Molecular Characterization of Chimeric Staphylococcus aureus Strains from Waterfowl.

Staphylococcus aureus is a versatile pathogen that does not only occur in humans but also in various wild and domestic animals, including several avian species. When characterizing S. aureus isolates from waterfowl, isolates were identified as atypical CC133 by DNA microarray analysis. They differed from previously sequenced CC133 strains in the presence of the collagen adhesin gene cna; some also showed a different capsule type and a deviant spa type. Thus, they were subjected to whole-genome sequencing. This revealed multiple insertions of large regions of DNA from other S. aureus lineages into a CC133-derived backbone genome. Three distinct strains were identified based on the size and extent of these inserts. One strain comprised two small inserts of foreign DNA up- and downstream of oriC; one of about 7000 nt or 0.25% originated from CC692 and the other, at ca. 38,000 nt or 1.3% slightly larger one was of CC522 provenance. The second strain carried a larger CC692 insert (nearly 257,000 nt or 10% of the strain's genome), and its CC522-derived insert was also larger, at about 53,500 nt or 2% of the genome). The third strain carried an identical CC692-derived region (in which the same mutations were observed as in the second strain), but it had a considerably larger CC522-like insertion of about 167,000 nt or 5.9% of the genome. Both isolates of the first, and two out of four isolates of the second strain also harbored a hemolysin-beta-integrating prophage carrying "bird-specific" virulence factors, ornithine cyclodeaminase D0K6J8 and a putative protease D0K6J9. Furthermore, isolates had two different variants of SCC elements that lacked mecA/mecC genes. These findings highlight the role of horizontal gene transfer in the evolution of S. aureus facilitated by SCC elements, by phages, and by a yet undescribed mechanism for large-scale exchange of core genomic DNA.

The History of Bovine Genital Campylobacteriosis in the Face of Political Turmoil and Structural Change in Cattle Farming in Germany.

Contagious bovine genital campylobacteriosis (BGC), also known as bovine venereal campylobacteriosis, is a disease relevant to international trade listed by the World Organization for Animal Health (WOAH). It is caused by Campylobacter fetus subsp. venerealis (Cfv), one of three subspecies of Campylobacter fetus. Bulls are the reservoir but BGC may also be spread by artificial insemination (AI). BGC is characterized by severe reproductive losses such as infertility, early embryonic death and abortion with considerable economic losses. This significant economic impact has prompted several countries to adopt stringent eradication and surveillance measures to contain the disease. While there are commercial and autologous vaccines available, scientific evidence for the effectiveness of vaccination is still lacking. In Germany, BCG was already found to be endemic in the 1920s, shortly after the agent and the disease had been described for the first time. It can be assumed that BCG had already circulated uncontrolled for a long time in the predecessor states of Germany, influenced only by the political situation and trading networks of the time. After WW II, BCG was eradicated in the German Democratic Republic due to industrialized cattle production based on AI but it was still endemic at low levels in the Federal Republic of Germany with its diverse cattle production. There has been a steady decline in BGC incidence in re-unified Germany over the past 28 years. A single genetic Cfv lineage was identified which probably emerged in the 19th century and diversified over time. Interestingly, no recurrent cross-border introduction became evident. This review gives insight into the history of bovine genital campylobacteriosis considering the structural change in cattle farming in Germany and reflecting on the political background of the time.

Characterisation of a Staphylococcus aureus Isolate Carrying Phage-Borne Enterotoxin E from a European Badger (Meles meles).

Staphylococcus (S.) aureus colonizes up to 30% of all humans and can occasionally cause serious infections. It is not restricted to humans as it can also often be found in livestock and wildlife. Recent studies have shown that wildlife strains of S. aureus usually belong to other clonal complexes than human strains and that they might differ significantly with regard to the prevalence of genes encoding antimicrobial resistance properties and virulence factors. Here, we describe a strain of S. aureus isolated from a European badger (Meles meles). For molecular characterisation, DNA microarray-based technology was combined with various next-generation sequencing (NGS) methods. Bacteriophages from this isolate were induced with Mitomycin C and characterized in detail by transmission electron microscopy (TEM) and NGS. The S. aureus isolate belonged to ST425 and had a novel spa repeat sequence (t20845). It did not carry any resistance genes. The uncommon enterotoxin gene see was detected in one of its three temperate bacteriophages. It was possible to demonstrate the induction of all three prophages, although only one of them was expected to be capable of excision based on its carriage of the excisionase gene xis. All three bacteriophages belonged to the family Siphoviridae. Minor differences in size and shape of their heads were noted in TEM images. The results highlight the ability of S. aureus to colonize or infect different host species successfully, which can be attributed to a variety of virulence factors on mobile genetic elements, such as bacteriophages. As shown in the strain described herein, temperate bacteriophages not only contribute to the fitness of their staphylococcal host by transferring virulence factors, but also increase mobility among themselves by sharing genes for excision and mobilization with other prophages.

Genomic insight into Campylobacter jejuni isolated from commercial turkey flocks in Germany using whole-genome sequencing analysis.

Campylobacter (C.) jejuni is a zoonotic bacterium of public health significance. The present investigation was designed to assess the epidemiology and genetic heterogeneity of C. jejuni recovered from commercial turkey farms in Germany using whole-genome sequencing. The Illumina MiSeq® technology was used to sequence 66 C. jejuni isolates obtained between 2010 and 2011 from commercial meat turkey flocks located in ten German federal states. Phenotypic antimicrobial resistance was determined. Phylogeny, resistome, plasmidome and virulome profiles were analyzed using whole-genome sequencing data. Genetic resistance markers were identified with bioinformatics tools (AMRFinder, ResFinder, NCBI and ABRicate) and compared with the phenotypic antimicrobial resistance. The isolates were assigned to 28 different sequence types and 11 clonal complexes. The average pairwise single nucleotide-polymorphisms distance of 14,585 SNPs (range: 0-26,540 SNPs) revealed a high genetic distinction between the isolates. Thirteen virulence-associated genes were identified in C. jejuni isolates. Most of the isolates harbored the genes flaA (83.3%) and flaB (78.8%). The wlaN gene associated with the Guillain-Barré syndrome was detected in nine (13.6%) isolates. The genes for resistance to ampicillin (bla OXA), tetracycline [tet(O)], neomycin [aph(3')-IIIa], streptomycin (aadE) and streptothricin (sat4) were detected in isolated C. jejuni using WGS. A gene cluster comprising the genes sat4, aph(3')-IIIa and aadE was present in six isolates. The single point mutation T86I in the housekeeping gene gyrA conferring resistance to quinolones was retrieved in 93.6% of phenotypically fluoroquinolone-resistant isolates. Five phenotypically erythromycin-susceptible isolates carried the mutation A103V in the gene for the ribosomal protein L22 inferring macrolide resistance. An assortment of 13 ß-lactam resistance genes (bla OXA variants) was detected in 58 C. jejuni isolates. Out of 66 sequenced isolates, 28 (42.4%) carried plasmid-borne contigs. Six isolates harbored a pTet-like plasmid-borne contig which carries the tet(O) gene. This study emphasized the potential of whole-genome sequencing to ameliorate the routine surveillance of C. jejuni. Whole-genome sequencing can predict antimicrobial resistance with a high degree of accuracy. However, resistance gene databases need curation and updates to revoke inaccuracy when using WGS-based analysis pipelines for AMR detection.

Sequence Analysis of Novel Staphylococcus aureus Lineages from Wild and Captive Macaques.

Staphylococcus aureus is a widespread and common opportunistic bacterium that can colonise or infect humans as well as a wide range of animals. There are a few studies of both methicillin-susceptible S. aureus (MSSA) and methicillin-resistant S. aureus (MRSA) isolated from monkeys, apes, and lemurs, indicating a presence of a number of poorly or unknown lineages of the pathogen. In order to obtain insight into staphylococcal diversity, we sequenced strains from wild and captive individuals of three macaque species (Macaca mulatta, M. assamensis, and M. sylvanus) using Nanopore and Illumina technologies. These strains were previously identified by microarray as poorly or unknown strains. Isolates of novel lineages ST4168, ST7687, ST7688, ST7689, ST7690, ST7691, ST7692, ST7693, ST7694, ST7695, ST7745, ST7746, ST7747, ST7748, ST7749, ST7750, ST7751, ST7752, ST7753, and ST7754 were sequenced and characterised for the first time. In addition, isolates belonging to ST2990, a lineage also observed in humans, and ST3268, a MRSA strain already known from macaques, were also included into the study. Mobile genetic elements, genomic islands, and carriage of prophages were analysed. There was no evidence for novel host-specific virulence factors. However, a conspicuously high rate of carriage of a pathogenicity island harbouring edinB and etD2/etE as well as a higher number of repeat units within the gene sasG (encoding an adhesion factor) than in human isolates were observed. None of the strains harboured the genes encoding Panton-Valentine leukocidin. In conclusion, wildlife including macaques may harbour an unappreciated diversity of S. aureus lineages that may be of clinical relevance for humans, livestock, or for wildlife conservation, given the declining state of many wildlife populations.

Occurrence, Phenotypic and Molecular Characteristics of Extended-Spectrum Beta-Lactamase-Producing Escherichia coli in Healthy Turkeys in Northern Egypt.

Poultry is one of the most important reservoirs for zoonotic multidrug-resistant pathogens. The indiscriminate use of antimicrobials in poultry production is a leading factor for development and dissemination of antimicrobial resistance. This study aimed to describe the prevalence and antimicrobial resistance of E. coli isolated from healthy turkey flocks of different ages in Nile delta region, Egypt. In the current investigation, 250 cloacal swabs were collected from 12 turkey farms in five governorates in the northern Egypt. Collected samples were cultivated on BrillianceTM ESBL agar media supplemented with cefotaxime (100 mg/L). The E. coli isolates were identified using MALDI-TOF-MS and confirmed by a conventional PCR assay targeting 16S rRNA-DNA. The phenotypic antibiogram against 14 antimicrobial agents was determined using the broth micro-dilution method. DNA-microarray-based assay was applied for genotyping and determination of both, virulence and resistance-associated gene markers. Multiplex real-time PCR was additionally applied for all isolates for detection of the actual most relevant Carbapenemase genes. The phenotypic identification of colistin resistance was carried out using E-test. A total of 26 E. coli isolates were recovered from the cloacal samples. All isolates were defined as multidrug-resistant. Interestingly, two different E. coli strains were isolated from one sample. Both strains had different phenotypic and genotypic profiles. All isolates were phenotypically susceptible to imipenem, while resistant to penicillin, rifampicin, streptomycin, and erythromycin. None of the examined carbapenem resistance genes was detected among isolates. At least one beta-lactamase gene was identified in most of isolates, where blaTEM was the most commonly identified determinant (80.8%), in addition to blaCTX-M9 (23.1%), blaSHV (19.2%) and blaOXA-10 (15.4%). Genes associated with chloramphenicol resistance were floR (65.4%) and cmlA1 (46.2%). Tetracycline- and quinolone-resistance-associated genes tetA and qnrS were detected in (57.7%) and (50.0%) of isolates, respectively. The aminoglycoside resistance associated genes aadA1 (65.4%), aadA2 (53.8%), aphA (50.0%), strA (69.2%), and strB (65.4%), were detected among isolates. Macrolide resistance associated genes mph and mrx were also detected in (53.8%) and (34.6%). Moreover, colistin resistance associated gene mcr-9 was identified in one isolate (3.8%). The class 1 integron integrase intI1 (84.6%), transposase for the transposon tnpISEcp1 (34.6%) and OqxB -integral membrane and component of RND-type multidrug efflux pump oqxB (7.7%) were identified among the isolates. The existing high incidence of ESBL/colistin-producing E. coli identified in healthy turkeys is a major concern that demands prompt control; otherwise, such strains and their resistance determinants could be transmitted to other bacteria and, eventually, to people via the food chain.

Description of Staphylococcal Strains from Straw-Coloured Fruit Bat (Eidolon helvum) and Diamond Firetail (Stagonopleura guttata) and a Review of their Phylogenetic Relationships to Other Staphylococci.

The phylogenetic tree of the Staphylococcus aureus complex consists of several distinct clades and the majority of human and veterinary S. aureus isolates form one large clade. In addition, two divergent clades have recently been described as separate species. One was named Staphylococcus argenteus, due to the lack of the "golden" pigment staphyloxanthin. The second one is S. schweitzeri, found in humans and animals from Central and West Africa. In late 2021, two additional species, S. roterodami and S. singaporensis, have been described from clinical samples from Southeast Asia. In the present study, isolates and their genome sequences from wild Straw-coloured fruit bats (Eidolon helvum) and a Diamond firetail (Stagonopleura guttata, an estrildid finch) kept in a German aviary are described. The isolates possessed staphyloxanthin genes and were closer related to S. argenteus and S. schweitzeri than to S. aureus. Phylogenetic analysis revealed that they were nearly identical to both, S. roterodami and S. singaporensis. We propose considering the study isolates, the recently described S. roterodami and S. singaporensis as well as some Chinese strains with MLST profiles stored in the PubMLST database as different clonal complexes within one new species. According to the principle of priority we propose it should be named S. roterodami. This species is more widespread than previously believed, being observed in West Africa, Southeast Asia and Southern China. It has a zoonotic connection to bats and has been shown to be capable of causing skin and soft tissue infections in humans. It is positive for staphyloxanthin, and it could be mis-identified as S. aureus (or S. argenteus) using routine procedures. However, it can be identified based on distinct MLST alleles, and "S. aureus" sequence types ST2470, ST3135, ST3952, ST3960, ST3961, ST3963, ST3965, ST3980, ST4014, ST4075, ST4076, ST4185, ST4326, ST4569, ST6105, ST6106, ST6107, ST6108, ST6109, ST6999 and ST7342 belong to this species.

Zoonotic bacteria in clinically healthy goats in petting zoo settings of zoological gardens in Germany.

Goats and other small ruminants are frequently used as contact animals in petting zoo settings of zoological gardens. However, they are capable to carry a broad spectrum of zoonotic pathogens without clinical signs. In this study, we analysed the presence of different zoonotic pathogens in 300 clinically healthy goats from 14 zoological gardens in Germany. Rectal and nasal swabs were investigated with a series of cultural and molecular techniques. In addition, vaginal swabs of the 230 female goats were investigated for the presence of Coxiella burnetii by real-time PCR. Antibodies against C. burnetii were tested in milk and serum by ELISA. Campylobacter spp. were found in 22.7%, Shiga-toxigenic Escherichia coli in 20.0% and Arcobacter spp. were found in 1.7% of the tested 300 goats after culture from rectal swabs and subsequent PCR. One sample contained an Escherichia fergusonii isolate with a blaCTX-M-1 -encoded extended-spectrum beta-lactamase phenotype. Neither Yersinia spp. nor Salmonella spp. were found. Nasal swabs of 20.7% of the goats yielded Staphylococcus aureus including one mecC-positive methicillin-resistant isolate. Neither Yersinia spp. nor Salmonella spp. were found, and none of the 230 vaginal swabs was positive for C. burnetii. Attempts to detect dermatophytes failed. In conclusion, a possible risk of transmission of zoonotic bacteria from goats in petting zoos to visitors should be considered. Appropriate information and facilities for hand washing and disinfection should be provided in all zoological gardens using goats as contact animals due to the regular presence of zoonotic bacteria in the collection.

Identification and characterization of multidrug-resistant ESBL-producing Salmonella enterica serovars Kentucky and Typhimurium isolated in Tunisia CTX-M-61/TEM-34, a novel cefotaxime-hydrolysing ß-lactamase of Salmonella.

AIMS: Molecular characterization of extended-spectrum ß-lactamases (ESBLs) among Salmonella Kentucky and Typhimurium isolates: partial sequence analysis of the types of ß-lactamases found in these isolates, clonality, resistance and supposed emergence of ESBL-producing strains. METHODS AND RESULTS: A retrospective study surveyed the ESBLs occurring in a total of 1404 Salmonella Kentucky and Typhimurium isolates collected over a 5-year period in Tunisia. Antimicrobial susceptibility tests, ESBL phenotype determination (double-disc synergy) were performed. Polymerase chain reaction assays were used for the detection of ß-lactamase genes (blaTEM , blaSHV , blaOXA-1 and blaCTX-M ), class 1 and class 2 integrases (intI1 and intI2) and the 3' conserved segment (3'-CS) of class 1 integron (qacEΔ1+sul1). Sequencing of amplicons of ß-lactamase genes was performed. Percentage of 9.8 of the isolates (S. Kentucky = 117, S. Typhimurium = 20) were either resistant to penicillin and had decreased susceptibility to cefotaxime or had a positive double-disc synergy test result. Polymerase chain reaction detected that these isolates harboured one or more ß-lactamase genes (blaTEM , blaSHV , blaOXA-1 or blaCTX-M ). TEM-1, TEM-34, CTX-M15, CTX-M9 and CTX-M61 type ESBLs were identified through sequencing. The novel Salmonella cefotaxime-hydrolysing ß-lactamase, CTX-M61/TEM-34, detected in this study showed the emergence of new CTX-M-type ESBLs in Tunisia. There were found 33 different multidrug resistance (MDR) patterns. CONCLUSION: These findings highlighted the proliferation of ESBLs and MDR in Salmonella Kentucky and Typhimurium isolates from numerous regions and sources in Tunisia, indicating an emerging public health concern. SIGNIFICANCE AND IMPACT OF THE STUDY: For the first time CTX-M-61/TEM-34, a novel cefotaxime-hydrolysing ß-lactamase of Salmonella had been detected.

Genomic epidemiology of Campylobacter fetus subsp. venerealis from Germany.

Campylobacter fetus subsp. venerealis (Cfv) causes bovine genital campylobacteriosis (BGC), a World Organization for Animal Health (WOAH)-listed trade-relevant disease characterized by severe reproductive losses, such as infertility, early embryonic death and abortion in cattle. BGC has significant economic implications that have prompted several countries to adopt stringent eradication and surveillance measures to contain the disease. In Germany, there has been a low incidence of BGC cases over the past 28 years. This study aimed to illustrate the genomic diversity of German Cfv strains isolated from different federal states in Germany. This study analyzed 63 Cfv strains, collected between 1985 and 2015, by whole-genome sequencing and compared them with genome data of 91 international Cfv isolates. The phylogenetic analysis showed that the Cfv population is genetically conserved and has geographic clusters. In Germany, one phylogenetic lineage comprising all strains was identified. This German lineage was part of a subclade that probably emerged in the nineteenth century and diversified over time. The results of this study point to a non-recurrent cross-border introduction of Cfv in Germany. The BGC control interventions in Germany can be considered successful as no outbreaks were reported since 2015.

Testing assembly strategies of Francisella tularensis genomes to infer an evolutionary conservation analysis of genomic structures.

BACKGROUND: We benchmarked sequencing technology and assembly strategies for short-read, long-read, and hybrid assemblers in respect to correctness, contiguity, and completeness of assemblies in genomes of Francisella tularensis. Benchmarking allowed in-depth analyses of genomic structures of the Francisella pathogenicity islands and insertion sequences. Five major high-throughput sequencing technologies were applied, including next-generation "short-read" and third-generation "long-read" sequencing methods. RESULTS: We focused on short-read assemblers, hybrid assemblers, and analysis of the genomic structure with particular emphasis on insertion sequences and the Francisella pathogenicity island. The A5-miseq pipeline performed best for MiSeq data, Mira for Ion Torrent data, and ABySS for HiSeq data from eight short-read assembly methods. Two approaches were applied to benchmark long-read and hybrid assembly strategies: long-read-first assembly followed by correction with short reads (Canu/Pilon, Flye/Pilon) and short-read-first assembly along with scaffolding based on long reads (Unicyler, SPAdes). Hybrid assembly can resolve large repetitive regions best with a "long-read first" approach. CONCLUSIONS: Genomic structures of the Francisella pathogenicity islands frequently showed misassembly. Insertion sequences (IS) could be used to perform an evolutionary conservation analysis. A phylogenetic structure of insertion sequences and the evolution within the clades elucidated the clade structure of the highly conservative F. tularensis.

Molecular Characterization of Staphylococcus aureus Isolated from Human and Food Samples in Northern Algeria.

Staphylococcus aureus is a commensal resident of the skin and nasal cavities of humans and can cause various infections. Some toxigenic strains can contaminate food matrices and cause foodborne intoxications. The present study aimed to provide relevant information (clonal complex lineages, agr types, virulence and antimicrobial resistance-associated genes) based on DNA microarray analyses as well as the origins and dissemination of several circulating clones of 60 Staphylococcus aureus isolated from food matrices (n = 24), clinical samples (n = 20), and nasal carriers (n = 16) in northern Algeria. Staphylococcus aureus were genotyped into 14 different clonal complexes. Out of 60 S. aureus, 13 and 10 isolates belonged to CC1-MSSA and CC97-MSSA, respectively. The CC 80-MRSA-IV was the predominant S. aureus strain in clinical isolates. The accessory gene regulator allele agr group III was mainly found among clinical isolates (70.4%). Panton-Valentine leukocidin genes lukF/lukS-PV were detected in 13.3% of isolates that all belonged to CC80-MRSA. The lukF/S-hlg, hlgA, and hla genes encoding for hemolysins and leucocidin components were detected in all Staphylococcusaureus isolates. Clinical and food isolates harbored more often the antibiotic resistance genes markers. Seventeen (28.3%) methicillin-resistant Staphylococcus aureus carrying the mecA gene localized on a SCCmec type IV element were identified. The penicillinase operon (blaZ/I/R) was found in 71.7% (43/60) of isolates. Food isolates belonging to CC97-MSSA carried several antibiotic resistance genes (blaZ, ermB, aphA3, sat, tetM, and tetK). The results of this study showed that all clones were found in their typical host, but interestingly, some nasal carriers had isolates assigned to CC705 thought to be absent in humans. The detection of MRSA strains among food isolates should be considered as a potential public health risk. Therefore, controlling the antibiotics prescription for a rational use in human and animal infections is mandatory.

Bacterial gastroenteritis caused by the putative zoonotic pathogen Campylobacter lanienae: First reported case in Germany.

Foodborne campylobacteriosis is the most common cause of human bacterial enteritis in Germany. Campylobacter jejuni and Campylobacter coli are the main causative agents for enteric disease, but a number of other species are involved, including rare ones. These rare Campylobacter spp. are emerging zoonotic pathogens in humans due to increasing international movement of supplies, livestock and people. Campylobacter lanienae was first isolated from healthy abattoir workers in Switzerland and at first its pathogenic potential for humans was considered to be low. Recently, the first case of Campylobacter lanienae -associated human enteritis was reported in Canada. Here, we describe a case of mild Campylobacter lanienae -associated enteritis with subsequent asymptomatic excretion in a butcher. The isolate is available at the TLV strain collection (no. TP00333/18). This first reported case of human Campylobacter lanienae campylobacteriosis in Germany demonstrates the agent's likely zoonotic pathogenicity.

Salmonella Derby: A Comparative Genomic Analysis of Strains From Germany.

Salmonella enterica subspecies enterica serovar Derby (S. Derby) is one of the most frequent causes for salmonellosis in humans and animals. Understanding the genetic diversity of S. Derby, as well as the nature and origin of its resistance to antimicrobial treatment are thus the key to epidemiological control and surveillance. Here, we report an analysis of 15 S. Derby strains isolated from pig and cattle in slaughterhouses across Germany (2000-2015), which belonged to multilocus sequence types (ST) ST39, ST40 and ST682. Strains were compared to publicly available S. Derby sequence data of these three STs from Germany, comprising 65 isolates collected between 2004 and 2018 from different sources (i.e., pigs, humans, cattle, wild boar, and poultry). A total of 80 sequences (ST39 = 34, ST40 = 21, and ST682 = 25) were analyzed to assess genetic diversity, to identify virulence-associated and antimicrobial resistance genes (ARGs), and to characterize plasmid content. Strains belonging to all three STs were identified in each source examined. Strains with the same ST were closely related regardless of origin. Altogether, 72.5% of the isolates carried at least one resistance gene, furthermore ST40 carried most of the ARGs and the plasmid replicons. The IncI1 replicon was detected in eleven isolates, four of them carried IncI1 plasmid ST26 with clonal complex 2. The comparison of these four isolates with an IncI1 ST26 plasmid reported in 2010 from a German pig (JX566770), showed only variations in a region carrying different ARGs and mobile genetic elements. The strains of our collection had similar genetic diversity as the strains taken from the public database. Moreover, we found that strains harboring multidrug resistant IncI plasmid were found in different animal species, indicating that S. Derby may be implicated in the spread of antimicrobial resistance among animal species. Results may contribute to the knowledge about the diversity in S. Derby in Germany, which may be useful for the future surveillance and antimicrobial resistance of this serovar.

Antimicrobial Susceptibility and Genomic Analysis of Aliarcobacter cibarius and Aliarcobacter thereius, Two Rarely Detected Aliarcobacter Species.

Aliarcobacter cibarius and Aliarcobacter thereius are two rarely detected Aliarcobacter species. In the study, we analyzed the antimicrobial susceptibility and provide detailed insights into the genotype and phylogeny of both species using whole-genome sequencing. Thermophilic Campylobacter species are the most common bacterial foodborne pathogens causing gastroenteritis in humans worldwide. The genus Aliarcobacter is part of the Campylobacteraceae family and includes the species Aliarcobacter butzleri, Aliarcobacter cryaerophilus, Aliarcobacter skirrowii, and the rarely described Aliarcobacter cibarius, Aliarcobacter faecis, Aliarcobacter lanthieri, Aliarcobacter thereius, and Acrobarter trophiarum. Aliarcobacter are emergent enteropathogens and potential zoonotic agents. Here, we generated, analyzed, and characterized whole-genome sequences of Aliarcobacter cibarius and Aliarcobacter thereius. They were isolated from water poultry farms in Germany, cultured and identified by MALDI-TOF MS. With PCR the identity was verified. Antibiotic susceptibility testing was carried out with erythromycin, ciprofloxacin, doxycycline, tetracycline, gentamicin, streptomycin, ampicillin, and cefotaxime using the gradient strip method (E-test). Whole-genome sequences were generated including those of reference strains. Complete genomes for six selected strains are reported. These provide detailed insights into the genotype. With these, we predicted in silico known AMR genes, virulence-associated genes, and plasmid replicons. Phenotypic analysis of resistance showed differences between the presence of resistance genes and the prediction of phenotypic resistance profiles. In Aliarcobacter butzleri, the nucleotide sequence of the gyrA gene (DQ464331) can show a signature mutation resulting in an amino acid change T85>I. Acrobarter cibarius and Acrobarter thereius showed the same gene as assessed by similarity annotation of the mutations 254C>G. Most of the isolates were found to be sensitive to ciprofloxacin. The ciprofloxacin-resistant Aliarcobacter thereius isolate was associated with the amino acid change T85>I. But this was not predicted with antibiotic resistance databases, before. Ultimately, a phylogenetic analysis was done to facilitate in future outbreak analysis.

Characterisation of S. aureus/MRSA CC1153 and review of mobile genetic elements carrying the fusidic acid resistance gene fusC.

While many data on molecular epidemiology of MRSA are available for North America, Western Europe and Australia, much less is known on the distribution of MRSA clones elsewhere. Here, we describe a poorly known lineage from the Middle East, CC1153, to which several strains from humans and livestock belong. Isolates were characterised using DNA microarrays and one isolate from the United Arab Emirates was sequenced using Nanopore technology. CC1153 carries agr II and capsule type 5 genes. Enterotoxin genes are rarely present, but PVL is common. Associated spa types include t504, t903 and t13507. PVL-positive CC1153-MSSA were found in Egyptian cattle suffering from mastitis. It was also identified among humans with skin and soft tissue infections in Saudi Arabia, France and Germany. CC1153-MRSA were mainly observed in Arabian Gulf countries. Some isolates presented with a previously unknown SCCmec/SCCfus chimeric element in which a mec B complex was found together with the fusidic acid resistance gene fusC and accompanying genes including ccrA/B-1 recombinase genes. Other isolates carried SCCmec V elements that usually also included fusC. Distribution and emergence of CC1153-MRSA show the necessity of molecular characterization of MRSA that are resistant to fusidic acid. These strains pose a public health threat as they combine resistance to beta-lactams used in hospitals as well as to fusidic acid used in the community. Because of the high prevalence of fusC-positive MRSA in the Middle East, sequences and descriptions of SCC elements harbouring fusC and/or mecA are reviewed. When comparing fusC and its surrounding regions from the CC1153 strain to available published sequences, it became obvious that there are four fusC alleles and five distinct types of fusC gene complexes reminiscent to the mec complexes in SCCmec elements. Likewise, they are associated with different sets of ccrA/B recombinase genes and additional payload that might include entire mec complexes or SCCmec elements.

Newly Isolated Animal Pathogen Corynebacterium silvaticum Is Cytotoxic to Human Epithelial Cells.

Corynebacterium silvaticum is a newly identified animal pathogen of forest animals such as roe deer and wild boars. The species is closely related to the emerging human pathogen Corynebacterium ulcerans and the widely distributed animal pathogen Corynebacterium pseudotuberculosis. In this study, Corynebacterium silvaticum strain W25 was characterized with respect to its interaction with human cell lines. Microscopy, measurement of transepithelial electric resistance and cytotoxicity assays revealed detrimental effects of C. silvaticum to different human epithelial cell lines and to an invertebrate animal model, Galleria mellonella larvae, comparable to diphtheria toxin-secreting C. ulcerans. Furthermore, the results obtained may indicate a considerable zoonotic potential of this newly identified species.

Characterization of Enterococci- and ESBL-Producing Escherichia coli Isolated from Milk of Bovides with Mastitis in Egypt.

This study aimed to investigate the prevalence and antimicrobial resistance of enterococci- and ESBL-producing E. coli isolated from milk of bovine mastitis cases in Egypt. Fifty milk samples of dairy animals were collected from localities in the Nile Delta region of Egypt. Isolates were identified using MALDI-TOF MS, and antibiotic susceptibility testing was performed by the broth microdilution method. PCR amplifications were carried out, targeting resistance-associated genes. Seventeen Enterococcus isolates and eight coliform isolates could be cultivated. Vancomycin resistance rate was high in Ent. faecalis. The VITEK 2 system confirmed all E. coli isolates as ESBL-producing. All Ent. faecalis isolates harbored erm(B), tetL and aac-aphD genes. The vanA gene was detected in Ent. faecalis isolate, vanB was found in other Enterococcus, while one isolate of E. casseliflavus exhibited the vanA gene. E. coli isolates exhibited high prevalence of erm(B) and tetL. E. coli isolates were analyzed by DNA microarray analysis. Four isolates were determined by O-serotyping as O8 (n = 1), O86 (n = 2) and O157 (n = 1). H-serotyping resulted in H11, H12, H21 (two isolates each) and one was of H16 type. Different virulence-associated genes were detected in E. coli isolates including lpfA, astA, celB, cmahemL, intI1 and intI2, and the iroN gene was identified by DNA microarray analysis.

Antimicrobial Resistance and in silico Virulence Profiling of Aliarcobacter butzleri Strains From German Water Poultry.

Aliarcobacter butzleri is an emerging foodborne and zoonotic pathogen that is usually transmitted via contaminated food or water. A. butzleri is not only the most prevalent Aliarcobacter species, it is also closely related to thermophilic Campylobacter, which have shown increasing resistance in recent years. Therefore, it is important to assess its resistance and virulence profiles. In this study, 45 Aliarcobacter butzleri strains from water poultry farms in Thuringia, Germany, were subjected to an antimicrobial susceptibility test using the gradient strip diffusion method and whole-genome sequencing. In the phylogenetic analysis, the genomes of the German strains showed high genetic diversity. Thirty-three isolates formed 11 subgroups containing two to six strains. The antimicrobial susceptibility testing showed that 32 strains were resistant to erythromycin, 26 to doxycycline, and 20 to tetracycline, respectively. Only two strains were resistant to ciprofloxacin, while 39 strains were resistant to streptomycin. The in silico prediction of the antimicrobial resistance profiles identified a large repertoire of potential resistance mechanisms. A strong correlation between a gyrA point mutation (Thr-85-Ile) and ciprofloxacin resistance was found in 11 strains. A partial correlation was observed between the presence of the bla3 gene and ampicillin resistance. In silico virulence profiling revealed a broad spectrum of putative virulence factors, including a complete lipid A cluster in all studied genomes.