Thus, clonal isolates were found across different One Health sectors (humans, animals, environment and food), time periods, and geographical locations, suggesting the lineage's stability and a universal environmental source. Importantly, this stability did not hinder the acquisition of AMR genes, emphasizing the adaptability of this bacterium to different selective pressures. Although only 2.4 % (41/1,735) of the studied genomes originated from non-human sources, such as animals, food, or the environment, we identified 9 cross-sectoral core genome multilocus sequence typing (cgMLST) clusters. Our study highlights the importance of ST8 as a prominent lineage of C. difficile with critical implications in the context of One Health. In addition, these findings strongly support the need for continued surveillance and investigation of non-human samples to gain a more comprehensive understanding of the epidemiology of C. difficile.

MRSA in bulk tank milk of dairy herds in Germany - changes over time.

OBJECTIVE: Methicillin-resistant Staphylococcus aureus (MRSA) have repeatedly been isolated from dairy herds. It was the purpose of this study to compare the results of 3 subsequent national scale cross-sectional investigations in dairy herds in Germany on the prevalence of MRSA in bulk tank milk and the characteristics of the isolates. MATERIAL AND METHODS: The investigations were carried out in 2010, 2014 and 2019, respectively. MRSA were isolated from 25 ml of bulk tank milk using a double selective enrichment protocol. Samples were distributed across the country according to the regional dairy cattle population. RESULTS: The prevalence of MRSA in bulk tank milk samples was lower in 2010 than in 2014 and tended to decrease until 2019. Prevalence was higher in samples from conventional than from organic herds and increased with herd size. Most isolates (75/78) were assigned to the clonal complex 398 and the spa-types t011 and t034. Resistance of the isolates to other antimicrobials than beta-lactams decreased over time. CONCLUSIONS: MRSA remain present in the German dairy population and are found more frequently in larger vs. smaller herds and in conventional vs. organic herds. CLINICAL RELEVANCE: MRSA should be considered in biosecurity protocols and with respect to occupational health of farm staff. Presence of MRSA in raw milk supports the recommendation not to drink unpasteurized raw milk.

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.

Evaluation of the Occurrence of Staphylococcaceae with Reduced Susceptibility to Cefoxitin in Wild Ungulates in Brandenburg, Germany, Based on Land Use-Related Factors.

Interactions between natural and human-used environments have a significant influence on the spread of antimicrobial resistance in wild ecosystems. Despite current knowledge, fundamental questions about the degree of impact of land use-related factors on the spread of antimicrobial-resistant staphylococci in European wild game animal populations have not yet been answered with certainty. In this study, we evaluated the occurrence of Staphylococcaceae showing reduced susceptibility to cefoxitin in nasal swabs of fallow deer (Dama dama), red deer (Cervus elaphus), roe deer (Capreolus capreolus), and wild boar (Sus scrofa) hunted in Brandenburg, Germany. Evaluations were focused on the use of open-source data regarding the extent as well as the degree of land use, especially for settlement or animal husbandry. Results showed that the detection rate of Staphylococcaceae showing a non-wild-type phenotype for cefoxitin differed between animal species of the studied hunting districts. Statistical analyses of results combined with data on land use features revealed that a high density of cattle or poultry in a county may be associated with an increased detection rate in roe deer or wild boar, respectively. Furthermore, positive correlations were determined between the prevalence of non-wild-type Staphylococcaceae in roe deer or fallow deer and the proportional extent of surface water bodies in the corresponding area. The presented approach establishes a general basis for a risk-oriented assessment of the effects of human activities on the epidemiology of transmissible microorganisms in the human-animal-environment interface, including antimicrobial-resistant bacteria. IMPORTANCE Intensive research regarding the impact of land use-related factors on the prevalence and distribution of antimicrobial-resistant Staphylococcaceae in game ungulate populations is necessary for adequately determining risks related to interactions between wild animals, domestic animals, and humans in common geographic locations. This systematic approach for the analysis of the observations in specific hunting districts of Brandenburg, Germany, adds an innovative value to the research strategy of antimicrobial resistance in wild game animals, which is in accordance with current recommendations worldwide. Thus, results and information obtained in this study build a relevant foundation for future risk assessment regarding the safety of game products. Furthermore, the data generated represent an important basis for improving existing guidelines in land use practices and hunting practices. The use of existing open source data collections provided by official governmental and nongovernmental entities increases not only the impact but also the applicability and comparability of information beyond the regional level.

Mammaliicoccus spp. from German Dairy Farms Exhibit a Wide Range of Antimicrobial Resistance Genes and Non-Wildtype Phenotypes to Several Antibiotic Classes.

Mammaliicocci might play a major role in antimicrobial resistance (AMR) gene transmission between organisms of the family Staphylococcaceae, such as the potentially pathogenic species Staphylococcus aureus. The interest of this study was to analyze AMR profiles of mammaliicocci from German dairy farms to evaluate the AMR transmission potential. In total, 65 mammaliicocci isolates from 17 dairy farms with a history of MRSA detection were analyzed for AMR genotypes and phenotypes using whole genome sequencing and antimicrobial susceptibility testing against 19 antibiotics. The various genotypic and phenotypic AMR profiles of mammaliicocci from German dairy farms indicated the simultaneous occurrence of several different strains on the farms. The isolates exhibited a non-wildtype phenotype to penicillin (58/64), cefoxitin (25/64), chloramphenicol (26/64), ciprofloxacin (25/64), clindamycin (49/64), erythromycin (17/64), fusidic acid (61/64), gentamicin (8/64), kanamycin (9/64), linezolid (1/64), mupirocin (4/64), rifampicin (1/64), sulfamethoxazol (1/64), streptomycin (20/64), quinupristin/dalfopristin (26/64), tetracycline (37/64), tiamulin (59/64), and trimethoprim (30/64). Corresponding AMR genes against several antimicrobial classes were detected. Linezolid resistance was associated with the cfr gene in the respective isolate. However, discrepancies between genotypic prediction and phenotypic resistance profiles, such as for fusidic acid and tiamulin, were also observed. In conclusion, mammaliicocci from dairy farms may carry a broad variety of antimicrobial resistance genes and exhibit non-wildtype phenotypes to several antimicrobial classes; therefore, they may represent an important source for horizontal gene transfer of AMR genes to pathogenic Staphylococcaceae.

Prevalence and diversity of Staphylococcus aureus in the Zambian dairy value chain: A public health concern.

Staphylococcus aureus is an important opportunistic pathogen of both humans and animals. It can cause several diseases, including mastitis, as well as food poisoning by production of heat-stable enterotoxins in food. The aim of this study was to determine the prevalence of S. aureus and the diversity of strains circulating in the Zambian dairy value chain, which have not been studied in detail before. Three provinces were covered by the study (Lusaka, Southern, and Western) and almost 2000 samples along the dairy value chain, covering both the informal and formal market sectors, were taken at two time points (dry and wet season), with a special focus on raw milk. Nearly 300 presumptive S. aureus isolates were confirmed by MALDI-TOF MS and real-time PCR. Raw milk from traditional and smallholder farms was widely contaminated with S. aureus; prevalence was 33-46% depending on the study province. Raw milk from milk collection centres, informal traders, traditional market sellers, and processors were also frequently contaminated with S. aureus. In addition, S. aureus was detected in several milk bucket swabs and nasal and hand swabs of milkers. From industrially processed (heat-treated) milk and dairy products, no S. aureus was isolated. Methicillin-resistant S. aureus (MRSA) were not detected, but around 10% of the S. aureus isolates carried lukS-PV, a marker gene for the virulence factor Pantone-Valentine leucocidin (PVL), which has been associated with severe diseases in human. Molecular typing identified a total of 44 spa types including 13 novel types: t18396, t18397, t18398, t18399, t18400, t18402, t18416, t20459, t20460, t20461, t20462, t20463, and t20464. Furthermore, 12 novel multi-locus sequence types were identified: ST7012, ST7100, ST7101, ST7177, ST7291, ST7304, ST7305, ST7344, ST7596, ST7597, ST7598, and ST7599, of which ST7012, ST7177, and ST7596 fall into the bovine-associated clonal linage CC97. The spa types t084, t267, t355, and the novel type t20464 were common in all three study provinces. The predominant spa type varied depending on the province. Whole genome sequencing (WGS) and core genome multi-locus sequence typing (cgMLST) indicates transmission of strains along the Zambian dairy chain with possible persistence in the chain over time. cgMLST also revealed a very close relatedness between some isolates from milkers and from raw milk or milk buckets. The high prevalence and wide spa type diversity of S. aureus, as well as possible direct or indirect transmission of (potentially highly virulent) S. aureus to humans along the Zambian dairy value chain, are of public health concern, particularly as milk and milk products are often consumed raw by the Zambian population.

A virulence factor as a therapeutic: the probiotic Enterococcus faecium SF68 arginine deiminase inhibits innate immune signaling pathways.

The probiotic bacterial strain Enterococcus faecium SF68 has been shown to alleviate symptoms of intestinal inflammation in human clinical trials and animal feed supplementation studies. To identify factors involved in immunomodulatory effects on host cells, E. faecium SF68 and other commensal and clinical Enterococcus isolates were screened using intestinal epithelial cell lines harboring reporter fusions for NF-κB and JNK(AP-1) activation to determine the responses of host cell innate immune signaling pathways when challenged with bacterial protein and cell components. Cell-free, whole-cell lysates of E. faecium SF68 showed a reversible, inhibitory effect on both NF-κB and JNK(AP-1) signaling pathway activation in intestinal epithelial cells and abrogated the response to bacterial and other Toll-like receptor (TLR) ligands. The inhibitory effect was species-specific, and was not observed for E. avium, E. gallinarum, or E. casseliflavus. Screening of protein fractions of E. faecium SF68 lysates yielded an active fraction containing a prominent protein identified as arginine deiminase (ADI). The E. faecium SF68 arcA gene encoding arginine deiminase was cloned and introduced into E. avium where it conferred the same NF-κB inhibitory effects on intestinal epithelial cells as seen for E. faecium SF68. Our results indicate that the arginine deiminase of E. faecium SF68 is responsible for inhibition of host cell NF-κB and JNK(AP-1) pathway activation, and is likely to be responsible for the anti-inflammatory and immunomodulatory effects observed in prior clinical human and animal trials. The implications for the use of this probiotic strain for preventive and therapeutic purposes are discussed.

Metabolic Characteristics of Porcine LA-MRSA CC398 and CC9 Isolates from Germany and China via Biolog Phenotype MicroArrayTM.

Livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) is an important zoonotic pathogen, often multi-resistant to antimicrobial agents. Among swine, LA-MRSA of clonal complex (CC) 398 dominates in Europe, Australia and the Americas, while LA-MRSA-CC9 is the main epidemic lineage in Asia. Here, we comparatively investigated the metabolic properties of rare and widespread porcine LA-MRSA isolates from Germany and China using Biolog Phenotype MicroArray technology to evaluate if metabolic variations could have played a role in the development of two different epidemic LA-MRSA clones in swine. Overall, we were able to characterize the isolates' metabolic profiles and show their tolerance to varying environmental conditions. Sparse partial least squares discriminant analysis (sPLS-DA) supported the detection of the most informative substrates and/or conditions that revealed metabolic differences between the LA-MRSA lineages. The Chinese LA-MRSA-CC9 isolates displayed unique characteristics, such as a consistently delayed onset of cellular respiration, and increased, reduced or absent usage of several nutrients. These possibly unfavorable metabolic properties might promote the ongoing gradual replacement of the current epidemic LA-MRSA-CC9 clone in China with the emerging LA-MRSA-CC398 lineage through livestock trade and occupational exposure. Due to the enhanced pathogenicity of the LA-MRSA-CC398 clone, the public health risk posed by LA-MRSA from swine might increase further.

Closely related Campylobacter jejuni strains from different sources reveal a generalist rather than a specialist lifestyle.

BACKGROUND: Campylobacter jejuni and Campylobacter coli are human intestinal pathogens of global importance. Zoonotic transmission from livestock animals or animal-derived food is the likely cause for most of these infections. However, little is known about their general and host-specific mechanisms of colonization, or virulence and pathogenicity factors. In certain hosts, Campylobacter species colonize persistently and do not cause disease, while they cause acute intestinal disease in humans. RESULTS: Here, we investigate putative host-specificity using phenotypic characterization and genome-wide analysis of genetically closely related C. jejuni strains from different sources. A collection of 473 fresh Campylobacter isolates from Germany was assembled between 2006 and 2010 and characterized using MLST. A subset of closely related C. jejuni strains of the highly prevalent sequence type ST-21 was selected from different hosts and isolation sources. PCR typing of strain-variable genes provided evidence that some genes differed between these strains. Furthermore, phenotypic variation of these strains was tested using the following criteria: metabolic variation, protein expression patterns, and eukaryotic cell interaction. The results demonstrated remarkable phenotypic diversity within the ST-21 group, which however did not correlate with isolation source. Whole genome sequencing was performed for five ST-21 strains from chicken, human, bovine, and food sources, in order to gain insight into ST-21 genome diversity. The comparisons showed extensive genomic diversity, primarily due to recombination and gain of phage-related genes. By contrast, no genomic features associated with isolation source or host were identified. CONCLUSIONS: The genome information and phenotypic data obtained in vitro and in a chicken infection model provided little evidence of fixed adaptation to a specific host. Instead, the dominant C. jejuni ST-21 appeared to be characterized by phenotypic flexibility and high genetic microdiversity, revealing properties of a generalist. High genetic flexibility might allow generalist variants of C. jejuni to reversibly express diverse fitness factors in changing environments.

Phylogenetic Tracking of LA-MRSA ST398 Intra-Farm Transmission among Animals, Humans and the Environment on German Dairy Farms.

Methicillin-resistant Staphylococcus aureus (MRSA) are a major threat to human and animal health, causing difficult-to-treat infections. The aim of our study was to evaluate the intra-farm transmission of livestock-associated (LA) MRSA sequence type (ST) 398 isolates on German dairy farms. A total of 115 LA-MRSA ST398 isolates originating from animals, humans and the environment of six dairy farms were analyzed by whole-genome sequencing and core genome multilocus sequence typing. Phylogenetic clusters of high allelic similarity were detected on all dairy farms, suggesting a MRSA transmission across the different niches. On one farm, closely related isolates from quarter milk samples (QMS), suckers of calf feeders and nasal cavities of calves indicate that MRSA may be transferred by feeding contaminated milk to calves. Detection of related MRSA isolates in QMS and teat cups (4/6 farms) or QMS and human samples (3/4 farms) pointed out a transmission of MRSA between cows during the milking process and a potential zoonotic risk. In conclusion, LA-MRSA ST398 isolates may spread between animals, humans and the environment on dairy farms. Milking time hygiene and other internal biosecurity measures on farms and pre-treatment of milk before feeding it to calves may reduce the risk of MRSA transmission.

Prevalence and phylogenetic relationship of Clostridioides difficile strains in fresh poultry meat samples processed in different cutting plants.

Clostridioides difficile is one of the most frequent causes of nosocomial infections in humans leading to (antibiotic-associated) diarrhea and severe pseudomembranous colitis. With an increasing frequency, C. difficile infections (CDI) are also observed independently of hospitalization and the age of the patients in an ambulant setting. One potential source of so-called community-acquired CDI is a zoonotic transmission to humans based on direct contact with animals or the consumption of food. To estimate the exposure of humans with C. difficile via food, we screened 364 different retail fresh poultry meat products purchased in Berlin and Brandenburg, Germany and further characterized the isolates. None of the 42 turkey or chicken meat samples without skin was contaminated. However, 51 (15.8%) of 322 tested fresh chicken meat samples with skin were C. difficile-positive. The vast majority (84.3%) of all isolates exhibited toxin genes tcdA and tcdB, whereas the binary toxin cdtA/B was absent. Most of the isolates (50/51) were susceptible to all six investigated antimicrobials. However, one non-toxigenic strain was multidrug resistant to the antimicrobials clindamycin and erythromycin. The isolates were mainly represented by PCR-ribotypes (RT) 001, RT002, RT005, and RT014, which were already associated with human CDI cases in Germany and were partially detected in poultry. The relatively high contamination rate of fresh retail chicken meat with skin purchased in Germany indicates chicken meat as a potential source of human infections. Moreover, we identified cutting plants with a higher rate of a C. difficile-contamination (21.4-32.8%). To compare the phylogenetic relationship of the isolated strains from certain cutting plants over several months in 2018 and 2019, we analyzed them using NGS followed by core genome MLST. Interestingly, highly related strains (0-3 alleles distance) of common clinical RT001 and RT002 isolates, as well as of the non-toxigenic RT205 isolates were detectable in same cutting plants over a period of three and 16 months, respectively.The continuous contamination with the same strain could be explained by the longterm persistence of this strain within the cutting plant (e.g., within the scalder), or with a recurring entry e.g. from the same fattening farm.

Multidrug-resistant Staphylococcus cohnii and Staphylococcus urealyticus isolates from German dairy farms exhibit resistance to beta-lactam antibiotics and divergent penicillin-binding proteins.

Non-aureus staphylococci are commonly found on dairy farms. Two rarely investigated species are Staphylococcus (S.) cohnii and S. urealyticus. Since multidrug-resistant S. cohnii and S. urealyticus are known, they may serve as an antimicrobial resistance (AMR) gene reservoir for harmful staphylococcal species. In our study, nine S. cohnii and six S. urealyticus isolates from German dairy farms were analyzed by whole-genome sequencing and AMR testing. The isolates harbored various AMR genes (aadD1, str, mecA, dfrC/K, tetK/L, ermC, lnuA, fexA, fusF, fosB6, qacG/H) and exhibited non-wildtype phenotypes (resistances) against chloramphenicol, clindamycin, erythromycin, fusidic acid, rifampicin, streptomycin, tetracycline, tiamulin and trimethoprim. Although 14/15 isolates lacked the blaZ, mecA and mecC genes, they showed reduced susceptibility to a number of beta-lactam antibiotics including cefoxitin (MIC 4-8 mg/L) and penicillin (MIC 0.25-0.5 mg/L). The specificity of cefoxitin susceptibility testing for mecA or mecC gene prediction in S. cohnii and S. urealyticus seems to be low. A comparison with penicillin-binding protein (PBP) amino acid sequences of S. aureus showed identities of only 70-80% with regard to PBP1, PBP2 and PBP3. In conclusion, S. cohnii and S. urealyticus from selected German dairy farms show multiple resistances to antimicrobial substances and may carry unknown antimicrobial resistance determinants.

Genomic Distinctions of LA-MRSA ST398 on Dairy Farms From Different German Federal States With a Low Risk of Severe Human Infections.

Methicillin-resistant Staphylococcus aureus (MRSA) have been found on German dairy farms and may be the cause of difficult-to-treat bovine mastitis. Considering the one health approach, MRSA might be transmitted from animals to humans raising the risk for severe infections. On 17 German dairy farms with a history of MRSA detection, MRSA strains were isolated from quarter milk, bulk tank milk, and swab samples of calves, heifers, pigs, and the environment. A selection of 33 isolates was analyzed using whole-genome sequencing and antimicrobial resistance testing. All detected MRSA strains were attributed to the livestock-associated sequence type 398. Methicillin-resistance was associated with the mecA gene in the staphylococcal cassette chromosome (SCC)mec types IVa (7/33) or V (26/33). The MRSA strains across the German federal states showed large allelic differences indicating independent development and distribution. On one farm, a clonal MRSA isolate was widely spread among different animals and the milking equipment. Moreover, MRSA transmission between two dairy farms in one federal state seems to be likely. In depth studies indicated that the resistance gene prediction and phenotypic resistance are in good agreement. Twenty eight strains were determined to exhibit a non-wildtype phenotype (resistant) against up to seven antimicrobial substances with an overall resistance to ß-lactams and tetracycline. Ten different phenotypic antimicrobial resistance patterns were found among the MRSA strains. The strains harbored a wide virulence gene repertoire, of which some of them are related to bovine mastitis. However, the isolates lacked typical human infection associated factors such as the immune evasion cluster genes, staphylococcal enterotoxin genes, or Panton-Valentine leukocidin genes leading to the assumption for a low risk for severe human infections and foodborne diseases.

Antimicrobial resistance pattern and virulence profile of S. aureus isolated from household cattle and buffalo with mastitis in Egypt.

Methicillin resistant S. aureus from cows with mastitis has received a growing interest worldwide. The present study aimed to provide a detailed description of the resistance and virulence traits of isolates from bovine mastitis samples. A total of 550 quarter milk samples were collected from 140 mastitic household dairy cows and buffalo from five herds at Dakahlia Governorate, Egypt, during 2017 and 2018. Staphylococcus spp. were isolated and differentiated using MALDI-TOF MS. A genotypic characterization was performed for S. aureus isolates using DNA-microarray and staphylococcal protein A (spa) typing. Furthermore, antibiotic resistances were phenotypically confirmed using broth microdilution. Six different clonal lineages (CC1-MRSA, CC5-MRSA, CC45-MRSA, CC97-MSSA, CC50-MSSA and CC1153-MSSA), including seven spa types (t127, t688, t132, t267, t521, t224 and t903) were identified. Spa type t267 was the most dominant among the investigated herds. This is the first report of the occurrence of clonal lineages CC97, CC1, CC45, CC50 and CC1153 from bovine mastitis in Egypt. All MRSA isolates and 33.3 % of MSSA were multi-resistant (i.e. resistant to more than three classes of compounds). Various virulence determinants were also observed including leukocidins, hemolysins, and enterotoxins. The study demonstrates a low diversity of S. aureus isolates recovered from several dairy herds. The findings of the observed virulotypes can be useful for future studies on anti-virulence therapies, immunogenicity and vaccine development.

Antimicrobial Resistance and Virulence of Methicillin-Resistant Staphylococcus aureus from Human, Chicken and Environmental Samples within Live Bird Markets in Three Nigerian Cities.

Background: Methicillin-resistant Staphylococcus aureus (MRSA) has emerged as a major threat to public health. This study investigated the occurrence of MRSA in humans, chickens, chicken meat and environmental samples within poultry farms and live bird markets in southwestern Nigeria. Methods: MRSA were isolated using selective culture and tested for antimicrobial susceptibility by broth microdilution. Selected isolates were characterized by whole genome sequencing (WGS). From WGS data, spa, dru, multilocus sequence typing (MLST) and SCCmec types, but also virulence and antimicrobial resistance genes, were identified. Results: Fifty-six MRSA isolates were detected in 734 samples. They showed resistance to ß-lactams (100%), tetracycline (60.7%), ciprofloxacin (33.9%), erythromycin (28.6%), gentamicin (32.1%), and trimethoprim/sulfamethoxazole (10.7%). All 30 isolates investigated by WGS carried mecA, dfrG, and tet(38) genes. Other resistance genes detected were blaZ (83.3%), fosB (73.3%), tet(K) (60.0%), aacA-aphD (36.6%), aphA3 (33.3%), msr(A) (30.0%), mph(C) (30.0%), dfrS1 (3.3%), and sat4 (3.3%). Seven spa types (t091, t314, t657, t1476, t2331, t4690 and t12236), four known (dt9aw, dt10ao, dt10cj, and dt11a) and two novel (dt10dr and dt11dw) dru types, as well as five sequence types (ST8, ST121, ST152, ST772 and ST789) were found among the MRSA isolates. All ST121 isolates carried an SCCmec type IV cassette and were not dru-typeable. ST152 and ST121 were found only in specific sample categories within defined locations, while ST8 and ST772 were distributed across most sample categories and locations. Three SCCmec types, IVa, V and Vc, were identified. All MRSA isolates possessed virulence genes including aur, clpP, coa, fnbA, esaA, hly, hla, ica, isdA, srtB, sspA, and vWbp, among others. The toxic shock syndrome toxin gene (tst) was not detected in any isolate, whereas the Pantone-Valentine leukocidin genes lukF-PV/lukS-PV were present in all ST121, all ST772, and all but one ST152 isolates. Conclusion: The results of this study (i) showed that chicken meat is contaminated by MRSA and (ii) suggested that live bird markets may serve as focal points for the dissemination of MRSA within the community.

The mycobacterial DNA-binding protein 1 (MDP1) from Mycobacterium bovis BCG influences various growth characteristics.

BACKGROUND: Pathogenic mycobacteria such as M. tuberculosis, M. bovis or M. leprae are characterised by their extremely slow growth rate which plays an important role in mycobacterial virulence and eradication of the bacteria. Various limiting factors influence the generation time of mycobacteria, and the mycobacterial DNA-binding protein 1 (MDP1) has also been implicated in growth regulation. Our strategy to investigate the role of MDP1 in mycobacterial growth consisted in the generation and characterisation of a M. bovis BCG derivative expressing a MDP1-antisense gene. RESULTS: The expression rate of the MDP1 protein in the recombinant M. bovis BCG containing the MDP1-antisense plasmid was reduced by about 50% compared to the reference strain M. bovis BCG containing the empty vector. In comparison to this reference strain, the recombinant M. bovis BCG grew faster in broth culture and reached higher cell masses in stationary phase. Likewise its intracellular growth in mouse and human macrophages was ameliorated. Bacterial clumping in broth culture was reduced by the antisense plasmid. The antisense plasmid increased the susceptibility of the bacteria towards Ampicillin. 2-D protein gels of bacteria maintained under oxygen-poor conditions demonstrated a reduction in the number and the intensity of many protein spots in the antisense strain compared to the reference strain. CONCLUSION: The MDP1 protein has a major impact on various growth characteristics of M. bovis BCG. It plays an important role in virulence-related traits such as aggregate formation and intracellular multiplication. Its impact on the protein expression in a low-oxygen atmosphere indicates a role in the adaptation to the hypoxic conditions present in the granuloma.