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Background: Bovine respiratory disease (BRD) is common in intensively raised cattle and is often treated with antibiotics. For practitioners, knowledge of the bacteria involved in an outbreak and their antibiotic susceptibility is warranted. To this end, samples from the upper or lower respiratory tract of calves can be submitted for bacteriological culture and susceptibility testing of relevant isolates. However, it is debated whether isolates from the upper respiratory tract are representative of bacteria causing infections in the lower respiratory tract. In this study, we used MALDI-TOF MS, multilocus sequence typing (MLST) and core-genome multilocus sequence typing (cgMLST) to compare culture results of 219 paired samples (sample pairs) of deep nasal swabs (DNS) and tracheobronchial lavage (TBL). The sample pairs came from 171 calves in 30 calf groups across 25 farms with 48 calves sampled twice. Results: The predominant bacterial pathogen was Pasteurella multocida, which was isolated from 37.4% of DNS and 22.4% of TBL. There was no statistically significant difference in isolation frequency of P. multocida between calves considered healthy and those suspected for BRD for DNS (P = 0.778) or TBL (P = 0.410). Among the 49 sample pairs where P. multocida was isolated from TBL, the same species was isolated from DNS in 29 sample pairs (59.2%). Isolates from 28 of these sample pairs were evaluated by MLST, and in 24 pairs (86.0%) P. multocida from DNS and TBL were of the same sequence type (ST). Moreover, cgMLST showed that the genetic distance between isolates within 21 of the 28 sample pairs (75.0%), was less than two alleles, and DNS and TBL isolates were considered identical. In seven sample pairs (25%), the genetic distance was greater, and DNS and TBL isolates were considered nonidentical. Conclusions: Pasteurella multocida was readily isolated from DNS and in calves where this species was isolated also from TBL, DNS and TBL isolates were identical in 75% of the sample pairs. This suggests that during an outbreak of BRD, submission of DNS samples from 4 to 6 calves could be a convenient approach for practitioners seeking guidance on P. multocida present in the lower respiratory tract and their antibiotic susceptibility. [ABSTRACT FROM AUTHOR]

Abstract Background Bovine respiratory disease (BRD) is common in intensively raised cattle and is often treated with antibiotics. For practitioners, knowledge of the bacteria involved in an outbreak and their antibiotic susceptibility is warranted. To this end, samples from the upper or lower respiratory tract of calves can be submitted for bacteriological culture and susceptibility testing of relevant isolates. However, it is debated whether isolates from the upper respiratory tract are representative of bacteria causing infections in the lower respiratory tract. In this study, we used MALDI-TOF MS, multilocus sequence typing (MLST) and core-genome multilocus sequence typing (cgMLST) to compare culture results of 219 paired samples (sample pairs) of deep nasal swabs (DNS) and tracheobronchial lavage (TBL). The sample pairs came from 171 calves in 30 calf groups across 25 farms with 48 calves sampled twice. Results The predominant bacterial pathogen was Pasteurella multocida, which was isolated from 37.4% of DNS and 22.4% of TBL. There was no statistically significant difference in isolation frequency of P. multocida between calves considered healthy and those suspected for BRD for DNS (P = 0.778) or TBL (P = 0.410). Among the 49 sample pairs where P. multocida was isolated from TBL, the same species was isolated from DNS in 29 sample pairs (59.2%). Isolates from 28 of these sample pairs were evaluated by MLST, and in 24 pairs (86.0%) P. multocida from DNS and TBL were of the same sequence type (ST). Moreover, cgMLST showed that the genetic distance between isolates within 21 of the 28 sample pairs (75.0%), was less than two alleles, and DNS and TBL isolates were considered identical. In seven sample pairs (25%), the genetic distance was greater, and DNS and TBL isolates were considered nonidentical. Conclusions Pasteurella multocida was readily isolated from DNS and in calves where this species was isolated also from TBL, DNS and TBL isolates were identical in 75% of the sample pairs. This suggests that during an outbreak of BRD, submission of DNS samples from 4 to 6 calves could be a convenient approach for practitioners seeking guidance on P. multocida present in the lower respiratory tract and their antibiotic susceptibility.

Aims: Our aim is to characterize through whole‐genome sequencing (WGS) the antimicrobial resistance (AMR) and heavy metal tolerance (HMT) genes content, plasmid presence, virulence potential and genomic diversity of the rare non‐typhoid Salmonella enterica serovar Orion (S. Orion) from 19 countries of the African, American, Eastern Mediterranean, European, Southeastern Asia and Western Pacific regions. Methods and Results: Totally 324 S. Orion genomes were screened for AMR, HMT and virulence genes, plasmids and Salmonella Pathogenicity Islands (SPIs). Genomic diversity was investigated using Multi‐Locus Sequence Typing (MLST) and core‐genome MLST (cgMLST). Efflux pump encoding genes mdsA and mdsB were present in all genomes analysed, while quinolone chromosomal point mutations and aminoglycoside, beta‐lactam, colistin, lincosamide, macrolide, phenicol, sulphonamide, trimethoprim, tetracycline and disinfectant resistance genes were found in 0.3%–5.9%. A total of 17 genomes (5.2%) from Canada, the United Kingdom, the USA and Tanzania showed a potential multi‐drug resistance profile. Gold tolerance genes golS and golT were detected in all genomes analysed, while arsenic, copper, mercury, silver and tellurium tolerance genes were found in 0.3%–35.5%. Col(MGD2) was the most frequently detected plasmid, in 15.4% of the genomes. Virulence genes related to adherence, macrophage induction, magnesium uptake, regulation, serum resistance, stress adaptation, type III secretion systems and six SPIs (1, 2, 3, 4, 5, 9, 12, 13, 14 and C63PI) were detected. ST639 was assigned to 89.2% of the S. Orion genomes, while cgMLST showed core‐genome STs and clusters of strains specific by countries. Conclusion: The high virulence factor frequencies, the genomic similarity among some non‐clinical and clinical strains circulating worldwide and the presence of a strain carrying a resistance gene against a last resource antimicrobial like colistin, highlight the potential risk of S. Orion strains for public health and food safety and reinforce the importance to not underestimate the potential hazard of rare non‐typhoid Salmonella serovars. [ABSTRACT FROM AUTHOR]

Abstract Background In most of Europe and especially in Germany, there is currently a concerning rise in the number of hospital-acquired infections due to vancomycin-resistant Enterococcus faecium (VREfm). Therefore, there is a need to improve our understanding of the way VREfm spreads in hospitals. In this study, we investigated the molecular epidemiology of VREfm isolates from the first appearance at our university hospital in 2004 until 2010. There is only very scarce information about the molecular epidemiology of VREfm from this early time in Germany. Methods Our analysis includes all available first VREfm isolates of each patient at our tertiary care center collected during the years 2004–2010. If available, additional consecutive VREfm isolates from some patients were analyzed. We used multilocus sequence typing (MLST) and core genome multilocus sequence typing (cgMLST) for the analysis and description of nosocomial transmission pathways as well as the detection of outbreaks. Results VREfm isolates from 158 patients and 76 additional subsequent patient isolates were included in the analysis. Until 2006, detections of VREfm remained singular cases, followed by a peak in the number of VREfm cases in 2007 and 2008 with a subsequent decline to baseline in 2010. MLST and cgMLST analysis show significant changes in the dominant sequence types (STs) and complex types (CTs) over the study period, with ST192 and ST17 being responsible for the peak in VREfm cases in 2007 and 2008. The four largest clusters detected during the study period are comprised of these two STs. Cluster analysis shows a focus on specific wards and departments for each cluster. In the early years of this study (2004–2006), all analyzed VREfm stemmed from clinical specimens, whereas since 2007, approximately half of the VREfm were detected by screening. Of the 234 VREfm isolates analyzed, 96% had a vanB and only 4% had a vanA resistance genotype. Conclusions This retrospective study contributes significant knowledge about regional VREfm epidemiology from this early VREfm period in Germany. One remarkable finding is the striking dominance of vanB-positive VREfm isolates over the entire study period, which is in contrast with countrywide data. Analysis of cgMLST shows the transition from sporadic VRE cases at our institution to a sharp increase in VRE numbers triggered by oligoclonal spread and specific outbreak clusters with the dominance of ST192 and ST17.

Background: In most of Europe and especially in Germany, there is currently a concerning rise in the number of hospital-acquired infections due to vancomycin-resistant Enterococcus faecium (VREfm). Therefore, there is a need to improve our understanding of the way VREfm spreads in hospitals. In this study, we investigated the molecular epidemiology of VREfm isolates from the first appearance at our university hospital in 2004 until 2010. There is only very scarce information about the molecular epidemiology of VREfm from this early time in Germany. Methods: Our analysis includes all available first VREfm isolates of each patient at our tertiary care center collected during the years 2004–2010. If available, additional consecutive VREfm isolates from some patients were analyzed. We used multilocus sequence typing (MLST) and core genome multilocus sequence typing (cgMLST) for the analysis and description of nosocomial transmission pathways as well as the detection of outbreaks. Results: VREfm isolates from 158 patients and 76 additional subsequent patient isolates were included in the analysis. Until 2006, detections of VREfm remained singular cases, followed by a peak in the number of VREfm cases in 2007 and 2008 with a subsequent decline to baseline in 2010. MLST and cgMLST analysis show significant changes in the dominant sequence types (STs) and complex types (CTs) over the study period, with ST192 and ST17 being responsible for the peak in VREfm cases in 2007 and 2008. The four largest clusters detected during the study period are comprised of these two STs. Cluster analysis shows a focus on specific wards and departments for each cluster. In the early years of this study (2004–2006), all analyzed VREfm stemmed from clinical specimens, whereas since 2007, approximately half of the VREfm were detected by screening. Of the 234 VREfm isolates analyzed, 96% had a vanB and only 4% had a vanA resistance genotype. Conclusions: This retrospective study contributes significant knowledge about regional VREfm epidemiology from this early VREfm period in Germany. One remarkable finding is the striking dominance of vanB-positive VREfm isolates over the entire study period, which is in contrast with countrywide data. Analysis of cgMLST shows the transition from sporadic VRE cases at our institution to a sharp increase in VRE numbers triggered by oligoclonal spread and specific outbreak clusters with the dominance of ST192 and ST17. [ABSTRACT FROM AUTHOR]

Although Campylobacter jejuni is the pathogen responsible for the most common foodborne illness, tracing of the infection source remains challenging due to its highly variable genome. Therefore, one of the aim of the study was to compare three genotyping methods (MLST, PFGE, and mP-BIT) to determine the most effective genotyping tool. C. jejuni strains were divided into 4 clusters based on strain similarity in the cgMLST dendrogram. Subsequently, the dendrograms of the 3 tested methods were compared to determine the accuracy of each method compared to the reference cgMLST method. Moreover, a cost-benefit analysis has showed that MLST had the highest inverse discrimination index (97%) and required less workflow, time, fewer consumables, and low bacterial sample quantity. PFGE was shown to be obsolete both because of its low discriminatory power and the complexity of the procedure. Similarly, mP‑BIT showed low separation results, which was compensated by its high availability. Therefore, our data showed that MLST is the optimal tool for genotyping C. jejuni. Another aim was to compare the antimicrobial resistance to ciprofloxacin, erythromycin, and tetracycline in C. jejuni strains isolated from human, water, air, food, and animal samples by two gene sequence-based prediction methods and to compare them with the actual susceptibility of C. jejuni strains using the disc diffusion method. Both tools, ResFinder and RGI, synchronously predict the antimicrobial susceptibility of C. jejuni and either can be used. [ABSTRACT FROM AUTHOR]

This study evaluated the contribution of cattle, sheep, poultry and pigs to the contamination of surface water from rivers by Campylobacter jejuni and C. coli using MLST, cgMLST and considered MALDI-TOF MS as an alternative technique. The 263 strains isolated from cattle (n = 61), sheep (n = 42), poultry (n = 65), pigs (n = 60) and surface water (n = 35) were distributed across 115 sequence types (STs), 49 for C. jejuni and 66 for C. coli. Considering MLST data, 14.2%, 11.4% and 2.8% of the surface water strains could be attributed to cattle, poultry and sheep, respectively, none to pigs, and 85.7% were non-attributed. Analysis of cg-MLST data with STRUCTURE indicated that C. jejuni strains from water were predominantly attributed to poultry (93.5%), weakly to sheep (<1%) and 6.3% non-attributed, and that conversely, C. coli strains from water were predominantly non-attributed (94.3%) and 5.7% attributed to poultry. Considering the protein profiles with a threshold of 94% and 97% of similarity, respectively, strains from surface water could be attributed to poultry (31.4% and 17.1%), and to cattle (17.1% and 5.7%); 54.1% and 77.1% were non-attributed. This study confirmed these livestock animals might contribute to the contamination of surface water, with a level of contribution depending on the typing technique and the method of analysis. MALDI-TOF could potentially be an alternative approach for source attribution. [ABSTRACT FROM AUTHOR]

Abstract Background Bovine mastitis accounts for significant economic losses to the dairy industry worldwide. Staphylococcus aureus is the most common causative agent of bovine mastitis. Investigating the prevalence of virulence factors and antimicrobial resistance would provide insight into the molecular epidemiology of mastitis-associated S. aureus strains. The present study is focused on the whole genome sequencing and comparative genomic analysis of 41 mastitis-associated S. aureus strains isolated from India. Results The results elucidate explicit knowledge of 15 diverse sequence types (STs) and five clonal complexes (CCs). The clonal complexes CC8 and CC97 were found to be the predominant genotypes comprising 21 and 10 isolates, respectively. The mean genome size was 2.7 Mbp with a 32.7% average GC content. The pan-genome of the Indian strains of mastitis-associated S. aureus is almost closed. The genome-wide SNP-based phylogenetic analysis differentiated 41 strains into six major clades. Sixteen different spa types were identified, and eight isolates were untypeable. The cgMLST analysis of all S. aureus genome sequences reported from India revealed that S. aureus strain MUF256, isolated from wound fluids of a diabetic patient, was the common ancestor. Further, we observed that all the Indian mastitis-associated S. aureus isolates belonging to the CC97 are mastitis-associated. We identified 17 different antimicrobial resistance (AMR) genes among these isolates, and all the isolates used in this study were susceptible to methicillin. We also identified 108 virulence-associated genes and discuss their associations with different genotypes. Conclusion This is the first study presenting a comprehensive whole genome analysis of bovine mastitis-associated S. aureus isolates from India. Comparative genomic analysis revealed the genome diversity, major genotypes, antimicrobial resistome, and virulome of clinical and subclinical mastitis-associated S. aureus strains.

ABSTRACT: Objectives: Carbapenem-resistant Acinetobacter baumannii (CRAB) is a prevalent pathogen contributing to hospital infections. Pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST) and core-genome MLST (cgMLST) are frequently used methods to illuminate the nosocomial transmission of CRAB. In this study, we compared the discriminatory power of the three typing methods. Methods: Antimicrobial susceptibility tests were performed by the broth microdilution and Vitek2 methods. PFGE, MLST and cgMLST were conducted to determine the clonality and phylogenetic relationship of the strains. Whole-genome sequence data were acquired by an Illumina HiSeq 2000, and cgMLST was analysed by the Ridom SeqSphere+ v.7.2.3 software. Results: A total of 149 carbapenem-resistant A. baumannii isolates had 15 different PFGE profiles (A-O type), and 73 of the isolates had related subtypes (A1 and A2), accounting for the majority of type A isolates. The maximum-likelihood phylogenetic analysis based on the cgMLST genes grouped the same PFGE clonal pattern A into nine different clusters. ST_Pasteur grouped all the strains into ST2, whereas ST_Oxford grouped the PFGE clonal pattern A isolates into six STs. In addition, the gdhB allele in the ST_Oxford scheme had two copies in five strains, which complicated the ST_Oxford typing. Conclusions: cgMLST was more discriminant than PFGE and MLST. CgMLST is the most suitable and comprehensive method for genotyping A. baumannii in surveillance and epidemiological research.

Background: Bovine mastitis accounts for significant economic losses to the dairy industry worldwide. Staphylococcus aureus is the most common causative agent of bovine mastitis. Investigating the prevalence of virulence factors and antimicrobial resistance would provide insight into the molecular epidemiology of mastitis-associated S. aureus strains. The present study is focused on the whole genome sequencing and comparative genomic analysis of 41 mastitis-associated S. aureus strains isolated from India. Results: The results elucidate explicit knowledge of 15 diverse sequence types (STs) and five clonal complexes (CCs). The clonal complexes CC8 and CC97 were found to be the predominant genotypes comprising 21 and 10 isolates, respectively. The mean genome size was 2.7 Mbp with a 32.7% average GC content. The pan-genome of the Indian strains of mastitis-associated S. aureus is almost closed. The genome-wide SNP-based phylogenetic analysis differentiated 41 strains into six major clades. Sixteen different spa types were identified, and eight isolates were untypeable. The cgMLST analysis of all S. aureus genome sequences reported from India revealed that S. aureus strain MUF256, isolated from wound fluids of a diabetic patient, was the common ancestor. Further, we observed that all the Indian mastitis-associated S. aureus isolates belonging to the CC97 are mastitis-associated. We identified 17 different antimicrobial resistance (AMR) genes among these isolates, and all the isolates used in this study were susceptible to methicillin. We also identified 108 virulence-associated genes and discuss their associations with different genotypes. Conclusion: This is the first study presenting a comprehensive whole genome analysis of bovine mastitis-associated S. aureus isolates from India. Comparative genomic analysis revealed the genome diversity, major genotypes, antimicrobial resistome, and virulome of clinical and subclinical mastitis-associated S. aureus strains. [ABSTRACT FROM AUTHOR]

Simple Summary: Strangles is a highly infectious disease of equines caused by Streptococcus equi ssp. equi (S. equi equi). The objective of this study was to characterize S. equi equi isolates obtained from suspected strangles cases in Indonesia using whole genome sequence-based analyses and antimicrobial susceptibility testing. Isolates were recovered from seven diseased horses on four farms in three different provinces in 2018. All these S. equi equi isolates were classified as ST179 and carried seM allele 166. Isolates differed from each other by only 2 to 14 core genome (cg) SNPs and built an exclusive sub-cluster in Bayesian Analysis of Population Structure cluster 2 (BAPS-2) of the cgMLST scheme. All isolates revealed predicted amino acid sequence identity to seven and high similarity to one of the eight antigen fragments contained in the Strangvac® recombinant subunit vaccine. Furthermore, all isolates were susceptible to beta-lactam antibiotics penicillin G, ampicillin, and ceftiofur. Our data suggest that the horses referred to in this study were affected by strains of the same novel sublineage within globally distributed BAPS-2 of S. equi equi. Nevertheless, penicillin G can be used as a first-choice antibiotic for treatment and Strangvac® may also be protective against these strains. Strangles, caused by Streptococcus equi ssp. equi (S. equi equi), is a highly infectious and frequent disease of equines worldwide. No data are available regarding the molecular epidemiology of strangles in Indonesia. This study aimed to characterize S. equi equi isolates obtained from suspected strangles cases in Indonesia in 2018. Isolates originated from seven diseased horses on four different farms located in three provinces of Indonesia. Whole genome sequences of these isolates were determined and used for seM typing, multilocus sequence typing (MLST), and core genome MLS typing (cgMLST). Genomes were also screened for known antimicrobial resistance genes and genes encoding for the recombinant antigens used in the commercial Strangvac® subunit vaccine. All seven S. equi equi isolates from Indonesia belonged to ST179 and carried seM allele 166. Isolates differed from each other by only 2 to 14 cgSNPs and built an exclusive sub-cluster within the Bayesian Analysis of Population Structure (BAPS) cluster 2 (BAPS-2) of the S. equi equi cgMLST scheme. All isolates revealed predicted amino acid sequence identity to seven and high similarity to one of the eight antigen fragments contained in Strangvac®. Furthermore, all isolates were susceptible to beta-lactam antibiotics penicillin G, ampicillin, and ceftiofur. Our data suggest that the horses from this study were affected by strains of the same novel sublineage within globally distributed BAPS-2 of S. equi equi. Nevertheless, penicillin G can be used as a first-choice antibiotic against these strains and Strangvac® may also be protective against Indonesian strains. [ABSTRACT FROM AUTHOR]

Methicillin-resistant Staphylococcus aureus (MRSA) isolates remain a serious threat to global health despite a decrease in MRSA infections since 2005. MRSA isolates exhibit great diversity worldwide, and their lineages show geographic variation. In this study, we used whole genome sequencing (WGS) to analyze antibiotic resistance genes and virulence genes, spa, staphylococcal cassette chromosome mec, sequence types (STs), and core genome multilocus sequence typing (cgMLST) of MRSA isolates from patients and environmental surface in hospitals in China to determine their prevalence and molecular traits. The highest number of infections by MRSA isolates was observed in patients aged ≥60 years (69.8%, P < 0.05). We identified a total of 19 STs from 162 MRSA isolates from patients. A significant increase was observed in the incidence of ST764-t002-II MRSA infection, which is replacing ST5-t002-II MRSA as the predominant ST. Similarly, isolates from environmental surface were predominantly ST764-t002-II (47%). Notably, most ST764 isolates (97.7%) carried seb, but not arginine catabolic mobile element (ACME), which differed from ST764 isolates in Japan and Thailand. The potential danger of spreading requires rigorous surveillance of emerging ST764 MRSA isolates. We also found higher resistance to seven antimicrobials [OXA, cefoxitin (FOX), ERY, CFZ, ciprofloxacin (CIP), levofloxacin (LEV), and moxifloxacin (MXF)]. Resistance to gentamicin (38.3%), tetracycline (55.9%), and minocycline (41.5%) were also common. Phenotypic resistance to antimicrobials was associated with resistance genes to its content, and cgMLST clustering suggested a strong link between these strains. Overall, our findings revealed the prevalence and molecular characteristics of MRSA isolates in Shanghai, China, providing a theoretical reference for preventing and controlling clonal transmission of MRSA isolates in hospitals in China. [ABSTRACT FROM AUTHOR]

This study evaluated the contribution of cattle, sheep, poultry and pigs to the contamination of surface water from rivers by Campylobacter jejuni and C. coli using MLST, cgMLST and considered MALDI-TOF MS as an alternative technique. The 263 strains isolated from cattle (n = 61), sheep (n = 42), poultry (n = 65), pigs (n = 60) and surface water (n = 35) were distributed across 115 sequence types (STs), 49 for C. jejuni and 66 for C. coli. Considering MLST data, 14.2%, 11.4% and 2.8% of the surface water strains could be attributed to cattle, poultry and sheep, respectively, none to pigs, and 85.7% were non-attributed. Analysis of cg-MLST data with STRUCTURE indicated that C. jejuni strains from water were predominantly attributed to poultry (93.5%), weakly to sheep (C. coli strains from water were predominantly non-attributed (94.3%) and 5.7% attributed to poultry. Considering the protein profiles with a threshold of 94% and 97% of similarity, respectively, strains from surface water could be attributed to poultry (31.4% and 17.1%), and to cattle (17.1% and 5.7%); 54.1% and 77.1% were non-attributed. This study confirmed these livestock animals might contribute to the contamination of surface water, with a level of contribution depending on the typing technique and the method of analysis. MALDI-TOF could potentially be an alternative approach for source attribution.

Campylobacter jejuni (C. jejuni), is considered among the most common bacterial causes of human bacterial gastroenteritis worldwide. The epidemiology and the transmission dynamics of campylobacteriosis in Egypt remain poorly defined due to the limited use of high‐resolution typing methods. In this pilot study, we evaluated the discriminatory power of multiple typing 'gene‐by‐gene based' techniques to characterize C. jejuni obtained from different sources and estimate the relative contribution of different potential sources of C. jejuni infection in Egypt. Whole genome sequencing (WGS) was performed on 90 C. jejuni isolates recovered from clinical samples, retail chicken, and dairy products in Egypt from 2017 to 2018. Comparative genomic analysis was performed using conventional seven‐locus multilocus sequence typing (MLST), ribosomal MLST (rMLST), core genome MLST (cgMLST), allelic variation in 15 host‐segregating (HS) markers, and comparative genomic fingerprinting (CGF40). The probabilistic source attribution was performed via STRUCTURE software using MLST, CGF40, cgMLST and allelic variation in HS markers. Comparison of the discriminatory power of the aforementioned genotyping methods revealed cgMLST to be the most discriminative method, followed by HS markers. The source attribution analysis showed the role of retail chicken as a source of infection among clinical cases in Egypt when HS and cgMLST were used (64.2% and 52.3% of clinical isolates were assigned to this source, respectively). Interestingly, the cattle reservoir was also identified as a contributor to C. jejuni infection in Egypt; 35.8% and 47.7% of clinical isolates were assigned to this source by HS and cgMLST, respectively. Here, we provided evidence of the importance of using WGS typing methods to facilitate source tracking of C. jejuni. Our findings suggest the importance of non‐poultry sources, together with the previously reported role of retail chicken in human campylobacteriosis in Egypt that can provide insights to inform national control measures. [ABSTRACT FROM AUTHOR]

Introduction. Listeriosis is a foodborne infection, especially dangerous for people in at-risk groups. Susceptibility to listeria infection is determined by a complex of reasons: environmental factors, host immune status, and pathogen virulence. The susceptibility to listeriosis can also be aggravated by previous infections, especially viral infections, which demonstrate a steadily increasing number of identified pathogens.The aim of our study was to present molecular and genetic characterization of pathogens causing sporadic invasive listeriosis in a megalopolis, primarily during the peak of influenza and ARVI incidence.Materials and methods. Listeria monocytogenes isolates were collected from 18 hospitalized patients at hospitals in Moscow, from November 2018 to October 2019. The first comparison group was represented by isolates from food products and fish preserves. The second comparison group included previously examined environmental isolates. The clinical isolates were examined by using multilocus sequence typing techniques, including the standard MLST scheme extended by loci of internalin genes. Isolates of the autochthonous genotype (ST7) were compared through whole-genome sequencing and subsequent analysis of the core genome (cgMLST).Results. In cases of invasive listeriosis, 44% of isolates were isolated from patients with listeriosis; 27% of isolates were obtained from patients with meningitis. L. monocytogenes of phylogenetic lineage II prevailed in these groups of cases that occurred when the epidemic threshold for influenza was crossed during the 2018/2019 season. Listeria pneumonia identified in the senior age group occurred during the season of autumn ARVI and was primarily caused by L. monocytogenes of phylogenetic lineage I. The examination of genomes of ST7 isolates demonstrated identity between the core genomes of bacteria isolated from the mother-infant pair. Out of ST7 food isolates most closely related to the clinical ones was the isolate from meat (23 locus differences, the common deletion in the MFS transporter locus). Analyzing invasive listeriosis, the comparison between the list of the identified genotypes and the data from European countries showed that each country had its own specific range of genotypes, though ST7 was detected in all the examined samples.Conclusions. Along with the monitoring of food manufacturing and storage, timely vaccination against seasonal respiratory infections and use of personal protective equipment in public spaces can reduce the risk of listeriosis incidence in at-risk groups.

Strangles, caused by Streptococcus equi ssp. equi (S. equi equi), is a highly infectious and frequent disease of equines worldwide. No data are available regarding the molecular epidemiology of strangles in Indonesia. This study aimed to characterize S. equi equi isolates obtained from suspected strangles cases in Indonesia in 2018. Isolates originated from seven diseased horses on four different farms located in three provinces of Indonesia. Whole genome sequences of these isolates were determined and used for seM typing, multilocus sequence typing (MLST), and core genome MLS typing (cgMLST). Genomes were also screened for known antimicrobial resistance genes and genes encoding for the recombinant antigens used in the commercial Strangvac® subunit vaccine. All seven S. equi equi isolates from Indonesia belonged to ST179 and carried seM allele 166. Isolates differed from each other by only 2 to 14 cgSNPs and built an exclusive sub-cluster within the Bayesian Analysis of Population Structure (BAPS) cluster 2 (BAPS-2) of the S. equi equi cgMLST scheme. All isolates revealed predicted amino acid sequence identity to seven and high similarity to one of the eight antigen fragments contained in Strangvac®. Furthermore, all isolates were susceptible to beta-lactam antibiotics penicillin G, ampicillin, and ceftiofur. Our data suggest that the horses from this study were affected by strains of the same novel sublineage within globally distributed BAPS-2 of S. equi equi. Nevertheless, penicillin G can be used as a first-choice antibiotic against these strains and Strangvac® may also be protective against Indonesian strains.

Salmonella enterica (S. enterica) is an important foodborne pathogen, causing food poisoning and human infection, and critically threatening food safety and public health. Salmonella typing is essential for bacterial identification, tracing, epidemiological investigation, and monitoring. Serotyping and multilocus sequence typing (MLST) analysis are standard bacterial typing methods despite the low resolution. Core genome MLST (cgMLST) is a high-resolution molecular typing method based on whole genomic sequencing for accurate bacterial tracing. We investigated 250 S. enterica isolates from poultry, livestock, food, and human sources in nine provinces of China from 2004 to 2019 using serotyping, MLST, and cgMLST analysis. All S. enterica isolates were divided into 36 serovars using slide agglutination. The major serovars in order were Enteritidis (31 isolates), Typhimurium (29 isolates), Mbandaka (23 isolates), and Indiana (22 isolates). All strains were assigned into 43 sequence types (STs) by MLST. Among them, ST11 (31 isolates) was the primary ST. Besides this, a novel ST, ST8016, was identified, and it was different from ST40 by position 317 C → T in dnaN. Furthermore, these 250 isolates were grouped into 185 cgMLST sequence types (cgSTs) by cgMLST. The major cgST was cgST235530 (11 isolates), and only three cgSTs contained isolates from human and other sources, indicating a possibility of cross-species infection. Phylogenetic analysis indicated that most of the same serovar strains were putatively homologous except Saintpaul and Derby due to their multilineage characteristics. In addition, serovar I 4,[5],12:i:- and Typhimurium isolates have similar genomic relatedness on the phylogenetic tree. In conclusion, we sorted out the phenotyping and genotyping diversity of S. enterica isolates in China during 2004–2019 and clarified the temporal and spatial distribution characteristics of Salmonella from different hosts in China in the recent 16 years. These results greatly supplement Salmonella strain resources, genetic information, and traceability typing data; facilitate the typing, traceability, identification, and genetic evolution analysis of Salmonella ; and therefore, improve the level of analysis, monitoring, and controlling of foodborne microorganisms in China. [ABSTRACT FROM AUTHOR]

Salmonella enterica (S. enterica) is an important foodborne pathogen, causing food poisoning and human infection, and critically threatening food safety and public health. Salmonella typing is essential for bacterial identification, tracing, epidemiological investigation, and monitoring. Serotyping and multilocus sequence typing (MLST) analysis are standard bacterial typing methods despite the low resolution. Core genome MLST (cgMLST) is a high-resolution molecular typing method based on whole genomic sequencing for accurate bacterial tracing. We investigated 250 S. enterica isolates from poultry, livestock, food, and human sources in nine provinces of China from 2004 to 2019 using serotyping, MLST, and cgMLST analysis. All S. enterica isolates were divided into 36 serovars using slide agglutination. The major serovars in order were Enteritidis (31 isolates), Typhimurium (29 isolates), Mbandaka (23 isolates), and Indiana (22 isolates). All strains were assigned into 43 sequence types (STs) by MLST. Among them, ST11 (31 isolates) was the primary ST. Besides this, a novel ST, ST8016, was identified, and it was different from ST40 by position 317 C → T in dnaN. Furthermore, these 250 isolates were grouped into 185 cgMLST sequence types (cgSTs) by cgMLST. The major cgST was cgST235530 (11 isolates), and only three cgSTs contained isolates from human and other sources, indicating a possibility of cross-species infection. Phylogenetic analysis indicated that most of the same serovar strains were putatively homologous except Saintpaul and Derby due to their multilineage characteristics. In addition, serovar I 4,[5],12:i:- and Typhimurium isolates have similar genomic relatedness on the phylogenetic tree. In conclusion, we sorted out the phenotyping and genotyping diversity of S. enterica isolates in China during 2004–2019 and clarified the temporal and spatial distribution characteristics of Salmonella from different hosts in China in the recent 16 years. These results greatly supplement Salmonella strain resources, genetic information, and traceability typing data; facilitate the typing, traceability, identification, and genetic evolution analysis of Salmonella; and therefore, improve the level of analysis, monitoring, and controlling of foodborne microorganisms in China.

Mycoplasma (M.) hyosynoviae is a commensal of the upper respiratory tract in swine, which has the potential to spread systemically, usually resulting in arthritis in fattening pigs and gilts. To date, very little is known about the epidemiology of M. hyosynoviae, mainly due to a lack of suitable typing methods. Therefore, this study aimed to develop both a conventional multi locus sequence typing (MLST) and a core genome (cg) MLST scheme. The development of the cgMLST was based on whole genome sequences of 64 strains isolated from pigs and wild boars during routine diagnostics as well as nine publicly available genomes. A cgMLST scheme containing 390 target genes was established using the Ridom© SeqSphere+ software. Using this scheme as a foundation, seven housekeeping genes were selected for conventional MLST based on their capability to reflect genome wide relatedness and subsequently, all 73 strains were typed by applying both methods. Core genome MLST results revealed a high diversity of the studied strain population and less than 100 allele differences between epidemiologically unrelated strains were only detected for four isolates from the US. On the other hand, seven clonal clusters (≤ 12 allele differences) comprising 20 isolates were identified. Comparison of the two typing methods resulted in highly congruent phylogenetic trees and an Adjusted Rand Coefficient of 0.893, while cgMLST showed marginally higher resolution when comparing closely related isolates, indicated by a slightly higher Simpson's ID (0.992) than conventional MLST (Simpson's ID = 0.990). Overall, both methods seem well suited for epidemiological analyses for scientific as well as diagnostic purposes. While MLST is faster and cheaper, cgMLST can be used to further differentiate closely related isolates. • first cgMLST and MLST schemes for typing Mycoplasma hyosynoviae. • cgMLST and MLST deliver highly congruent results. • MLST features sufficient discriminatory power reflecting core genome wide relationship. • cgMLST has marginally better resolution when comparing closely related isolates. • cgMLST revealed high diversity and limited clonality among Mycoplasma hyosynoviae strains. [ABSTRACT FROM AUTHOR]

The state of Florida reports a high burden of non-typhoidal Salmonella enterica with approximately two times higher than the national incidence. We retrospectively analyzed the population structure and molecular epidemiology of 1,709 clinical isolates from 2017 and 2018. We found 115 different serotypes. Rarefaction suggested that the serotype richness did not differ between children under 2 years of age and older children and adults and, there are ~22 well-characterized dominant serotypes. There were distinct differences in dominant serotypes between Florida and the USA as a whole, even though S. Enteritidis and S. Newport were the dominant serotypes in Florida and nationally. S. Javiana, S. Sandiego, and S. IV 50:z4, z23:- occurred more frequently in Florida than nationally. Legacy Multi Locus Sequence Typing (MLST) was of limited use for differentiating clinical Salmonella isolates beyond the serotype level. We utilized core genome MLST (cgMLST) hierarchical clusters (HC) to identify potential outbreaks and compared them to outbreaks detected by Pulse Field Gel Electrophoresis (PFGE) surveillance for five dominant serotypes (Enteritidis, Newport, Javiana, Typhimurium, and Bareilly). Single nucleotide polymorphism (SNP) phylogenetic-analysis of cgMLST HC at allelic distance 5 or less (HC5) corroborated PFGE detected outbreaks and generated well-segregated SNP distance-based clades for all studied serotypes. We propose “combination approach” comprising “HC5 clustering,” as efficient tool to trigger Salmonella outbreak investigations, and “SNP-based analysis,” for higher resolution phylogeny to confirm an outbreak. We also applied this approach to identify case clusters, more distant in time and place than traditional outbreaks but may have been infected from a common source, comparing 176 Florida clinical isolates and 1,341 non-clinical isolates across USA, of most prevalent serotype Enteritidis collected during 2017–2018. Several clusters of closely related isolates (0–4 SNP apart) within HC5 clusters were detected and some included isolates from poultry from different states in the US, spanning time periods over 1 year. Two SNP-clusters within the same HC5 cluster included isolates with the same multidrug-resistant profile from both humans and poultry, supporting the epidemiological link. These clusters likely reflect the vertical transmission of Salmonella clones from higher levels in the breeding pyramid to production flocks.

Methicillin-resistant Staphylococcus aureus (MRSA) presenting spa type t899 is commonly associated with sequence type 9 (ST9) but is also increasingly linked to ST398. This study provides genomic insight into the diversity of t899 isolates using core genome multilocus sequence typing (cgMLST), single nucleotide polymorphism (SNP)-based phylogeny, and the description of selected antimicrobial resistance and virulence markers. The SNP-based phylogenic tree showed that isolates sharing the same spa type (t899) but different STs highly diverged in their core and accessory genomes, revealing discriminant antimicrobial resistance (AMR) and virulence markers. Our results highlighted the idea that in a surveillance context where only spa typing is used, an additional multiplex PCR for the detection of the tet(M), sak, and seg genes would be valuable in helping distinguish ST9 from ST398 isolates on a routine basis. IMPORTANCE This study showed the genetic diversity and population structure of S. aureus presenting the same spa type, t899, but belonging to different STs. Our findings revealed that these isolates vary deeply in their core and accessory genomes, contrary to what is regularly inferred from studies using spa typing only. Given that identical spa types can be associated with different STs and that spa typing only is not appropriate for S. aureus isolates that have undergone major recombination events which include the passage of the spa gene (such as in t899-positive MRSA), the combination of both MLST and spa typing methods is recommended. However, spa typing alone is still largely used in surveillance studies and basic characterization. Our data suggest that additional markers, such as tet(M), sak, and seg genes, could be implemented in an easy and inexpensive manner in order to identify S. aureus lineages with a higher accuracy. [ABSTRACT FROM AUTHOR]

aim of this study was to investigate the clinical and microbiological features of community-onset CRE (CO-CRE) obtained from outpatients at a tertiary hospital in China. Patients and Methods: We isolated 64 CRE strains from outpatients and divided them into three groups: 36 hospital-acquired CRE (HA-CRE), 28 CO-CRE including 15 community-acquired CRE (CA-CRE) and 13 healthcare-associated CRE (HCA-CRE). Clinical information was collected. The antibiotic susceptibilities of the 28 CO-CRE strains were tested. Whole-genome sequencing (WGS) was conducted, and then drug resistance gene analysis was performed. CgMLST and SNP comparisons were used to analyze the genomic relationship with E. coli and K. pneumoniae strains, respectively. Results: In this study, the 28 CO-CRE isolates included K. pneumoniae (53.6%), E. coli (28.6%), E. cloacae (7.1%), C. freundii (7.1%) and E. asburiae (3.6%). The CO-CRE isolates were mainly isolated from urine samples (75%). The ceftazidime/avibactam resistance rate of community-onset E. coli was significantly higher than that of community-onset K. pneumoniae, while the aztreonam, ciprofloxacin, levofloxacin, and chloramphenicol resistance rates were significantly lower (P< 0.05). Thirteen of the 15 K. pneumoniae strains belonged to ST11 containing blaKPC-2. Correspondingly, 8 E. coli strains belonged to 7 STs, and they all were NDM producers. K. pneumoniae belonged to two major clusters, while E. coli was sporadic. The number of SNPs separating ST11 K. pneumoniae isolates ranged from 7 to 2154. Conclusion: Community-onset CRE is not rare, and the dissemination of E. coli was sporadic while K. pneumoniae was clonal spread with similar STs as HA-CRE. Active surveillance of CRE in the community setting is in demand. [ABSTRACT FROM AUTHOR]

Keywords: Acinetobacter baumannii; whole genome sequencing; cgMLST; mlst; genotyping EN Acinetobacter baumannii whole genome sequencing cgMLST mlst genotyping 1 5 5 05/02/20 20200429 NES 200429 Recently, discussions arose about the accuracy of the two I Acinetobacter baumannii i multilocus sequence typing (MLST) Schemes (Hamidian et al., [5]; Castillo-Ramirez and Grana-Miraglia, [1]; Gaiarsa et al., [4]). [Extracted from the article]

Mycoplasma iowae is a worldwide spread and economically important avian pathogen that mostly infects turkeys. Currently, multi-locus sequence typing (MLST) serves as the gold standard method for strain identification in M. iowae. However, additional robust genotyping methods are required to effectively monitor M. iowae infections and conduct epidemiological investigations. The first aim of this study was to develop genotyping assays with high resolution, that specifically target M. iowae , namely a multiple-locus variable number of tandem-repeats analysis (MLVA) and a core genome multi-locus sequence typing (cgMLST) schema. The second aim was the determination of relationships among a diverse selection of M. iowae strains and clinical isolates with a previous and the newly developed assays. The MLVA was designed based on the analyses of tandem-repeat (TR) regions in the six serotype reference strains (I, J, K, N, Q and R). The cgMLST schema was developed based on the coding sequences (CDSs) common in 95% of the examined 99 isolates. The samples were submitted for a previously published MLST assay for comparison with the developed methods. Out of 94 TR regions identified, 17 alleles were selected for further evaluation by PCR. Finally, seven alleles were chosen to establish the MLVA assay. Additionally, whole genome sequence analyses identified a total of 676 CDSs shared by 95% of the isolates, all of which were included into the developed cgMLST schema. The MLVA discriminated 19 distinct genotypes (GT), while with the cgMLST assay 79 sequence types (ST) could be determined with Simpson's diversity indices of 0.810 (MLVA) and 0.989 (cgMLST). The applied assays consistently identified the same main clusters among the diverse selection of isolates, thereby demonstrating their suitability for various genetic analyses and their ability to yield congruent results. • cgMLST and MLVA genotyping assays specific for M. iowae were developed. • Relationships in a diverse set of 99 M. iowae strains were determined. • The established methods are suitable for epidemiological investigations. • Low genetic variability was found among the M. iowae strains. [ABSTRACT FROM AUTHOR]

In the absence of consistent national molecular typing data to enhance the surveillance of Salmonella Enteritidis, it was considered useful to collect baseline information on the genetic diversity and antibiotic susceptibility of strains isolated in Romania between January 2016 and April 2020 and compare them to strains described in major international outbreaks of the same period. A collection of 245 clinical isolates were genotyped by a standardised multiple-locus variable-number of tandem repeats analysis (MLVA) 5-loci protocol and screened for antimicrobial resistance against 15 compounds. Twenty strains were further subjected to whole genome sequencing (WGS) and compared to epidemiologically relevant high-throughput sequencing data available in European databases. Twenty-seven MLVA genotypes were identified, of which three, commonly reported in Europe between 2016–2020, covered 72% of the collection. Antibiotic resistance was detected in 30% of the strains, with resistance to nalidixic acid and ciprofloxacin as the most common phenotype, and also associated with two prevalent MLVA clones. WGS-derived multilocus sequence typing (MLST) revealed a single sequence type (ST11) further resolved into 10 core-genome MLST complex types. The minimum spanning tree constructed from the cgMLST data clustered Romanian and international strains, which shared more than 95% of the core genes, revealing links with a contemporaneous multi-country outbreak. This study could be regarded as a forerunner to the advent of using this integrative approach in the public health practice at a national level and thus contribute to the concerted actions at a European level to stop outbreaks.

There is a need for active molecular surveillance of human and veterinary Campylobacter infections. However, sequencing of all isolates is associated with high costs and a considerable workload. Thus, there is a need for a straightforward complementary tool to prioritize isolates to sequence. In this study, we proposed to investigate the ability of MALDI-TOF MS to pre-screen C. jejuni genetic diversity in comparison to MLST and cgMLST. A panel of 126 isolates, with 10 clonal complexes (CC), 21 sequence types (ST) and 42 different complex types (CT) determined by the SeqSphere+ cgMLST, were analysed by a MALDI Biotyper, resulting into one average spectra per isolate. Concordance and discriminating ability were evaluated based on protein profiles and different cut-offs. A random forest algorithm was trained to predict STs. With a 94% similarity cut-off, an AWC of 1.000, 0.933 and 0.851 was obtained for MLSTCC, MLSTST and cgMLST profile, respectively. The random forest classifier showed a sensitivity and specificity up to 97.5% to predict four different STs. Protein profiles allowed to predict C. jejuni CCs, STs and CTs at 100%, 93% and 85%, respectively. Machine learning and MALDI-TOF MS could be a fast and inexpensive complementary tool to give an early signal of recurrent C. jejuni on a routine basis.

[Display omitted] • All strains were divided into 5 different species. • The qnrD1 genes detected in 8 samples were all located on the Col3M plasmid. • The icm/dot type IVB-related genes were first reported in Yersinia massiliensis. • 19 sequence types were newly discovered. • Some strains had high similarity to strains from South Korea and Denmark. Yersinia spp. vary significantly in their ability to cause diseases that threaten public health. Their pathogenicity is frequently associated with increasing antimicrobial resistance (AMR) and various virulence factors. The aim of the study was to investigate the AMR genes, virulence factors, and genetic diversity of Yersinia strains isolated from meats and fish in Wenzhou in 2020 by using whole-genome sequencing (WGS). A total of 50 isolates were collected. The phylogenetic relationships among the Yersinia species were also analyzed using multilocus sequence typing (MLST), core genome multi-locus sequence typing (cgMLST), and single nucleotide polymorphism (SNP) analysis. According to the results, all the strains could be classified into five species, with most isolated from beef, followed by poultry, pork, and fish. AMR genes were identified in 23 strains. And the qnrD1 genes were all located in the Col3M plasmid. Virulence genes, such as yaxA , ystB , pla , and yplA , were also found in the 15 Y. enterocolitica strains. And this study also found the presence of icm/dot type IVB-related genes in one Yersinia massiliensis isolate. MLST analysis identified 43 sequence types (STs), 19 of which were newly detected in Yersinia. Moreover, cgMLST analysis revealed that no dense genotype clusters were formed (cgMLST 5341, 5344, 5346–5350, 5353–5390). Instead, the strains appeared to be dispersed over large distances, except when multiple isolates shared the same ST. Isolates Y4 and Y26 were closely related to strains originating from South Korea and Denmark. This study showed considerable diversity in Yersinia spp. isolated from local areas (Wenzhou City). The data generated in our study may enrich the molecular traceability database of Yersinia and provide a basis for the development of more effective antipathogen control strategies. [ABSTRACT FROM AUTHOR]

In Finland, Taylorella equigenitalis , the causative agent of contagious equine metritis (CEM), was first detected in 1992. The aim of this study was to genotype Finnish T. equigenitalis isolates to investigate the epidemiology of the infection in the Finnish horse population. A total of 34 T. equigenitalis isolates from 24 horses obtained during 1992–2021 were subjected to whole genome sequencing (WGS) and subsequent local ad hoc core genome multi-locus sequence typing (cgMLST) targeting 1259 loci. Classical MLST profiles were extracted from the whole-genome sequence data. Three novel MLST types, ST81, ST82 and ST83, and four previously described sequence types, ST16, ST17, ST50 and ST63 were detected among the isolates. cgMLST minimum spanning tree analysis using 12 allele difference as threshold, resulted in five clusters and three singletons. cgMLST clusters were congruent with the MLST-defined groups, except for the ST83 isolates which were divided into two clusters. However, the high discriminatory power cgMLST allowed differentiation between isolates of the same MLST type as each isolate had a unique core genome ST. Our study suggests that cgMLST has the prospective for being a standardised typing method for T. equigenitalis in the future, and further contributes to worldwide phylogenetic and spatio-temporal analyses needed to better understand the epidemiology of the bacterium. • A new ad-hoc cgMLST scheme for T. equigenitalis was developed • The 34 isolates were divided into 34 cgMLST sequence types • Isolates with the same MLST type mainly clustered together in the cgMLST analysis • Isolates from 24 horses fell into 3 novel and 4 previously described MLST types • New sequence data from isolates from Finnhorses and Fjord horses are provided [ABSTRACT FROM AUTHOR]

Poultry is considered a major reservoir of human campylobacteriosis. It also been reported that not only poultry, but also wild birds, are capable of carrying C. jejuni, thus demonstrating to be a risk of spreading the bacteria in the environment. To gain insight into the population structure and investigate the antimicrobial resistance genotypes and phenotypes, we analyzed a collection of 135 C. jejuni from 15 species of wild birds in Italy. MLST revealed the presence of 41 sequence types (STs) and 13 clonal complexes (CCs). ST-179 complex and the generalist ST-45 complex were the most prevalent. Core genome MLST revealed that C. jejuni from ST-45 complex clustered according to the bird species, unlike the ST-179 complex which featured 3 different species in the same cluster. Overall we found a moderate prevalence of resistance to tetracycline (12.5%), ciprofloxacin and nalidixic acid (10%). The novel ST isolated from one pigeon showed resistance to all the antibiotics tested. The ST-179 complex (33.3%) was identified with significantly higher nalidixic acid resistance relative to other tested STs. Nine AMR genes (tet(O), cmeA, cmeB, cmeC, cmeR, aad, blaOXA-61, blaOXA-184 and erm(B)) and 23S rRNA and gyrA-associated point mutations were also described, indicating a concordance level between genotypic and phenotypic resistance of 23.3%, 23.4% and of 37.5% for streptomycin, tetracycline and quinolones/fluoroquinolones, respectively. We recommend that particular attention should be given to wild birds as key sentinel animals for the ecosystem contamination surveillance.

We investigated the epidemiology of cholera in Taiwan during 2002-2018. Vibrio cholerae sequence type (ST) 75 clone emerged in 2009 and has since become more prevalent than the ST69 clone from a previous pandemic. Closely related ST75 strains have emerged in 4 countries and may now be widespread in Asia. [ABSTRACT FROM AUTHOR]

Mycoplasma synoviae (MS) is a poultry pathogen with reported increased prevalence and virulence in recent years. MS strain identification is essential for prevention, control efforts and epidemiological outbreak investigations. Multiple multilocus based sequence typing schemes have been developed for MS, yet the resolution of these schemes could be limited for outbreak investigation. The cost of whole genome sequencing became close to that of sequencing the seven MLST targets; however, there is no standardized method for typing MS strains based on whole genome sequences. In this paper, we propose a core genome multilocus sequence typing (cgMLST) scheme as a standardized and reproducible method for typing MS based whole genome sequences. A diverse set of 25 MS whole genome sequences were used to identify 302 core genome genes as cgMLST targets (35.5% of MS genome) and 44 whole genome sequences of MS isolates from six countries in four continents were used for typing applying this scheme. cgMLST based phylogenetic trees displayed a high degree of agreement with core genome SNP based analysis and available epidemiological information. cgMLST allowed evaluation of two conventional MLST schemes of MS. The high discriminatory power of cgMLST allowed differentiation between samples of the same conventional MLST type. cgMLST represents a standardized, accurate, highly discriminatory, and reproducible method for differentiation between MS isolates. Like conventional MLST, it provides stable and expandable nomenclature, allowing for comparing and sharing the typing results between different laboratories worldwide. [ABSTRACT FROM AUTHOR]

Numerous genotyping techniques based on different principles and with different costs and levels of resolution are currently available for understanding the transmission dynamics of brucellosis worldwide. We aimed to compare the population structure of the genomes of 53 Brazilian Brucella abortus isolates using eight different genotyping methods: multiple-locus variable-number tandem-repeat analysis (MLVA8, MLVA11, MLVA16), multilocus sequence typing (MLST9, MLST21), core genome MLST (cgMLST) and two techniques based on single nucleotide polymorphism (SNP) detection (parSNP and NASP) from whole genomes. The strains were isolated from six different Brazilian states between 1977 and 2008 and had previously been analyzed using MLVA8, MLVA11, and MLVA16. Their whole genomes were sequenced, assembled, and subjected to MLST9 MLST21, cgMLST, and SNP analyses. All the genotypes were compared by hierarchical grouping method based on the average distances between the correlation matrices of each technique. MLST9 and MLST21 had the lowest level of resolution, both revealing only four genotypes. MLVA8, MLVA11, and MLVA16 had progressively increasing levels of resolution as more loci were analyzed, identifying 6, 16, and 44 genotypes, respectively. cgMLST showed the highest level of resolution, identifying 45 genotypes, followed by the SNP-based methods, both of which had 44 genotypes. In the assessed population, MLVA was more discriminatory than MLST and was easier and cheaper to perform. SNP techniques and cgMLST provided the highest levels of resolution and the results from the two methods were in close agreement. In conclusion, the choice of genotyping technique can strongly affect one's ability to make meaningful epidemiological conclusions but is dependent on available resources: while the VNTR based techniques are more indicated to high prevalence scenarios, the WGS methods are the ones with the best discriminative power and therefore recommended for outbreaks investigation. • MLST had the lowest level of resolution for the Brucella abortus strains accessed. • MLVA16 showed a good discriminatory power and is a good strategy for endemic areas. • SNP techniques are the most indicated for B. abortus outbreak investigations. [ABSTRACT FROM AUTHOR]

• Most of the carbapenemase-producing Acinetobacter baumannii isolates were from males. • The most prevalent carbapenemase gene was bla OXA-23. • Most isolates belonged to international clone IC2. • A new international clone, proposed to be named IC11, was described. This study aimed to characterise carbapenemase-producing Acinetobacter baumannii (A. baumannii) isolates from Danish patients using whole genome sequencing (WGS). It also compared typing and epidemiological data for further investigation of the spread and origin of the carbapenemase-producing A. baumannii isolates. From 1 January 2014 to 30 September 2021, 141 carbapenemase-producing A. baumannii isolates, received at the national reference laboratory at Statens Serum Institut, were investigated using WGS. Multilocus sequence typing (MLST) and cgMLST data, obtained by SeqSphere+ software, were linked to data related to source of isolation, patient age and sex, hospital admission and travel history. Most of the carbapenemase-producing A. baumannii isolates were from males (n = 100, 71%). Most patients (n = 88, 63%) had travelled outside Scandinavia before admission to a Danish hospital. The most prevalent carbapenemase gene was bla OXA-23 (n = 124). Isolates belonging to the dominating international clone IC2 accounted for 78% of all isolates. A new international ST164/OXA-91 clone, proposed to be named IC11, was recognised and described. The cgMLST analysis revealed 17 clusters, reflecting both sporadic travel to similar geographical areas and confirmed outbreaks in Danish hospitals. The occurrence of carbapenemase-producing A. baumannii in Denmark was still low; however, isolates belonging to major international clones with a high potential to spread within hospitals, mainly IC2, dominated. OXA-23 was by far the most prevalent carbapenemase detected. Sporadic and travel-related introductions to Danish hospitals, also intra-hospital transmission, could be confirmed, emphasising the need for continuing vigilance. [ABSTRACT FROM AUTHOR]

Human infections with Brucella melitensis are occasionally reported in Sweden, despite the fact that the national flocks of sheep and goats are officially free from brucellosis. The aim of our study was to analyze 103 isolates of B. melitensis collected from patients in Sweden between 1994 and 2016 and determine their putative geographic origin using whole genome sequencing (WGS)-based tools. The majority of the strains were assigned to East Mediterranean and African lineages. Both in silico Multiple Loci VNTR (Variable Number of Tandem Repeats) Analysis (MLVA) and core genome Multilocus Sequence Typing (cgMLST) analyses identified countries of the Middle East as the most probable source of origin of the majority of the strains. Isolates collected from patients with travel history to Iraq or Syria were often associated with genotypes from Turkey, as the cgMLST profiles from these countries clustered together. Sixty strains were located within a distance of 20 core genes to related genotypes from the publicly available database, and for eighteen isolates, the closest genotype was different by more than 50 loci. Our study showed that WGS based tools are effective in tracing back the geographic origin of infection of patients with unknown travel status, provided that public sequences from the location of the source are available.

Salmonella Typhimurium (S. Typhimurium) is an important food-borne and zoonotic pathogen that causes salmonellosis. With the development of whole genome sequencing (WGS), genome-based typing has been widely applied to bacteriology. In this study, we investigated genotyping and phylogenetic clusters of S. Typhimurium isolates from humans and animals in different provinces (including Beijing, Shandong, Guangxi, Shaanxi, Henan, and Shanghai) of China during 2009–2018 using multi locus sequence typing (MLST), core genome MLST (cgMLST), whole genome MLST (wgMLST) and single nucleotide polymorphism (SNP) based on WGS. 29 S. Typhimurium isolates from chicken (n = 22), sick pigeon (n = 2), patients (n = 4) and sick swine (n = 1) were tested. MLST analysis showed S. Typhimurium strains were divided into four STs, namely ST19 (n = 14), ST34 (n = 12), ST128 (n = 2) and ST1544 (n = 1). cgMLST and wgMLST divided 29 strains into 27 cgSTs and 29 wgST, respectively. Phylogenetic clustering showed that all isolates were divided into 4 clusters and 4 singletons. SNP analysis was used to examine MLST, cgMLST, wgMLST analysis. Finally, comparisons of MLST, cgMLST, wgMLST, and SNP were analyzed and the results showed their precision increased in order. In summary, genomic typing and phylogenetic relationships of 29 S. Typhimurium strains from different sources in China were analyzed. These findings were beneficial to investigate molecular pathogenesis, bacterial diversity, and traceability analysis of Salmonella. • We investigated genome-based typing of Salmonella Typhimurium isolates from animals and humans in China during 2004–2019, including multi locus sequence typing (MLST), core genome MLST (cgMLST), whole genome MLST (wgMLST) and single nucleotide polymorphism (SNP). • In-silico MLST analysis of S. Typhimurium isolates showed all of the tested strains were divided into four STs, involving ST19 (n = 14), ST34 (n = 12), ST128 (n = 2) and ST1544 (n = 1). The genetic differences between isolates of ST19 were larger. • cgMLST analysis results showed that 29 S. Typhimurium isolates were divided into 27 cgSTs. Among them, 25 cgSTs only included one strain, but cgST236374 and cgST236376 respectively contained 2 isolates. • 29 S. Typhimurium isolates were divided into 29 wgSTs, and each wgST contained single S. Typhimurium isolate. • Phylogenetic minimum spanning tree base on MLST showed that 29 S. Typhimurium isolates were divided into 4 clusters, however, they were clustered into 4 clusters and 4 singletons in the minimum spanning tree based on cgMLST, wgMLST and SNP, respectively. The genetic differences among isolates from human at the same time and place were large. [ABSTRACT FROM AUTHOR]

ABSTRACT Klebsiella pneumoniae is among the most common causes of hospital-acquired infections and has emerged as an urgent threat to public health due to carbapenem antimicrobial resistance. K. pneumoniae commonly colonizes hospitalized patients and causes extraintestinal infections such as urinary tract infection, bloodstream infection (septicemia), and pneumonia. If colonization is an intermediate step before infection, then detection and characterization of colonizing isolates could enable strategies to prevent or empirically treat K. pneumoniae infections in hospitalized patients. However, the strength of the association between colonization and infection is unclear. To test the hypothesis that hospitalized patients become infected with their colonizing strain, 1,765 patients were screened for rectal colonization with K. pneumoniae, and extraintestinal isolates from these same patients were collected over a 3-month period in a cohort study design. The overall colonization prevalence was 23.0%. After adjustment for other patient factors, colonization was significantly associated with subsequent infection: 21 of 406 (5.2%) colonized patients later had extraintestinal infection, compared to 18 of 1,359 (1.3%) noncolonized patients (adjusted odds ratio [OR], 4.01; 95% confidence interval, 2.08 to 7.73; P < 0.001). Despite a high diversity of colonizing isolates, 7/7 respiratory, 4/4 urinary, and 2/5 bloodstream isolates from colonized patients matched the patient corresponding rectal swab isolates, based on wzi capsular typing, multilocus sequence typing (MLST), and whole-genome sequence analysis. These results suggest that K. pneumoniae colonization is directly associated with progression to extraintestinal infection. IMPORTANCE K. pneumoniae commonly infects hospitalized patients, and these infections are increasingly resistant to carbapenems, the antibiotics of last resort for life-threatening bacterial infections. To prevent and treat these infections, we must better understand how K. pneumoniae causes disease and discover new ways to predict and detect infections. This study demonstrates that colonization with K. pneumoniae in the intestinal tract is strongly linked to subsequent infection. This finding helps to identify a potential time frame and possible approach for intervention: the colonizing strain from a patient could be isolated as part of a risk assessment, and antibiotic susceptibility testing could guide empirical therapy if the patient becomes acutely ill.

Background: Next-generation sequencing provides a powerful means of molecular characterization. However, methods such as single-nucleotide polymorphism detection or whole-chromosome sequence analysis are computationally expensive, prone to errors, and are still less accessible than traditional typing methods. Here, we present the Listeria monocytogenes core-genome sequence typing method for molecular characterization. This method uses a high-confidence core (HCC) genome, calculated to ensure accurate identification of orthologs. We also developed an evolutionarily relevant nomenclature based upon phylogenetic analysis of HCC genomes. Finally, we created a pipeline (LmCGST; https://sourceforge.net/projects/lmcgst/files/) that takes in raw next-generation sequencing reads, calculates a subject HCC profile, compares it to an expandable database, assigns a sequence type, and performs a phylogenetic analysis. Results: We analyzed 29 high-quality, closed Listeria monocytogenes chromosome sequences and identified loci that are reliable targets for automated molecular characterization methods. We identified 1013 open-reading frames that comprise our high-confidence core (HCC) genome. We then populated a database with HCC profiles from 114 taxa. We sequenced 84 randomly selected isolates from the Listeriosis Reference Service for Canada's collection and analysed them with the LmCGST pipeline. In addition, we generated pulsed-field gel electrophoresis, ribotyping, and in silico multi-locus sequence typing (MLST) data for the 84 isolates and compared the results to those obtained using the CGST method. We found that all of the methods yielded results that are generally congruent. However, due to the increased numbers of categories, the CGST method provides much greater discriminatory power than the other methods tested here. Conclusions: We show that the CGST method provides increased discriminatory power relative to typing methods such as pulsed-field gel electrophoresis, ribotyping, and multi-locus sequence typing while it addresses several shortcomings of other methods of molecular characterization with next-generation sequence data. It uses discrete, well-defined groupings (types) of organisms that are phylogenetically relevant and easily interpreted. In addition, the CGST scheme can be expanded to include additional loci and HCC profiles in the future. In total, the CGST method provides an approach to the molecular characterization of Listeria monocytogenes with next-generation sequence data that is highly reproducible, easily standardized, portable, and accessible. [ABSTRACT FROM AUTHOR]

Listeria monocytogenes is a foodborne pathogen ubiquitously found in nature and which has been isolated from food and food processing environments. This study aimed to characterize L. monocytogenes strains isolated from the production and processing environments of frozen sliced mushrooms (Agaricus bisporus). An analysis was executed along the mushroom processing chain including one mushroom grower and two mushroom processing factories. A total of 153 L. monocytogenes strains were isolated, which could be grouped in three PCR serogroups, namely, serogroup 1/2a-3a (39.2%), serogroup 1/2b-3b-7 (34.0%) and serogroup 4b-4d-4e (26.8%). A selection of 44 L. monocytogenes strains isolated from the processing environment after cleaning and disinfection (C&D) and from frozen sliced mushrooms was genotyped by whole genome sequencing (WGS), because these strains pose a potential risk for product contamination after C&D and for human consumption. Multilocus sequence typing (MLST) revealed 11 clonal complexes (CCs), with strains belonging to CC1, CC4, CC37 and CC87 being detected in both processing factories. Comparative WGS analysis of the 44 strains showed the presence of Listeria pathogenicity island 1 (LIPI-1) with a disrupted version of actA in all CC1, CC4, CC5, CC59 strains, and all but one CC224 strains. Notably, both inlA and inlB were detected as full-length loci in every strain, except for inlA in a CC6 strain that harbored a three amino acid deletion. LIPI-3 was detected in all CC1, CC4, CC6 and CC224 strains, while LIPI-4 was detected in all CC4 and CC87 strains. In addition, antibiotic susceptibility tests showed susceptibility towards fourteen antibiotics tested. The bcrABC operon was found in one CC5 strain, that showed a higher tolerance towards benzalkonium chloride than any other strain tested with confluent growth till 12.5 μg/ml for the CC5 strain compared to 2.5 μg/ml for the other strains. This study highlights that the ecology of L. monocytogenes in the frozen sliced mushroom production chain is highly diverse, and shows the importance of hygienic measures to control L. monocytogenes along the frozen sliced mushroom production chain. • L. monocytogenes from frozen sliced mushroom industry showed high genomic diversity. • Important virulence determinants were found across strains. • Resistance towards benzalkonium chloride was present in one of the 44 strains. • All strains were found susceptible to a wide range of antibiotics. [ABSTRACT FROM AUTHOR]