MLST Typing Research Articles

Clinical and molecular characteristics of carbapenem non-susceptible Escherichia coli: A nationwide survey from Oman.

The prevalence of carbapenem-resistant Enterobacterales (CRE) in the Arabian Peninsula is predicted to be high, as suggested from published case reports. Of particular concern, is carbapenem-resistant E. coli (CR-EC), due to the importance of this species as a community pathogen. Herein, we conducted a comprehensive molecular characterization of putative CR-EC strains from Oman. We aim to establish a baseline for future molecular monitoring. We performed whole-genome sequencing (WGS) for 35 putative CR-EC. Isolates were obtained from patients at multiple centers in 2015. Genetic relatedness was investigated using several typing approaches such as MLST, SNP calling, phylogroup and CRISPR typing. Maxiuium likelihood SNP-tree was performed by RAxML after variant calling and removal of recombination regions with Snippy and Gubbins, respectively. Resistance genes, plasmid replicon types, virulence genes, and prophage were also characterised. The online databases CGE, CRISPRcasFinder, Phaster and EnteroBase were used for the in silico analyses. Screening for mutations in genes regulating the expression of porins and efflux pump as well as mutations lead to fluoroquinolones resistance were performed with CLC Genomics Workbench. The genetic diversity suggests a polyclonal population structure with 21 sequence types (ST), of which ST38 being the most prevalent (11%). SNPs analysis revealed possible transmission episodes. Whereas, CRISPR typing helped to spot outlier strains belonged to phylogroups other than B2 which was CRISPR-free. The virulent phylogroups B2 and D were detected in 4 and 9 isolates, respectively. In some strains bacteriophages acted as vectors for virulence genes. Regarding resistance to β-lactam, 22 were carbapenemase producers, 3 carbapenem non-susceptible but carbapenemase-negative, 9 resistant to expanded-spectrum cephalosporins, and one isolate with susceptibility to cephalosporins and carbapenems. Thirteen out of the 22 (59%) carbapenemase-producing isolates were NDM and 7 (23%) were OXA-48-like which mirrors the situation in Indian subcontinent. Two isolates co-produced NDM and OXA-48-like enzymes. In total, 80% (28/35) were CTX-M-15 producers and 23% (8/35) featured AmpC. The high-risk subclones ST131-H30Rx/C2, ST410-H24RxC and ST1193-H64RxC were detected, the latter associated with NDM. To our knowledge, this is the first report of ST1193-H64Rx subclone with NDM. In conclusion, strains showed polyclonal population structure with OXA-48 and NDM as the only carbapenemases in CR-EC from Oman. We detected the high-risk subclone ST131-H30Rx/C2, ST410-H24RxC and ST1193-H64RxC. The latter was reported with carbapenemase gene for the first time here.

Temporal Changes in Patient-Matched Staphylococcus epidermidis Isolates from Infections: towards Defining a 'True' Persistent Infection.

Staphylococcus epidermidis is found naturally on the skin but is a common cause of persistent orthopaedic device-related infections (ODRIs). This study used a pan-genome and gene-by-gene approach to analyse the clonality of whole genome sequences (WGS) of 115 S. epidermidis isolates from 55 patients with persistent ODRIs. Analysis of the 522 gene core genome revealed that the isolates clustered into three clades, and MLST analysis showed that 83% of the isolates belonged to clonal complex 2 (CC2). Analysis also found 13 isolate pairs had different MLST types and less than 70% similarity within the genes; hence, these were defined as re-infection by a different S. epidermidis strain. Comparison of allelic diversity in the remaining 102 isolates (49 patients) revealed that 6 patients had microevolved infections (>7 allele differences), and only 37 patients (77 isolates) had a ‘true’ persistent infection. Analysis of the core genomes of isolate pairs from 37 patients found 110/841 genes had variations; mainly in metabolism associated genes. The accessory genome consisted of 2936 genes; with an average size of 1515 genes. To conclude, this study demonstrates the advantage of using WGS for identifying the accuracy of a persistent infection diagnosis. Hence, persistent infections can be defined as ‘true’ persistent infections if the core genome of paired isolates has ≤7 allele differences; microevolved persistent infection if the paired isolates have >7 allele differences but same MLST type; and polyclonal if they are the same species but a different MLST type.

Control of Listeria monocytogenes Biofilms in a Simulated Food-Processing Environment.

Biofilm-forming ability may vary significantly among different Listeria (L.) monocytogenes strains. This interstrain variation is also observed in L. monocytogenes biofilm resistance to antimicrobial compounds commonly used in the food-processing environment. The screening of a large set of L. monocytogenes strains with specific characteristics, such as serotype, MLST type, and other genetic characteristics under various environmental conditions, may lead to a better understanding of the mechanisms underlying the establishment of the pathogen on food contact surfaces. In this chapter, traditional methods for L. monocytogenes strains characterization with regard to biofilm formation and novel biofilm control methods will be described.

Molecular Characteristics and Zoonotic Potential of Salmonella Weltevreden From Cultured Shrimp and Tilapia in Vietnam and China.

Salmonella Weltevreden is increasingly reported from aquatic environments, seafood, and patients in several Southeast Asian countries. Using genome-wide analysis, we characterized S. Weltevreden isolated from cultured shrimp and tilapia from Vietnam and China to study their genetic characteristics and relatedness to clinical isolates of S. Weltevreden ST-365. The phylogenetic analysis revealed up to 312 single-nucleotide polymorphism (SNP) difference between tilapia isolates, whereas isolates from shrimp were genetically more closely related. Epidemiologically unrelated isolates from Vietnam were closely related to isolates from China, e.g., 20 SNPs differences between strains 28V and 75C. In comparison with strains from other parts of the world, our environmental isolates predominantly clustered within the continental South Asia lineage, constituted mostly of strains from human stool with as low as seven SNPs difference, e.g., 30V versus Cont_ERR495254. All sequenced isolates were MLST type ST-365 and contained the major virulence-related genes encoded by the Salmonella Pathogenicity Islands 1–5. Ten of the isolates harbored the IncFII(S) plasmid similar to the virulence genes-mediated plasmid pSPCV of S. Paratyphi C, and one isolate had the IncQ1 plasmid on the same contig with strA/B, sul2, and tetA resistance genes similar to the IncQ1 type, pNUC of S. Typhimurium. A pangenomic analysis yielded 7891 genes including a core genome of 4892 genes, with a closely related accessory genome content between clinical and environmental isolates (Benjamini p > 0.05). In a search for differences that could explain the higher prevalence of S. Weltevreden in aquatic samples, genomes were compared with those of other Salmonella enterica serovars. S. Weltevreden revealed specific regions harboring glpX (Fructose-1;6-bisphosphatase; class II), rfbC (dTDP-4-dehydrorhamnose 3;5-epimerase), and cmtB (PTS Mannitol-specific cryptic phosphotransferase enzyme IIA component) involved in carbohydrate biosynthesis pathways. Our study builds grounds for future experiments to determine genes or pathways that are essential when S. Weltevreden are in aquatic environments and microbial interactions providing survival advantages to S. Weltevreden in such environments.

Molecular epidemiological characteristics of methicillin-resistant Staphylococcus aureus during 2017-2018 at a hospital in Shanghai

Objective: To identify the epidemic clones of MRSA isolates at a hospital in shanghai. Methods: A total of 72 MRSA isolates have been isolated from a second grade hospital between 2017 and 2018, including 32 CA-MRSA isolates, 13 HA-MRSA isolates and 26 MRSA isolates from environment. In this study, MLST and PFGE typing methods were used to analyze the molecular epidemiology of the MRSA isolates. Results: A total of 72 MRSA isolates have been obtained including 46 isolates from clinical specimens, 26 isolates from environments. The 46 MRSA isolates from clinical specimens consisted of 33 CA-MRSA (community-acquired MRSA) and 13 HA-MRSA (hospital-acquired MRSA). Furthermore, these patients infected with MRSA isolates were mostly distributed in the department of geriatrics (34.8%, 16/46), internal medicine (26.1%, 12/46) and surgery (26.1%, 12/46). MLST typing results showed that ST764 was predominant in isolates from both clinical specimens and hospital environments. Furthermore, PFGE typing results showed that most ST764 MRSA had high homolog (>90%). Conclusion: ST764 MRSA isolates might spread in community, hospital and environments. Therefore, continuous monitoring of MRSA and its variation may be useful in understanding the involvement of epidemic clone, and in searching new strategies to control MRSA infection.

Prevalence, Virulence Feature, Antibiotic Resistance and MLST Typing of Bacillus cereus Isolated From Retail Aquatic Products in China.

Bacillus cereus is one of the most important foodborne pathogenic microorganisms, which can lead to gastrointestinal and non-gastrointestinal diseases. However, the potential risk of B. cereus in aquatic products in China has not been comprehensively evaluated yet. In this study, a total of 860 aquatic samples from three types of retail aquatic products were collected from 39 major cities in China from 2011 to 2016. The contamination, distribution of virulence genes, antibiotic resistance and genetic diversity of B. cereus isolates were measured and analyzed. Of all the samples, 219 (25.47%) were positive for B. cereus and 1.83% (4/219) of the samples had contamination levels of more than 1,100 most probable number (MPN)/g. Different isolates had virulence potential, within which 59.6% (164/275) contained all three kinds of enterotoxin genes (nhe, hbl, and cytK-2) and 5.1% (14/275) possessed cereulide encoding gene cesB. The antimicrobial resistance profiles revealed the universal antibiotic resistance to rifampin and most β-lactams, suggesting the necessity to continuously monitor the antibiotic resistance of B. cereus in aquatic products and to control drug use in aquaculture. In sum, our study indicates the potential hazards of B. cereus isolated from aquatic products to customers and may provide a reference for clinical treatment caused by B. cereus.

Genomic Analysis of Staphylococcus aureus Strains Originating from Hungarian Rabbit Farms Reinforce the Clonal Origin of Various Virulence Types.

Simple SummaryStaphylococcosis is a major disease in both human and veterinary medicine. In commercial rabbit production, highly virulent variants cause significant economic losses. This study describes the comparative analysis of the whole genome of 51 strains sequenced by our group, and 12 sequences derived from online repositories. The investigation was based on whole-genome sequencing and whole-genome multilocus sequence typing. The typical highly virulent strains showed great similarity with ST121/t645 isolates from Italy, Spain and the UK. The most prevalent genotype in Hungary was an atypical highly virulent variant; these strains form a novel sequence type (ST5993), and belong to three different spa types (t4770, t711 and t2407). Low virulence (LV) strains grouped into two main clusters, two ST1 LV strains formed a separate cluster from the majority forming a clone-like group, despite the five different multilocus sequence typing (MLST) patterns and seven different spa types. Some strains in this survey showed genetic polymorphism on more than 50% of the examined loci, however, even within the same MLST ST group, hundreds of loci showed polymorphism, which could facilitate the very fine differentiation of the strains.Staphylococcosis is one of the most important infectious diseases in rabbit medicine, especially in commercial farming. Previous studies revealed the existence of virulent variants adapted to rabbits. Typical and atypical, highly virulent as well as low virulent variants have been isolated and reported from industrial units in all major rabbit-meat-producing countries. Preceding the research focused on detecting defined nucleotide sequences, the genome of these organisms as a whole was rarely subjected to scientific investigations. The authors sequenced 51 Staphylococcus strains originating from industrial rabbit farms in Hungary. Another 12 draft genomes of rabbit isolates were constructed from read sequences available in digital repositories, and were compared based on whole-genome multilocus sequence typing. The clonal origin of highly virulent variants is confirmed, the strains from Hungary were closely related with the strains isolated in the UK, Italy, and Spain. Atypical highly virulent strains are the most prevalent in Hungary, they form a separate clonal cluster. The low virulent strains were genetically similar, but more heterogeneous than the highly virulent (HV) and aHV strains even by the traditional MLST typing scheme. Other “non-aureus” Staphylococcus species were also identified.

Marked Presence of Methicillin-Resistant Staphylococcus aureus in Wild Lagomorphs in Valencia, Spain.

Simple SummaryThe presence of bacteria resistant to antibiotics, such as Staphylococcus aureus resistant to methicillin (MRSA), is becoming an increasing everyday concern for their implications for human and animal health. This is even more alarming when such bacteria are isolated in wild animals which, in principle, should not come into contact with antibiotics. This work studied 353 rabbits and 10 hares hunted in rabbit high-density areas. Of these, 41.3% carried S. aureus in some sampled locations, of which 63.3% were MRSA. These are surprising results given the unexpected high presence of MRSA in the studied animals. This finding is very worrying because these animals tend to enter the food chain with no veterinary control, which implies a risk for human health. Thus it is necessary to extend this study to other, less-populated areas with other animal species (ruminants, rabbit predators, hunting dogs, etc.) or even water sources to obtain further knowledge about the origin of bacterial resistances in nature.The appearance of methicillin-resistant strains of Staphylococcus aureus (MRSA) in several animal species (including rabbits) has set off alarms for their capacity to act as reservoirs for this bacterium. This is especially important in wild animals given its epidemiological implications. The objectives of this study were to identify and characterize S. aureus, specifically MRSA, strains in wild lagomorph high-density areas. Ten hares and 353 wild rabbits from 14 towns with a high rabbit density in the Valencian region (eastern Spanish coast) were sampled. Swabs from the nasal cavity, ears, perineum and lesions (when present) were taken for microbiological studies. The detection of different genes and antibiotic susceptibility studies were also carried out. Of all the animals, 41.3% were positive for S. aureus, of which 63.3% were MRSA. Ears were the anatomical location with more S. aureus and MRSA strains. The more frequently identified MLST type was ST1945 (97.1%, 136/140). The mecA gene was found only in one sample. The rest (n = 139) carried the mecC gene and were included in CC130, except one. Penicillin resistance was detected in 28 mec-negative isolates and, in one case, bacitracin resistance. mecA isolate presented resistance to enrofloxacin and tetracycline, and 10 mecC isolates also showed bacitracin resistance. No MRSA isolate was positive for genes chp, sea, tst and PVL. Two ST1945 isolates contained IEC type E (comprising genes scn and sak). mecA-isolate was positive for blaZ. Of the 28 MSSA strains showing resistance to penicillin, 22 carried the blaZ gene. These surprising results highlight the marked presence of MRSA strains in wild rabbits in high-density areas.

Risk Factors for Subsequential Carbapenem-Resistant Klebsiella pneumoniae Clinical Infection Among Rectal Carriers with Carbapenem-Resistant Klebsiella pneumoniae.

PurposeCarbapenem-resistant Klebsiella pneumoniae (CRKP) infection has become a critical clinical concern for its high mortality. Rectal carriage of CRKP has been reported playing an important role in CRKP infection; however, the extent to which carrier develops clinical CRKP infection is unclear. This study aimed to identify risk factors for developing subsequential CRKP clinical infection in rectal carriers with CRKP.Patients and MethodsPatients were screened for rectal carriage of CRKP in a tertiary university hospital; then, rectal CRKP carriers were divided into case group (those who developed subsequential clinical infection) and control group. Demographics, comorbid conditions, invasive procedures, antimicrobial exposure and other clinical parameters of those two groups were compared and analyzed using univariate and multivariate logistic regression analyses. Antimicrobial susceptibility profile and carbapenemase phenotype/genotype of those CRKP isolates were determined. MLST was applied to elucidate the molecular epidemiology of rectal CRKP isolates and clinical infection ones.ResultsEight hundred and thirty-five patients were screened for rectal CRKP carriage. A total of 62 CRKP rectal carriers were identified; among them, 37.1% (23/62) developed CRKP clinical infection. CRKP isolates were resistant to most of the tested antimicrobial agents. ST11 was the dominant MLST type in rectal CRKP isolates (71.0%), and all the 23 clinical infection isolates were ST11. Multivariate analysis revealed that admission to the intensive care unit (ICU) (OR, 6.753; P=0.006), being in coma condition (OR, 11.085; P=0.015) and receiving central venous catheter (OR, 8.628; P=0.003) were independent risk factors for progressing to subsequential CRKP infection among those rectal carriers.ConclusionThis study identified independent risk factors for developing subsequential CRKP clinical infection among CRKP rectal carriers, with being in coma condition as a new finding. It would help clinician target those high-risk rectal CRKP-colonized patients for prevention of subsequential clinical infection.

Investigation of biofilm production and its association with genetic and phenotypic characteristics of OM (osteomyelitis) and non-OM orthopedic Staphylococcus aureus

BackgroundStaphylococcus aureus is a primary pathogen of orthopedic infections. By mediating antimicrobial resistance, S. aureus biofilm plays an important role in the recalcitrance of orthopedic infections, especially for the intractable osteomyelitis (OM). This study investigated the relationship between biofilm production and various genetic or phenotypic characteristics among orthopedic S. aureus strains.MethodsA total of 137 orthopedic S. aureus isolates were enrolled and divided into OM and non-OM groups. Biofilm production was evaluated using the crystal violet assay. Genetic and phenotypic characteristics including MRSA identification, MLST and spa typing, carriage of virulence genes, drug resistance, and patients’ inflammatory responses indicators were characterized. The relationship between biofilm production and above-mentioned features was respectively analyzed among all isolates and compared between OM and non-OM isolates.ResultsBiofilm production presented no significant difference between OM (including 9 MRSA isolates) and non-OM (including 21 MRSA isolates) strains. We found that ST88, t377 and ST630-MSSA-t377 strains produced very strong biofilms, while MLST types of ST15, ST25, ST398, ST5, ST59 and spa types of t002, t2325, t437 tended to produce weaker biofilms. Strains with the following profiles produced stronger biofilms: fib(+)-hlgv(+)-lukED(+)-sei(-)-sem(-)-seo(-) for all isolates, sei(-)-sem(-)-seo(-) for OM isolates, and cna (+)-fib (+)-hlgv (+)-lukED (+)-seb(-)-sed(-) for non-OM isolates. In addition, not any single drug resistance was found to be related to biofilm production. We also observed that, among OM patients, strains with stronger biofilms caused weaker inflammatory responses.ConclusionSome genetic or phenotypic characteristics of orthopedic strains were associated with biofilm production, and this association could be different among OM and non-OM strains. The results are of great significance for better understanding, evaluating and managing different kinds of biofilm-associated orthopedic infections, and provide potential targets for biofilm clearance.

Characterization of the genotype and the phenotype of nontoxigenic strains of Corynebacterium diphtheriae subsp. lausannense isolated in Russian residents

In 2018, a few sequencing studies were published revealing the existence of two monophyletic clusters within the C. diphtheriae species, meaning that this species can be divided into two subspecies: C. diphtheriae subsp. diphtheriae and C. diphtheriae subsp. lausannense. The objective of our study was to describe the genotype and the phenotype of 2 nontoxigenic C. diphtheriae strains isolated in Russia in 2017–2018, which were classified by us as C. diphtheriae subsp. lausannense based on the aggregated data yielded by a variety of techniques, including microbiological and molecular genetic techniques, as well as a bioinformatic search for subspecies-specific genes in the publicly available genomes of C. diphtheriae. The isolated strains had morphological and biochemical characteristics of C. diphtheriae. The strains were assigned to the MLST type ST199 included in the clonal complex associated with subsp. lausannense. PCR revealed that both analyzed strains of C. diphtheriae subsp. lausannense carried the ptsI gene encoding phosphoenolpyruvate-protein phosphotransferase and did not carry the narG gene encoding the synthesis of nitrate reductase subunits, whereas the strains of C. diphtheriae subsp. diphtheriae had the narG gene and did not have ptsI. We experimentally proved the ability of lausannense strains to ferment N-acetylglucosamine. Our findings expand the knowledge of the biological diversity of C. diphtheriae and indicate the need for estimating the spread of these microorganisms in Russia, as well as their pathogenic potential.

Association between vancomycin-resistant Enterococcus faecium colonization and subsequent infection: a retrospective WGS study.

Since 2012, the incidence of vancomycin-resistant Enterococcus faecium (VREfm) has increased dramatically in Copenhagen and vanA E. faecium has become endemic and polyclonal. To examine whether a patient with a positive VRE clinical sample had the same VREfm in a preceding screening sample (within 60 days). We performed a 30 month retrospective study. From our laboratory information system (LIS), we identified all patients with an invasive VREfm isolate and a VREfm rectal screening isolate within 60 days before infection. VREfm pairs (screening isolate and invasive isolate) were whole-genome sequenced. All isolates were analysed using SeqSphere and core-genome MLST (cgMLST) types were determined. We examined all isolates for the presence of the three most dominant vanA plasmids in the Capital Region of Denmark. Two novel vanA plasmids were closed by Nanopore/Illumina sequencing. We found a total of 19 VREfm pairs. Of these, 13 patients had pairs with matching cgMLST types and vanA plasmids and a median number of 6 days from identification of carriage to clinical infection. One patient had a pair with non-matching cgMLST types but matching vanA plasmids and 24 days between identification of carriage to clinical infection. Five patients had pairs with non-matching cgMLST types and non-matching vanA plasmids and a median number of 18 days from identification of carriage to clinical infection. Of our 19 pairs, 13 were a match regarding cgMLST types (68%) and 1 more (5%) had matching vanA plasmids. Infection was thus preceded by colonization with the same isolates in 13 out of 19 patients. The five mismatches (26%) could be explained by the longer interval between colonization and infection.

Whole genome sequencing reveals extended natural transformation in Campylobacter impacting diagnostics and the pathogens adaptive potential

Campylobacter is the major bacterial agent of human gastroenteritis worldwide and represents a crucial global public health burden. Species differentiation of C. jejuni and C. coli and phylogenetic analysis is challenged by inter-species horizontal gene transfer. Routine real-time PCR on more than 4000 C. jejuni and C. coli field strains identified isolates with ambiguous PCR results for species differentiation, in particular, from the isolation source eggs. K-mer analysis of whole genome sequencing data indicated the presence of C. coli hybrid strains with huge amounts of C. jejuni introgression. Recombination events were distributed over the whole chromosome. MLST typing was impaired, since C. jejuni sequences were also found in six of the seven housekeeping genes. cgMLST suggested that the strains were phylogenetically unrelated. Intriguingly, the strains shared a stress response set of C. jejuni variant genes, with proposed roles in oxidative, osmotic and general stress defence, chromosome maintenance and repair, membrane transport, cell wall and capsular biosynthesis and chemotaxis. The results have practical impact on routine typing and on the understanding of the functional adaption to harsh environments, enabling successful spreading and persistence of Campylobacter.

Antibiotic resistance and molecular characteristics of methicillin-resistant Staphylococcus epidermidis recovered from hospital personnel in China

ObjectivesStaphylococcus epidermidis is a major nosocomial pathogen predominantly associated with indwelling medical device infections. Studies reporting on S. epidermidis recovered from hospital personnel in China are scarce. The aim of this study was to evaluate the carriage and antibiotic resistance of S. epidermidis among the hospital personnel in Tianjin, China and provide insights into their genetic diversity. MethodsOne hundred and seven S. epidermidis isolates were recovered from 68 hospital personnel in two public hospitals in Tianjin between March 2018 and May 2018. Staphylococcal cassette chromosome mec (SCCmec) types were determined by the combination of mec and ccr complexes. Multi-locus sequence typing was used to determine the sequence types (ST) of S. epidermidis isolates. ResultsSixty-two (76.5%) isolates were determined to be methicillin-resistant S. epidermidis (MRSE). Thirty-five (51%) of 68 hospital personnel carried S. epidermidis, of which 32 (91%) were carriers of MRSE. All 62 MRSE isolates had high levels of resistance to penicillin (90%) and cefoxitin (100%). Thirty-seven (60%) isolates carried SCCmec type IV, followed by 15 (24%) carrying SCCmec V, and 4 (6%) SCCmec II. Novel STs were assigned to four S. epidermidis isolates (ST832, ST833, ST834 and ST835). ConclusionsIn this study, the majority of MRSE belonged to cluster II domain of CC2. The ST59-IV was a dominant clone among isolates recovered from hospital personnel. Determination of new MLST types confirmed the genetic diversity of these isolates. These observations highlight the need to review the infection control strategies to reduce the carriage of MRSE among hospital personnel.

Genomic Stability of Composite SCCmec ACME and COMER-Like Genetic Elements in Staphylococcus epidermidis Correlates With Rate of Excision.

The epidemiological success of methicillin-resistant Staphylococcus aureus USA300 has been associated with the presence of two mobile elements, the arginine catabolic mobile element (ACME) and the copper and mercury resistance (COMER) element. These two mobile elements are associated with resistance to copper, which has been related to host fitness and survival within macrophages. Several studies found that ACME is more prevalent, and exhibits greater diversity, in Staphylococcus epidermidis while COMER has not been identified in S. epidermidis or any other staphylococcal species. We aimed in this study to evaluate the presence and diversity of ACME and COMER-like elements in our S. epidermidis clinical isolates. The genomes of 58 S. epidermidis clinical isolates, collected between 2009 and 2018 in a Scottish hospital, were sequenced. A core-genome phylogenetic tree and genome based MLST typing showed that more than half of the isolates belong to the clinically predominant sequence type2 (ST2) and these isolates have been found to split into two lineages within the phylogenetic tree. Analysis showed the presence of SCCmec in the majority of isolates. Comparative analysis identified a cluster of ACME-positive isolates with most of them belonging to ST48. ACME showed high variation even between isolates of the same ACME type and ST. COMER-like elements have been identified in one of the two major hospital adapted drug resistant ST2 lineages; and showed high stability. This difference in stability at the genomic level correlates well with the up to one hundred times higher excision frequency found for the SCCmec elements in ACME-containing isolates compared to COMER-like element containing isolates. ACME/COMER-like element positive isolates did not show a significant phenotype of decreased copper susceptibility, while resistance to mercury was over-represented in COMER-like element positive isolates. To the best of our knowledge, this is the first molecular characterization of COMER-like elements in S. epidermidis isolates. The presence of the COMER-like elements is the most prominent accessory genome feature of these successful lineages suggesting that this chromosomal island contributes to the success and wide clinical distribution of ST2 S. epidermidis.

Detection of Streptococcus pyogenes Virulence Factors.

Streptococcus pyogenes encodes multiple virulence factors and their presence is often related to the severity of the disease. We designed the system of four low-volume multiplex PCR reactions to detect genes encoding 20 virulence factors: spd3, sdc, sdaB, sdaD, speB, spyCEP, scpA, mac, sic, speL, speK, speM, speC, speI, speA, speH, speG, speJ, smeZ, and ssa. Classification of strains based on the virulence factors absence or presence correlates with PFGE MLST and emm typing results. The typing/detection system is fast and cost-effective, can be used to detect GAS virulence factors and as a rapid tool to effectively differentiate between strains.

Unidirectional animal-to-human transmission of methicillin-resistant Staphylococcus aureus ST398 in pig farming; evidence from a surveillance study in southern Italy

BackgroundLivestock-associated methicillin-resistant Staphylococcus aureus (MRSA) belonging to clonal complex 398 is recognized as an occupational hazard for workers employed in intensive animal husbandry, especially in the swine-breeding chain. In this study, we compared the prevalence and epidemiological type of MRSA isolates from swine and farm workers in a large area of southern Italy.MethodsBetween January and March 2018, 88 workers from 32 farms where we had previously performed a survey for MRSA colonization of farmed pigs, were sampled by nasal swabbing. A follow-up investigation was conducted on seven workers 1 year after primary screening. MRSA isolates were characterized by MLST, spa and SCCmec typing, and tested for susceptibility to 15 antimicrobials. Epidemiological correlations between human and swine MRSA isolates were supported by Rep-MP3 and RAPD PCR fingerprinting, and whole-genome sequencing.ResultsThe overall colonization rate of MRSA in swine farm workers was 21.6%, being significantly higher in intensive farms and in workers with direct animal contact. All human MRSA isolates were multi-drug resistant, belonged to the ST398 livestock clade, and did not carry Panton-Valentine leukocidin and enterotoxin genes. Notably, 94.1% of human MRSA isolates belonged to the same epidemiological type as swine MRSA isolates from the corresponding farm. Persistent MRSA carriage was documented in some workers 1 year after primary sampling.ConclusionsWe report a high prevalence of MRSA among swine farm workers, with higher colonization rates associated with intensive breeding and animal exposure. Our findings suggest unidirectional animal-to-human transmission of LA-MRSA and denote the high zoonotic transmissibility of the ST398 livestock clade.

Comparative assessment of genotypic and phenotypic correlates of Staphylococcus pseudintermedius strains isolated from dogs with otitis externa and healthy dogs

Staphylococcus pseudintermedius is considered a primary pathogen of canine skin and soft tissue infections, and the rapid emergence of methicillin-resistant S. pseudintermedius worldwide is a major issue. In the current study, genotypic and phenotypic correlates associated with S. pseudintermedius causing canine otitis externa were evaluated using 41 S. pseudintermedius strains isolated from dogs with otitis externa (n = 26) and healthy dogs (n = 15). The S. pseudintermedius strains were subjected to a comparative analysis of (i) genotypes (multilocus sequence typing, agr, and spa types), (ii) methicillin resistance and SCCmec types, (iii) multidrug resistance (MDR), (iv) biofilm formation, and (v) susceptibility to canine cathelicidin (K9CATH). A high degree of genetic diversity was observed in both groups of S. pseudintermedius strains, regardless of methicillin resistance. Almost all methicillin-resistant strains (>95%) harbored SCCmec V and displayed MDR. Although there was no difference in biofilm formation, S. pseudintermedius strains derived from otitis externa exhibited enhanced resistance to cationic antimicrobial peptide (K9CATH) compared with strains from healthy dogs. The high degree of heterogeneity in MLST, agr, and spa types prevented the identification of correlations between any specific genotype and virulence phenotype in otitis externa caused by S. pseudintermedius, These findings provide an important basis for monitoring and treating canine skin and soft tissue infections in Korea.

Retrospective investigation of listeriosis outbreaks in small ruminants using different analytical approaches for whole genome sequencing-based typing of Listeria monocytogenes

Listeria monocytogenes is the causative agent of listeriosis, a serious disease affecting both humans and animals. While listeriosis outbreaks in humans are commonly investigated in detail, routine typing of L. monocytogenes is generally not performed in animal outbreaks. Here, seven presumable listeriosis outbreaks in small ruminants were retrospectively identified based on the pulsed-field gel electrophoresis (PFGE) profiles. Outbreaks were further characterised using three different analytical approaches based on the whole-genome sequencing (WGS) data: core-genome multilocus sequence typing (cgMLST), whole-genome MLST (wgMLST) and whole-genome single nucleotide polymorphism (wgSNP) typing. A monoclonal pattern of all seven outbreaks was identified using all three approaches, indicating common-source outbreaks. The outbreak strains belonged to sequence types (STs) 1 (n = 3), ST18 (n = 1), ST21 (n = 2) and ST184 (n = 1). Two epidemiologically linked ST1 outbreaks with indistinguishable PFGE profiles showed a polyphyletic nature and differed in >78 SNPs; thus, they were classified as separate outbreaks according to WGS. In ST184, the outbreak strain was also found in faeces of apparently healthy ruminants, silage and water collected from the trough, which were the most likely source(s) of infection. The outbreak-associated isolates differed in 0–7 cgMLST alleles, 0–12 wgMLST alleles and 1–13 SNPs. The minimum genetic diversity between outbreak-associated isolates and epidemiologically unrelated isolates of the same ST was low in all analysed cases, approaching the maximum diversity within the outbreak cluster. The results suggest that a fixed threshold to define the outbreak cluster should only be considered as a guide and highlight the role of epidemiological data for outbreak confirmation. The identified cgMLST clusters may be further investigated by wgMLST and/or wgSNP typing to increase confidence during investigations of outbreaks caused by highly clonal L. monocytogenes groups. This study gives an overview of the inter- and intra-outbreak genetic diversity of L. monocytogenes strains involved in animal outbreaks, hence improving their investigation.

Epidemiology of invasive meningococcal disease in Greece, 2006-2016.

The present study describes the epidemiology of invasive meningococcal disease (IMD) in Greece for the period 2006-2016. Combined data from notified and laboratory-confirmed IMD cases were obtained from the two involved National Centres (Epidemiology and Reference Laboratory). Laboratory identification and typing was carried out by both conventional (culture) and molecular methods (PCR, MLST, PorA, and FetA typing). A total of 796 IMD cases were notified; of those, 720 (91%) were laboratory confirmed. Overall, a decline on the annual incidence of confirmed cases was observed, ranging from 0.91 (2006) to 0.47 (2016) /100,000. A similar trend was observed in most age groups especially in children 0-4years (7.7 to 2.9/100,000), with the exception of an increase in the incidence rate in adults > 20years (0.21 to 0.32/100,000). The overall case fatality rate was 6.5% (52/796), annual range 2-13%. Among 658 strains which were typed by sero/genogroup, 80% were identified as MenB (annual range 65-92%); however, a decline was observed in MenB incidence from 5.3 (2006) to 2.7 (2016), among infants and toddlers, while MenW (1%), MenY (2%), and MenA (1%) remained low. During the 11years, the annual incidence of IMD declined by 50%, especially in the 0-4-year age group, due mainly to MenB. Continuous surveillance of IMD is important for the development of future vaccination and public health policies.