Clonal Complex CC398 Research Articles

Whole genome analysis of Neisseria meningitidis isolates from invasive meningococcal disease collected in the Czech Republic over 28 years (1993-2020).

Invasive meningococcal disease belongs among the most dangerous infectious diseases in the world. Several polysaccharide conjugate vaccines against serogroups A, C, W and Y are available and two recombinant peptide vaccines against serogroup B (MenB vaccines) have been developed: MenB-4C (Bexsero) and MenB-fHbp (Trumenba). The aim of this study was to define the clonal composition of the Neisseria meningitidis population in the Czech Republic, to determine changes in this population over time and to estimate the theoretical coverage of isolates by MenB vaccines. This study presents the analysis of whole genome sequencing data of 369 Czech N. meningitidis isolates from invasive meningococcal disease covering 28 years. Serogroup B isolates (MenB) showed high heterogeneity and the most common clonal complexes were cc18, cc32, cc35, cc41/44, and cc269. Isolates of clonal complex cc11 were predominately serogroup C (MenC). The highest number of serogroup W isolates (MenW) belonged to clonal complex cc865, which we described as exclusive to the Czech Republic. Our study supports the theory that this cc865 subpopulation originated in the Czech Republic from MenB isolates by a capsule switching mechanism. A dominant clonal complex of serogroup Y isolates (MenY) was cc23, which formed two genetically quite distant subpopulations and which showed constant representation throughout the observed period. The theoretical coverage of isolates by two MenB vaccines was determined using the Meningococcal Deduced Vaccine Antigen Reactivity Index (MenDeVAR). Estimated Bexsero vaccine coverage was 70.6% (for MenB) and 62.2% (for MenC, W, Y). For Trumenba vaccine, estimated coverage was 74.6% (for MenB) and 65.7% (for MenC, W, Y). Our results demonstrated sufficient coverage of Czech heterogeneous population of N. meningitidis with MenB vaccines and, together with surveillance data on invasive meningococcal disease in the Czech Republic, were the basis for updating recommendations for vaccination against invasive meningococcal disease.

Cfr and fexA genes in methicillin-resistant Staphylococcus aureus from humans and livestock in the Netherlands

BackgroundAlthough the Netherlands is a country with a low endemic level of methicillin-resistant Staphylococcus aureus (MRSA), a national MRSA surveillance has been in place since 1989. In 2003 livestock emerged as a major reservoir of MRSA and currently livestock-associated MRSA (clonal complex CC398) make up 25% of all surveillance isolates. To assess possible transfer of resistant strains or resistance genes, MRSA obtained from humans and animals were characterized in detail.MethodsThe sequenced genomes of 6327 MRSA surveillance isolates from humans and from 332 CC398 isolates from livestock-related samples were analyzed and resistance genes were identified. Several isolates were subjected to long-read sequencing to reconstruct chromosomes and plasmids.ResultsHere we show the presence of the multi-resistance gene cfr in seven CC398 isolates obtained from humans and in one CC398 isolate from a pig-farm dust sample. Cfr induces resistance against five antibiotic classes, which is true for all but two isolates. The isolates are genetically unrelated, and in seven of the isolates cfr are located on distinct plasmids. The fexA gene is found in 3.9% surveillance isolates and in 7.5% of the samples from livestock. There is considerable sequence variation of fexA and geographic origin of the fexA alleles.ConclusionsThe rare cfr and fexA resistance genes are found in MRSA from humans and animals in the Netherlands, but there is no evidence for spread of resistant strains or resistance plasmids. The proportion of cfr-positive MRSA is low, but its presence is worrying and should be closely monitored.

Microbiome profiling and characterization of virulent and vancomycin-resistant Enterococcus faecium from treated and untreated wastewater, beach water and clinical sources

Vancomycin-resistant Enterococcus faecium (VREfm) is an opportunistic pathogen among the highest global priorities regarding public and environmental health. Following One Health approach, we determined for the first time the antibiotic resistance and virulence genes, and sequence types (STs) affiliation of VREfm recovered simultaneously from marine beach waters, submarine outfall of a wastewater treatment plant and an offshore discharge of untreated sewage, and compared them with the surveillance VREfm from regional university hospital in Croatia to assess the hazard of their transmission and routes of introduction into the natural environment. Importantly, VREfm recovered from wastewater, coastal bathing waters and hospital shared similar virulence, multidrug resistance, and ST profiles, posing a major public health threat. All isolates carried the vanA gene, while one clinical isolate also possessed the vanC2/C3 gene. The hospital strains largely carried the aminoglycoside-resistance genes aac(6′)-Ie-aph(2″)-Ia, and aph(2″)-Ib and aph(2″)-Id, which were also predominant in the environmental isolates. The hyl gene was the most prevalent virulence gene. The isolates belonged to 10 STs of the clonal complex CC17, a major epidemic lineage associated with hospital infections and outbreaks, with ST117 and ST889 common to waterborne and hospital isolates, pointing to their sewage-driven dissemination.To gain better insight into the diversity of accompanying taxons in the surveyed water matrices, microbiome taxonomic profiling was carried out using Illumina-based 16S rDNA sequencing and their resistome features predicted using the PICRUSt2 bioinformatics tool. An additional 60 pathogenic bacterial genera were identified, among which Arcobacter, Acinetobacter, Escherichia-Shigella, Bacteroides and Pseudomonas were the most abundant and associated with a plethora of antibiotic resistance genes and modules, providing further evidence of the hazardous effects of wastewater discharges, including the treated ones, on the natural aquatic environment that should be adequately addressed from a sanitary and technological perspective.

Reactive vaccination as control strategy for an outbreak of invasive meningococcal disease caused by Neisseria meningitidis C:P1.5-1,10-8:F3-6:ST-11(cc11), Bergamo province, Italy, December 2019 to January 2020

In Italy, serogroup C meningococci of the clonal complex cc11 (MenC/cc11) have caused several outbreaks of invasive meningococcal disease (IMD) during the past 20 years. Between December 2019 and January 2020, an outbreak of six cases of IMD infected with MenC/cc11 was identified in a limited area in the northern part of Italy. All cases presented a severe clinical picture, and two of them were fatal. This report is focused on the microbiological and molecular analysis of meningococcal isolates with the aim to reconstruct the chain of transmission. It further presents the vaccination strategy adopted to control the outbreak. The phylogenetic evaluation demonstrated the close genetic proximity between the strain involved in this outbreak and a strain responsible for a larger epidemic that had occurred in 2015 and 2016 in the Tuscany Region. The rapid identification and characterisation of IMD cases and an extensive vaccination campaign contributed to the successful control of this outbreak caused by a hyperinvasive meningococcal strain.

Beyond CC398: Characterisation of Other Tetracycline and Methicillin-Resistant Staphylococcus aureus Genetic Lineages Circulating in Spanish Hospitals.

Tetracycline resistance (TetR) has been evidenced as a good phenotypic marker for detection of livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) isolates of the clonal complex CC398. The aim of this study was to characterise a collection of 95 TetR-MRSA isolates, not belonging to the lineage CC398, that were obtained in a previous multicentre study, to detect other MRSA clonal complexes that could be associated with this phenotypic TetR marker. The TetR-MRSA isolates were recovered from 20 Spanish hospitals during 2016 and they were characterised to determine their antimicrobial resistance and virulence phenotypes/genotypes as well as the presence of the immune evasion cluster (IEC). A high proportion of isolates belonging to the CC1 lineage (46%) were observed, as well as to the CC5, CC8 and CC45 lineages (11% each one). Thirty-two different spa-types were identified, being predominantly CC1-t127 (40%) and CC45-t1081 (11%). The IEC system (with the gene scn as marker) was present in 73% of isolates and 16% produced the Panton Valentine leucocidin (PVL). A high proportion of MRSA-CC1 isolates were scn-negative (38.6%) and 52.9% of them were blaZ-negative. A multidrug resistance (MDR) phenotype was identified in 86% of MRSA isolates. The knowledge of other TetR-MRSA genetic lineages, in addition to CC398, is highly relevant, since most of them were MDR and some of them presented important virulence factors. Strains potentially associated with livestock (as the subpopulation CC1-t127-scn-negative) or with humans (as the CC45 lineage or the subpopulation CC1-scn-positive) have been found in this study. The use of tetracycline-resistance for detection, not only of CC398 but also of other LA-MRSA lineages should be tracked in the future.

Staphylococcus aureus genotype variation among and within periprosthetic joint infections.

Staphylococcus aureus is a common organism in orthopedic infections, but little is known about the genetic diversity of strains during an infectious process. Using periprosthetic joint infection (PJI) as a model, a prospective study was designed to quantify genetic variation among S. aureus strains both among and within patients. Whole genome sequencing and multilocus sequence typing was performed to genotype these two populations at high resolution. In nasal cultures, 78% of strains were of clonal complexes CC5, CC8, and CC30. In PJI cultures, only 63% could be classified in these common clonal complexes. The PJI cultures had a larger proportion of atypical strains, and these atypical strains were associated with poor host status and compromised immune conditions. Mutations in genes involved in fibronectin binding (ebh, fnbA, clfA, and clfB) systematically distinguished later PJI isolates from the first PJI isolate from each patient. Repeated mutations in S. aureus genes associated with extracellular matrix binding were identified, suggesting adaptive, parallel evolution of S. aureus during the development of PJI.

Plasmid-Chromosome Crosstalk in Staphylococcus aureus: A Horizontally Acquired Transcription Regulator Controls Polysaccharide Intercellular Adhesin-Mediated Biofilm Formation.

Livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) of clonal complex CC398 typically carry various antimicrobial resistance genes, many of them located on plasmids. In the bovine LA-MRSA isolate Rd11, we previously identified plasmid pAFS11 in which resistance genes are co-localized with a novel ica-like gene cluster, harboring genes required for polysaccharide intercellular adhesin (PIA)-mediated biofilm formation. The ica genes on pAFS11 were acquired in addition to a pre-existing ica locus on the S. aureus Rd11 chromosomal DNA. Both loci consist of an icaADBC operon and icaR, encoding a corresponding icaADBC repressor. Despite carrying two biofilm gene copies, strain Rd11 did not produce PIA and transformation of pAFS11 into another S. aureus strain even slightly diminished PIA-mediated biofilm formation. By focusing on the molecular background of the biofilm-negative phenotype of pAFS11-carrying S. aureus, we identified the pAFS11-borne ica locus copy as functionally fully active. However, transcription of both plasmid- and core genome-derived icaADBC operons were efficiently suppressed involving IcaR. Surprisingly, although being different on the amino acid sequence level, the two IcaR repressor proteins are mutually replaceable and are able to interact with the icaA promoter region of the other copy. We speculate that this regulatory crosstalk causes the biofilm-negative phenotype in S. aureus Rd11. The data shed light on an unexpected regulatory interplay between pre-existing and newly acquired DNA traits in S. aureus. This also raises interesting general questions regarding functional consequences of gene transfer events and their putative implications for the adaptation and evolution of bacterial pathogens.

Spread of Linezolid-Resistant Enterococcus spp. in Human Clinical Isolates in the Czech Republic.

The aim of this study was to map and investigate linezolid resistance mechanisms in linezolid-resistant enterococci in the Czech Republic from 2009 to 2019. Altogether, 1442 isolates of Enterococcus faecium and Enterococcus faecalis were examined in the National Reference Laboratory for Antibiotics. Among them, 8% of isolates (n = 115) were resistant to linezolid (E. faecium/n = 106, E. faecalis/n = 9). Only three strains of E. faecium were resistant to tigecycline, 72.6% of isolates were resistant to vancomycin. One isolate of E. faecium harbored the cfr gene. The majority (87%, n = 11) of E. faecium strains were resistant to linezolid because of the mutation G2576T in the domain V of the 23S rRNA. This mutation was detected also in two strains of E. faecalis. The presence of the optrA gene was the dominant mechanism of linezolid resistance in E. faecalis isolates. None of enterococci contained cfrB, poxtA genes, or any amino acid mutation in genes encoding ribosomal proteins. No mechanism of resistance was identified in 4 out of 106 E. faecium linezolid resistant isolates in this study. Seventeen sequence types (STs) including four novel STs were identified in this work. Clonal complex CC17 was found in all E. faecium isolates.

Vancomycin-resistant Enterococcus faecium in Algeria: phenotypic and genotypic characterization of clinical isolates.

vancomycin-resistant Enterococcus faecium (VREfm) is a major public health problem worldwide. The aim of our study was to determine the microbiological, epidemiological and molecular characteristics of VREfm isolated in north-central, eastern and western Algeria. a collection of 48 VREfm isolated from September 2010 to April 2017 in several Algerian hospitals were studied. Minimum inhibitory concentrations (MICs) were determined by E-test method according to CLSI guidelines. the detection of van genotype of all strains was performed by PCR. Clonal relationship of five VREfm targeted by region were characterized using multilocus sequence typing (MLST). All isolates have multidrug-resistance (MDR) and were resistant to at least five classes of antibiotics; however, all were susceptible to tigecycline and daptomycin with MIC50 at 0.094 µg/mL and 2 µg/mL respectively. All strains belonged to vanA genotype and have high level of resistance to vancomycin and teicoplanin. MLST revealed two sequence types (STs): ST80 (from the four regions of Algeria) and ST789, both belonging to the former hospital-adapted clonal complex CC17. the alarming dissemination of MDR E. faecium vanA and the ST80 in several regions of Algeria suggest a clonal spread of VREfm strains, which urgently require implementation of adequate infection control measures.

Genetic diversity of vancomycinresistant Enterococcus faecium isolated from blood culture in patients with hematological malignancies

Objective. To study the genetic diversity of vancomycin-resistant Enterococcus faecium (VR-E. faecium) isolated from the blood culture in patients with hematological malignancies by multilocus sequence typing (MLST). Materials and Methods. VR-E. faecium isolated from the blood culture in hematological patients in 6 hospitals of 4 Russian cities (2003–2019) were evaluated. Susceptibility to vancomycin was tested by the broth microdilution method (CLSI, 2018). Vancomycin-resistance genes (vanA, vanB) were identified by polymerase chain reaction. Genotyping of VR-E. faecium was performed by MLST. Results. A total of 83 VR-E. faecium were examined. The vanA genes were detected in 71.1% (n = 59) VR-E. faecium, vanB genes – in 28.9% (n = 24). A total of 22 sequence types (STs) belonging to epidemic clonal complex CC17 were detected. The dominant sequence types were ST17 (19.3%), ST78 (18.1%), ST80 (16.9%), and comprised 54.3% VR-E. faecium. Other sequence types included 1 to 4 strains. VR-E. faecium carrying vanA, in comparison with VR-E. faecium vanB, significantly more often belonged to ST78 (23.7% vs. 4.2%, p = 0.0559, respectively) and ST80 (23.7% versus 0%, p = 0.0079, respectively) and less frequently to ST17 (6,8% versus 50%, р < 0.0001). Circulation of 9 STs including «high-risk» clones ST17 and ST78 was detected during two study periods (2003–2011 and 2012–2019). Conclusions. This study showed a genetic diversity of VR-E. faecium that was represented by 22 STs. All VR-E. faecium belonged to epidemic clonal complex CC17 and comprised «high-risk» clones ST17, ST78 and less common STs.

Genetic Relatedness and Diversity of Staphylococcus aureus from Different Reservoirs: Humans and Animals of Livestock, Poultry, Zoo, and Aquaculture.

The main aim of this study was the characterization of antibiotic resistance mechanisms in 82 Staphylococcus aureus strains isolated from humans and animals. Antibiotic susceptibility testing was performed on all S. aureus isolates accordingly, and antibiotic-resistant genes were investigated by genotypic methods. The genetic diversity of S. aureus was studied through spa, multilocus sequence typing (MLST), and agr typing methods. The majority of S. aureus from human sources were resistant to cefoxitin (and harbor the mecA gene) and fluoroquinolones, whereas only four strains of S. aureus from animal sources revealed resistance to ciprofloxacin. In the set of S. aureus isolated from humans, the most frequent spa, MLST, and agr group were t032, ST22, and I, respectively. In strains from animal origin the most common spa, MLST, and agr group found were t2383, ST398, and III/not typable, respectively. S. aureus from humans and animals were identified either in clonal complexes CC5, CC30, and CC398, suggesting that they have the same putative founder in their evolution. Considering the three CCs encompassing strains from human and animal reservoirs with different spa-types, we can hypothesize that this might reflect an adaptation to different phylogenetic lineages in those reservoirs (host species) probably associated to genetic diversification of pre-existing strains.

Prevalence of methicillin-resistant Staphylococcus aureus colonization among healthcare workers at a tertiary care hospital in northeastern Brazil

SummaryBackgroundStaphylococcus aureus is a human pathogen of clinical importance related to a variety of infections.AimThe objective of this study was to analyze the molecular and epidemiological characteristics of S. aureus obtained from healthcare professionals (HCP) of a hospital in southwestern Bahia, Brazil.MethodsSamples were collected from hands, nasal cavity, and laboratory coats of 80 HCP. The bacterial isolates recovered from 240 samples were identified as S. aureus, and then analyzed for their antimicrobial resistance profile, genotypic characterization, and pathogenicity.Findings178 isolates were identified as S. aureus, being mostly isolated from the nasal cavity. Thirty isolates (16.8%) were characterized as MRSA. The virulence gene frequency varied according to isolate source. All virulence genes were identified in at least one hand isolate. Isolates from laboratory coats did not show seb and pvl. Isolates from the nasal cavity did not exhibit pvl. The SCCmec type I was identified in 56.7% of MRSA isolates. Among MRSA isolates, 14 PFGE pulsotypes were characterized, with profile A being predominant (nine isolates). Clonal complexes CC5, CC45, and CC398 were found. MRSA isolates induced cytokine gene expression in macrophages, with IL-10 and IL-17 being expressed more often.ConclusionWe found a high colonization rate for S. aureus among HCP. Moreover, we observed that MRSA strains presented different virulence factors and could induce cytokine gene expression, indicating an urgent need to control colonization rates of HCP by MRSA isolates in order to protect hospital patients and the general public.

Meningococcal Urethritis: an additional Source of Meningococcal Disease?

Background . Beginning with the middle of the XX century some observations about cases of meningococcal urethritis have appeared. The number of articles for such subjects has promptly increased in the last decade of XXI. The epidemiological features of meningococcal urethritis are slightly understood. Aims . A review of the latest publications on meningococcal urethritis is presented. Particular attention is given to outbreaks of meningococcal urethritis caused by the new genetic clade USNmCU of N.meningitidis, which occurred in several US states in 2013-2016. Conclusions . The isolates from patients were non-capsulated and belonged to the CC-11 clonal complex, the most virulent among N.meningitidis strains of serogroups B, C, Y, W. The orogenital transmission of clade strains has been proven. It is assumed that N.meningitidis acquired genes for anaerobic growth as a result of genetic recombination during co-presence with N. gonorrhea on the mucous membranes and became to be able to colonize the urogenital tract. The urethritogenic meningococcal clade is the result of the adaptation of the capsular strain of meningococcus serogroup C of the clonal complex CC-11, which is responsible for outbreaks of generalized meningococcal infection in MSM communities. The urethritogenic strains are considered as representatives of the emergent clade of N. gonorhoeae. Their ability to be transmitted through direct sexual contact is unknown. The epidemiological role of the clade in the spread of meningococcal infection is discussed. It is necessary to develop the researches concerning the epidemiological features of meningococcal urethritis and its additional role as the source of meningococcal disease.

The Characterization of OXA-232 Carbapenemase-Producing ST437 Klebsiella pneumoniae in China

Carbapenem-resistant Klebsiella pneumoniae (CRKP) was epidemic around the world and become a global threat to public health. The most important carbapenem-resistant mechanism is producing carbapenemases, especially Klebsiella pneumoniae carbapenemase (KPC), which is prevalent in the international clonal complex CC11. The high-risk multidrug-resistant CC11 is widespread worldwide, and KPC-producing and (New Delhi metallo) NDM-producing strains had been reported in this clonal complex before; moreover, cases with the CC11 strain faced more severe forms of drug resistance and treatment challenges than other clonal complexes. In this study, we identified an OXA-232-producing ST437 Klebsiella pneumoniae isolate in China, which belonged to CC11. The isolate was resistant to β-lactams, aminoglycosides, and fluoroquinolones but susceptible to fosfomycin, tigecycline, and colistin. The blaOXA-232 gene was located on a 6141 bp ColKP3-type nonconjugative plasmid, and the plasmid was transformed by chemical transformation successfully. This is the first report of OXA-232-producing ST437 K. pneumoniae in China, a new clone of high-risk multidrug-resistant CC11.

Epidemiology of bacteria and viruses in the respiratory tract of humans and domestic pigs.

Bacteria and viruses were analysed in the upper respiratory tract of symptomatic pig farmers and their domestic pigs. Eighty six human nasal and 495 (50 pools) porcine snout swabs were collected in Schleswig-Holstein, Germany. Staphylococcus (S.) aureus (62.8%, 54/86), human rhino- and coronaviruses (HRV, 29.1%, 25/86; HCoV, 16.3%, 14/86) were frequently detected in humans, while Haemophilus parasuis (90.0%, 45/50), Mycoplasma hyorhinis (78.6%, 11/14), Enterovirus G (EV-G, 56.0%, 28/50) and S.aureus (36.0%, 18/50), respectively, were highly prevalent in pigs. The detection of S.aureus in human follow-up samples indicates a carrier status. The methicillin-resistant phenotype (MRSA) was identified in 33.3% (18/54) of nasal swabs and in one of 18 (5.6%) pooled snout swabs that were tested positive for S.aureus. Strains were indicative of the livestock-associated clonal complex CC398, with t011 being the most common staphylococcal protein A type. Enterobacterales and non-fermenters were frequently isolated from swabs. Their detection in follow-up samples suggests a carrier status. All were classified as being non-multiresistant. There was no example for cross-species transmission of viruses. In contrast, transmission of S.aureus through occupational contact to pigs seems possible. The study contributes to the 'One Health' approach.

High persister cell formation by clinical Staphylococcus aureus strains belonging to clonal complex 30.

Bacterial persisters form a subpopulation of cells that survive lethal concentrations of antibiotics without being genetically different from the susceptible population. They are generally considered to be phenotypic variants that spontaneously have entered a dormant state with low ATP levels or reduced membrane potential. In Staphylococcus aureus, a serious opportunistic human pathogen, persisters are believed to contribute to chronic infections that are a major global healthcare problem. While S. aureus persisters have mostly been studied in laboratory strains, we have here investigated the ability of clinical strains to form persisters. For 44 clinical strains belonging to the major clonal complexes CC5, CC8, CC30 or CC45, we examined persister cell formation in stationary phase when exposed to 100 times the MIC of ciprofloxacin, an antibiotic that targets DNA replication. We find that while all strains are able to form persisters, those belonging to CC30 displayed on average 100-fold higher persister cell frequencies when compared to strains of other CCs. Importantly, there was no correlation between persister formation and the cellular ATP content of the individual strains, but the group of CC30 strains displayed slightly lower membrane potential compared to the non-CC30 group. CC30 strains have previously been associated with chronic and reoccuring infections and we hypothesize that there could be a correlation between lineage-specific characteristics displayed via in vitro persister assays and the observed clinical spectrum of disease.

Emergence of vanA-Type Vancomycin-Resistant Enterococcus faecium ST 78 Strain with a rep2-Type Plasmid Carrying a Tn1546-Like Element Isolated from a Urinary Tract Infection in China.

PurposeThe emergence of vancomycin-resistant enterococci (VRE) dramatically narrows therapeutic options. Although the prevalence of VRE in China has maintained a low level, VRE outbreaks have been reported in some tertiary hospitals in the developed areas of China. The clonal background of vanA-positive Enterococcus faecium strains has not been well characterized in China. Here, we report the whole-genome sequence of a vanA-type vancomycin-resistant E. faecium belonging to sequence type (ST) 78 isolated from a urinary tract infection in China.Patients and MethodsA vancomycin-resistant E. faecium was isolated from a 66-year-old male patient diagnosed with brainstem hemorrhage. Antibiotic susceptibility assays were performed according to the guidelines of the Clinical and Laboratory Standards Institute (CLSI). Complete genome sequencing was performed using both the HiSeqTM 4000 platform and the MinION platform. Plasmid, genomic and phylogenetic relationship analysis were further performed.ResultsE. faecium VRE1 was resistant to all antimicrobials tested except for tetracyclines and oxazolidinones. The whole genome of E. faecium VRE1 was composed of one chromosomal DNA and four plasmids. Two virulence genes and five antimicrobial resistance genes were identified. In silico multilocus sequence typing (MLST) showed that it belonged to ST78 (clonal complex CC17), a well-known epidemic clone that is widespread in Europe and the United States. Three antimicrobial resistance genes, including aminoglycoside resistance genes ant(6)-Ia and aph(3ʹ)-III; and glycopeptide resistance gene vanA, were located on a rep2-type plasmid carrying a Tn1546-like element that has not been reported. The most closely related strain harboring a similar plasmid backbone was recovered from fodder sample in China that differed by 178 cgMLST loci.ConclusionOur study characterizes the genomic feature of a vancomycin-resistant E. faecium ST78 strain harboring a vanA-carrying plasmid in China. The ST78 clonal group possessed the potential to emerge as a successful vanA-carrying epidemic lineage in China.

Occurrence and Characteristics of Livestock-Associated Methicillin-Resistant Staphylococcus aureus in Quarter Milk Samples From Dairy Cows in Germany.

The aim of this study was to identify and characterize LA-MRSA in a very recent collection of staphylococci isolated from bovine quarter milk samples. All milk samples (n = 14,924) sent to the MBFG in March 2017 were included into this study. The samples originated from 3,887 cows with 3,367 samples from 2,280 animals being positive for bacteria, prototheca and/or yeast. The second most common infectious agent was Staphylococcus and 659 isolates were investigated. Staphylococcus aureus was confirmed by PCR for the spa gene. A CC398-specific PCR was performed for all S. aureus isolates. Susceptibility to penicillin was tested for all isolates by agar disk diffusion. All oxacillin resistant isolates were analyzed by microarray and tested for their susceptibility to 30 antimicrobial agents. Of the isolates 372 were S. aureus from Germany with 214 isolates being not epidemiologically related. Among the independent isolates nine were identified as oxacillin resistant. In addition five isolates epidemiologically related to these nine were MRSA. One of them showed differences to the other MRSA isolate from the same farm resulting in altogether ten different MRSA isolates. All ten belonged to the clonal complex CC398. These ten LA-MRSA isolates had three to six antimicrobial resistance genes. The gene mecA was in all cases located on a SCCmec V element. Among the remaining S. aureus seven independent isolates belonged to CC398. In conclusion this study showed a high detection rate of staphylococci in bovine quarter milk samples. In contrast MRSA was rarely detected and belonged in all cases to CC398. Only 7/214 MSSA (3.3%) belonged to this CC.

Bacterial content and characterization of antibiotic resistant Staphylococcus aureus in Danish sushi products and association with food inspector rankings

This study examined the prevalence and phenotypic and genotypic antibiotic resistance patterns of Staphylococcus aureus in sushi from 20 Danish outlets. Microbial quality of sushi products and food inspector ranking of outlets were assessed and results for thirteen of the outlets were compared with findings from a previous study in 2012. Inspector rankings were similar in the two studies. The mesophilic aerobic counts were slightly lower (p = 0.0296) in 2017 than in 2012 with average values of the 13 shops of 5.2 log CFU/g and 5.7 log CFU/g, respectively. In both studies E. coli was only found in the products from outlets that did not have consistently superior rankings. On the other hand prevalence and average counts of Staphylococcus spp. were slightly higher in 2017 (p = 0.0286) but no methicillin-resistant S. aureus (MRSA) were observed in the present study. Methicillin-sensitive S. aureus (MSSA) were, however, isolated from 18.7% of sushi products with an average count below 2 log CFU/g. Based on spa and multilocus sequence typing (MLST), isolates belonged to clonal complex CC7 (t2016), CC20 (t7836), CC45 (t065, t127, t362), CC88 (t1998) and CC398 (t164, t331, t1451). The Panton-Valentine leukocidin (PVL)-encoding gene lukF was detected only in isolates of the t065 spa-type whereas the scn gene from the ΦSa3 prophage was detected in 76.5% of the isolates, supporting that the majority of isolates were of likely human origin. Thirty-six isolates (70.6%) were resistant to at least one of the antibiotic compounds tested. Antibiotic resistance genes that confer resistance to β-lactams (blaZ) and macrolides (ermC) were detected in 33.3% and 9.8% of isolates, respectively. The tet(K) gene that encode tetracycline resistance was only found in a t7836 strain. Overall, this study indicates that S. aureus in sushi products in Denmark do not represent a major food safety hazard due to, firstly, the low temperature and limited time of storage of product may prevent significant growth and production of toxic levels of enterotoxin of this species. Secondly, the S. aureus isolates obtained did not include MRSA variants and none of them encoded PVL that constitute one of the virulence factors in pathogenesis. Several MSSA isolates contained however genes encoding antibiotic resistance, which emphasize the potential role of foods as vehicles for transmission of such variants.

Detection and quantification of methicillin-resistant Staphylococcus aureus in fresh broiler meat at retail in Germany

The aim of this study was to detect, to quantify and to characterize MRSA in broiler meat samples with skin. Furthermore, we compared an isolation method using a second selective enrichment step (method A) with a simpler method omitting this step (method B). For quantification we used a direct plating method on selective agar plates and a “Most probable number” (MPN) technique for estimation of low numbers of MRSA. Presumptive MRSA colonies were confirmed by MALDI-TOF and by PCR. After confirmation the isolated MRSA were characterized by spa-typing and, if necessary, by multi-locus sequence typing.Method B detected more MRSA-positive samples (16.7%, n = 215) than method A (12.1%). However, method B also produced more false positive results (28.4%).The highest estimated number of MRSA in fresh broiler meat with skin was 1100 MPN/g, but in most positive samples (80.1%) the estimated numbers of MRSA were lower than 10 MPN/g. Thus, the numbers of MRSA in the samples were too low to detect using the spread plate technique. Ten different spa-types were identified. Six of these with 69% of the isolates were assigned to the clonal complex CC398 (t034; t011; t2576; t571; t5452; t1457). Spa-types t1430, t13177 and t899 can be assigned to CC9. Spa-type t304 was identified as MLST-type ST6.In conclusion, we provide quantitative data on low level contamination of fresh broiler meat with MRSA. Most isolated MRSA were from livestock associated spa-types. Omitting the second enrichment step was associated with an increase in sensitivity but lower specificity of the cultural method.