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ABSTRACT Previously, we demonstrated that the majority of vancomycin-resistant Enterococcus faecium (VREfm) strains from in-patients of the University Hospital Erlangen, Germany, belonged to only three clonal lineages, namely ST117/CT71 vanB and two novel ST1299 vanA lineages classified as CT3109 and CT1903. The goal of the current study was (i) to investigate whether VREfm is also detectable in wastewater of the city of Erlangen, (ii) to identify their molecular features, and (iii) to clarify whether VREfm could arise from the community of the city of Erlangen or can be (directly) connected to nosocomial infections in the hospital setting. From April to May 2023, a total of 244 VREfm strains from raw wastewater of the city of Erlangen were analyzed by core genome multilocus sequence typing (cgMLST). Moreover, 20 of them were further investigated for single nucleotide polymorphisms (SNPs). The molecular characterization of the wastewater VREfm strains revealed a high prevalence (27.9%) of the recently identified clonal lineage ST1299/CT3109 vanA, which is mainly characterized by the presence of the tetracycline-resistance determinant tet(M) and the virulence genes pilA and prpA. The SNPs analysis revealed the presence of two major clusters, namely cluster I (≤65 SNPs), which included well-known hospital-adapted vanB clonal lineages such as ST117/CT71 and ST80/CT1065 and cluster II (≤70 SNPs), which were mainly characterized by the lineage ST1299/CT3109 vanA. Based on the concomitant resistance to vancomycin and tetracycline, we propose that ST1299/CT3109 vanA primarily originated and spread outside of hospital settings.IMPORTANCEThis study provides a detailed genomic analysis of vancomycin-resistant Enterococcus faecium (VREfm) strains isolated from municipal wastewater with a particular focus on clonal lineages, antimicrobial resistance, and the presence of virulence genes. The high wastewater prevalence of the recently identified clonal lineage ST1299/CT3109 vanA, which has been previously detected in hospitals, suggests an enormous potential for future spread in Germany.

ABSTRACT Since 2019, we have observed a remarkable rise of vancomycin resistance among invasive strains of Enterococcus (E.) faecium at the University Hospital Erlangen (UKER), a tertiary-care hospital in Germany. Here, we analyzed the genetic diversity of E. faecium isolated from blood cultures of in-patients by core genome multilocus sequence typing to see whether the occurrence of vancomycin resistance was associated with the spread of new clonal lineages. Between January and December 2022, 37 consecutive non-duplicate isolates were included in this study. Resistance to vancomycin was detected in 15 isolates (40.5%). The affected patients had a median age of 65 years, and 26 of them (70.3%) were males. Patients with bloodstream infection caused by vancomycin-resistant E. faecium (VREfm) or vancomycin-susceptible E. faecium (VSEfm) showed a similar in-hospital mortality rate (33.3% vs 27.3%, P = 0.692), indicating that VREfm were not more virulent than VSEfm. In contrast, the genetic diversity was higher in VSEfm (15 clonal lineages) than in VREfm isolates (seven clonal lineages). The majority of VREfm isolates (73.2%) belonged to only three clonal lineages: ST117/CT71 vanB (n = 4), which is the most commonly detected vancomycin-resistant lineage in Germany, and two novel ST1299 vanA lineages classified as CT3109 (n = 4) and CT1903 (n = 3). We conclude that the high rate of vancomycin resistance among invasive E. faecium isolates at UKER could be associated with the emergence of novel ST1299 vanA lineages, which is likely to be relevant also for other hospitals and countries. Future studies will focus on the prevalence of ST1299 vanA in the general population. IMPORTANCE The proportion of VREfm among all Enterococcus faecium isolated from blood cultures in German hospitals has increased in the period 2015–2020 from 11.9% to 22.3% with a country-wide spread of the clonal lineage ST117/CT71 vanB. In this study, we provided useful information about the genetic diversity of invasive strains of E. faecium. Moreover, our findings confirm the nosocomial spread of novel ST1299 vanA lineages, which recently had a rapid expansion in Austria and the south-eastern part of Germany.

Acinetobacter baumannii is an opportunistic pathogen responsible for healthcare-associated infections (HAIs) and outbreaks. Antimicrobial resistance mechanisms and virulence factors allow it to survive and spread in the hospital environment. However, the molecular mechanisms of these traits and their association with international clones are frequently unknown in low- and middle-income countries. Here, we analyze the phenotype and genotype of seventy-six HAIs and outbreak-causing A. baumannii isolates from a Mexican hospital over ten years, with special attention to the carbapenem resistome and biofilm formation. The isolates belonged to the global international clone (IC) 2 and the Latin America endemic IC5 and were predominantly extensively drug-resistant (XDR). Oxacillinases were identified as a common source of carbapenem resistance. We noted the presence of the blaOXA-143-like family (not previously described in Mexico), the blaOXA-72 and the blaOXA-398 found in both ICs. A low prevalence of efflux pump overexpression activity associated with carbapenem resistance was observed. Finally, strong biofilm formation was found, and significant biofilm-related genes were identified, including bfmRS, csuA/BABCDE, pgaABCD and ompA. This study provides a comprehensive profile of the carbapenem resistome of A. baumannii isolates belonging to the same pulse type, along with their significant biofilm formation capacity. Furthermore, it contributes to a better understanding of their role in the recurrence of infection and the endemicity of these isolates in a Mexican hospital. [ABSTRACT FROM AUTHOR]

Abstract Background Yersinia enterocolitica has been sporadically recovered from animals, foods, and human clinical samples in various regions of Ningxia, China. However, the ecological and molecular characteristics of Y. enterocolitica, as well as public health concerns about infection in the Ningxia Hui Autonomous Region, remain unclear. This study aims to analyze the ecological and molecular epidemiological characteristics of Y. enterocolitis in order to inform the public health intervention strategies for the contains of related diseases. Methods A total of 270 samples were collected for isolation [animals (n = 208), food (n = 49), and patients (n = 13)], then suspect colonies were isolated and identified by the API20E biochemical identification system, serological tests, biotyping tests, and 16S rRNA-PCR. Then, we used an ecological epidemiological approach combined with machine learning algorithms (general linear model, random forest model, and eXtreme Gradient Boosting) to explore the associations between ecological factors and the pathogenicity of Y. enterocolitis. Furthermore, average nucleotide identity (ANI) estimation, single nucleotide polymorphism (SNP), and core gene multilocus sequence typing (cgMLST) were applied to characterize the molecular profile of isolates based on whole genome sequencing. The statistical test used single-factor analysis, Chi-square tests, t-tests/ANOVA-tests, Wilcoxon rank-sum tests, and Kruskal–Wallis tests. Results A total of 270 isolates of Yersinia were identified from poultry and livestock (n = 191), food (n = 49), diarrhoea patients (n = 13), rats (n = 15), and hamsters (n = 2). The detection rates of samples from different hosts were statistically different (χ 2 = 22.636, P

Background: Yersinia enterocolitica has been sporadically recovered from animals, foods, and human clinical samples in various regions of Ningxia, China. However, the ecological and molecular characteristics of Y. enterocolitica, as well as public health concerns about infection in the Ningxia Hui Autonomous Region, remain unclear. This study aims to analyze the ecological and molecular epidemiological characteristics of Y. enterocolitis in order to inform the public health intervention strategies for the contains of related diseases. Methods: A total of 270 samples were collected for isolation [animals (n = 208), food (n = 49), and patients (n = 13)], then suspect colonies were isolated and identified by the API20E biochemical identification system, serological tests, biotyping tests, and 16S rRNA-PCR. Then, we used an ecological epidemiological approach combined with machine learning algorithms (general linear model, random forest model, and eXtreme Gradient Boosting) to explore the associations between ecological factors and the pathogenicity of Y. enterocolitis. Furthermore, average nucleotide identity (ANI) estimation, single nucleotide polymorphism (SNP), and core gene multilocus sequence typing (cgMLST) were applied to characterize the molecular profile of isolates based on whole genome sequencing. The statistical test used single-factor analysis, Chi-square tests, t-tests/ANOVA-tests, Wilcoxon rank-sum tests, and Kruskal–Wallis tests. Results: A total of 270 isolates of Yersinia were identified from poultry and livestock (n = 191), food (n = 49), diarrhoea patients (n = 13), rats (n = 15), and hamsters (n = 2). The detection rates of samples from different hosts were statistically different (χ2 = 22.636, P < 0.001). According to the relatedness clustering results, 270 isolates were divided into 12 species, and Y. enterocolitica (n = 187) is a predominated species. Pathogenic isolates made up 52.4% (98/187), while non-pathogenic isolates made up 47.6% (89/187). Temperature and precipitation were strongly associated with the pathogenicity of the isolates (P < 0.001). The random forest (RF) prediction model showed the best performance. The prediction result shows a high risk of pathogenicity Y. enterocolitica was located in the northern, northwestern, and southern of the Ningxia Hui Autonomous Region. The Y. enterocolitica isolates were classified into 54 sequence types (STs) and 125 cgMLST types (CTs), with 4/O:3 being the dominant bioserotype in Ningxia. The dominant STs and dominant CTs of pathogenic isolates in Ningxia were ST429 and HC100_2571, respectively. Conclusions: The data indicated geographical variations in the distribution of STs and CTs of Y. enterocolitica isolates in Ningxia. Our work offered the first evidence that the pathogenicity of isolates was directly related to fluctuations in temperature and precipitation of the environment. CgMLST typing strategies showed that the isolates were transmitted to the population via pigs and food. Therefore, strengthening health surveillance on pig farms in high-risk areas and focusing on testing food of pig origin are optional strategies to prevent disease outbreaks. [ABSTRACT FROM AUTHOR]

ABSTRACT Corynebacterium striatum has recently received increasing attention due to its multiple antimicrobial resistances and its role as an invasive infection/outbreak agent. Recently, whole-genome sequencing (WGS)-based core genome multilocus sequence typing (cgMLST) has been used in epidemiological studies of specific human pathogens. However, this method has not been reported in studies of C. striatum. In this work, we aim to propose a cgMLST scheme for C. striatum. All publicly available C. striatum genomes, 30 C. striatum strains isolated from the same hospital, and 1 epidemiologically unrelated outgroup C. striatum strain were used to establish a cgMLST scheme targeting 1,795 genes (hereinafter referred to as 1,795-cgMLST). The genotyping results of cgMLST showed good congruence with core genome-based single-nucleotide polymorphism typing in terms of tree topology. In addition, the cgMLST provided a greater discrimination than the MLST method based on 6 housekeeping genes (gyrA, gyrB, hsp65, rpoB, secA1, and sodA). We established a clonal group (CG) threshold based on 104 allelic differences; a total of 56 CGs were identified from among 263 C. striatum strains. We also defined an outbreak threshold based on seven allelic differences that is capable of identifying closely related isolates that could give clues on hospital transmission. According to the results of analysis of drug-resistant genes and virulence genes, we identified CG4, CG5, CG26, CG28, and CG55 as potentially hypervirulent and multidrug-resistant CGs of C. striatum. This study provides valuable genomic epidemiological data on the diversity, resistance, and virulence profiles of this potentially pathogenic microorganism. IMPORTANCE Recently, WGS of many human and animal pathogens has been successfully used to investigate microbial outbreaks. The cgMLST schema are powerful genotyping tools that can be used to investigate potential epidemics and provide classification of the strains precise and reliable. In this study, we proposed the development of a cgMLST typing scheme for C. striatum, and then we evaluated this scheme for its applicability to hospital transmission investigations. This report describes the first cgMLST schema for C. striatum. The analysis of hospital transmission of C. striatum based on cgMLST methods has important clinical epidemiological significance for improving nosocomial infection monitoring of C. striatum and in-depth understanding of its nosocomial transmission routes.

ABSTRACT Listeria monocytogenes is a life-threatening foodborne pathogen. Here, we report the genomic characterization of a nationwide dataset of 411 clinical and 82 food isolates collected in Taiwan between 2014 and 2019. The observed incidence of listeriosis increased from 0.83 to 7 cases per million population upon implementation of mandatory notification in 2018. Pregnancy-associated cases accounted for 2.8% of human listeriosis and all-cause 7-day mortality was of 11.9% in nonmaternal-neonatal listeriosis. L. monocytogenes was isolated from 90% of raw pork and 34% of chicken products collected in supermarkets. Sublineages SL87, SL5, and SL378 accounted for the majority (65%) of clinical cases. SL87 and SL378 were also predominant (57%) in food products. Five cgMLST clusters accounted for 57% clinical cases, suggesting unnoticed outbreaks spanning up to 6 years. Mandatory notification allowed identifying the magnitude of listeriosis in Taiwan. Continuous real-time genomic surveillance will allow reducing contaminating sources and disease burden. IMPORTANCE Understanding the phylogenetic relationship between clinical and food isolates is important to identify the transmission routes of foodborne diseases. Here, we performed a nationwide study between 2014 and 2019 of both clinical and food Listeria monocytogenes isolates and sequenced their genomes. We show a 9-fold increase in listeriosis reporting upon implementation of mandatory notification. We found that sublineages SL87 and SL378 predominated among both clinical (50%) and food (57%) isolates, and identified five cgMLST clusters accounting for 57% of clinical cases, suggestive of potential protracted sources of contamination over up to 6 years in Taiwan. These findings highlight that mandatory declaration is critical in identifying the burden of listeriosis, and the importance of genome sequencing for a detailed characterization of the pathogenic L. monocytogenes genotypes circulating in Asia.

ABSTRACT Campylobacter is the leading bacterial cause of diarrheal illnesses worldwide. Campylobacter jejuni and C. coli are the most common species accounting for campylobacteriosis. Although the proportion of campylobacteriosis caused by C. coli is increasing rapidly in China, the underlying mechanisms of this emergence remain unclear. In this study, we analyzed the whole-genome sequences and associated environments of 1,195 C. coli isolates with human, poultry, or porcine origins from 1980 to 2021. C. coli isolates of human origin were closely related to those from poultry, suggesting that poultry was the main source of C. coli infection in humans. Analysis of antimicrobial resistance determinants indicated that the prevalence of multidrug-resistant C. coli has increased dramatically since the 2010s, coinciding with the shift in abundance from C. jejuni to C. coli in Chinese poultry. Compared with C. jejuni, drug-resistant C. coli strains were better adapted and showed increased proliferation in the poultry production environment, where multiple antimicrobial agents were frequently used. This study provides an empirical basis for the molecular mechanisms that have enabled C. coli to become the dominant Campylobacter species in poultry; we also emphasize the importance of poultry products as sources of campylobacteriosis caused by C. coli in human patients. IMPORTANCE The proportion of campylobacteriosis caused by C. coli is increasing rapidly in China. Coincidentally, the dominant species of Campylobacter occurring in poultry products has shifted from C. jejuni to C. coli. Here, we analyzed the whole-genome sequences of 1,195 C. coli isolates from different origins. The phylogenetic relationship among C. coli isolates suggests that poultry was the main source of C. coli infection in humans. Further analysis indicated that antimicrobial resistance in C. coli strains has increased dramatically since the 2010s, which could facilitate their adaptation in the poultry production environment, where multiple antimicrobial agents are frequently used. Thus, our findings suggest that the judicious use of antimicrobial agents could mitigate the emergence of multidrug-resistant C. coli strains and enhance clinical outcomes by restoring drug sensitivity in Campylobacter.

ABSTRACT Over the past decade, whole-genome sequencing (WGS) has overtaken traditional bacterial typing methods for studies of genetic relatedness. Further, WGS data generated during epidemiologic studies can be used in other clinically relevant bioinformatic applications, such as antibiotic resistance prediction. Using commercially available software tools, the relatedness of 38 clinical isolates of multidrug-resistant Pseudomonas aeruginosa was defined by two core genome multilocus sequence typing (cgMLST) methods, and the WGS data of each isolate was analyzed to predict antibiotic susceptibility to nine antibacterial agents. The WGS typing and resistance prediction data were compared with pulsed-field gel electrophoresis (PFGE) and phenotypic antibiotic susceptibility results, respectively. Simpson’s Diversity Index and adjusted Wallace pairwise assessments of the three typing methods showed nearly identical discriminatory power. Antibiotic resistance prediction using a trained analytical pipeline examined 342 bacterial-drug combinations with an overall categorical agreement of 92.4% and very major, major, and minor error rates of 3.6, 4.1, and 4.1%, respectively. IMPORTANCE Multidrug-resistant Pseudomonas aeruginosa isolates are a serious public health concern due to their resistance to nearly all or all of the available antibiotics, including carbapenems. Utilizing molecular approaches in conjunction with antibiotic susceptibility prediction software warrants investigation for use in the clinical laboratory workflow. These molecular tools coupled with antibiotic resistance prediction tools offer the opportunity to overcome the extended turnaround time and technical challenges of phenotypic susceptibility testing.

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Whole-genome sequencing was carried out on 30 Staphylococcus (S.) aureus isolates from dairy cows with subclinical mastitis from all five provinces of Rwanda. Twenty-five of the isolates produced enough sequence to be analyzed using core genome multilocus sequence typing (cg-MLST). The isolates group into three main clusters. The largest cluster contain isolates of sequence type (ST) 152 (n = 6) and the closely related ST1633 (n = 2). These sequence types have previously mainly been encountered in humans. The isolates of the second-largest cluster belong to ST5477 (n = 5),so far exclusively isolated from cows in Rwanda. The third cluster consists of isolates of ST97 (n = 4), which is a well-known bovine-adapted sequence type. These three clusters were all widespread over the country. Isolates of the usually human-adapted sequence types 1 (n = 2) and 5 (n= 1) were found and a single isolate of ST2430, previously found among humans in Africa. Finally, four isolates of novel sequence types were found: ST7108 (n = 2), ST7109 (n = 1), and ST7110 (n = 1). The blaZ penicillin resistance gene was found in 84% of the isolates and was in all cases corroborated by phenotypic resistance determination. Five (20%) of the isolates carried a tetracycline resistance gene, tet(K) or tetM, and three of these five also displayed phenotypic resistance while two isolates carried a tetM-gene but were yet tetracycline susceptible. Seven (28%) isolates carried the dfrG gene conferring resistance to trimethoprim. Four of these isolates indeed were resistant to trimethoprim while three isolates were sensitive. The str gene conferring resistance to aminoglycosides was found in three isolates; however, none of these displayed resistance to gentamycin. Our data revealed a high diversity of the sequence types of S. aureus isolates from cows with subclinical mastitis in Rwanda. Two major clusters of ST97 and ST5477 are likely to be bovine adapted and cause mastitis while the third cluster of ST152 usually have been found in humans and may signify a recent transmission of these types from human to cows, for example from hand milking. The high prevalence of this sequence type among dairy cows may pose zoonotic threat. The sequence types were widely distributed without any geographic correlation. Penicillin resistance, the most common type of resistance with a prevalence over 80%, but also tetracycline and trimethoprim resistance were displayed by several isolates.

Public health and regulatory agencies worldwide sequence all Listeria monocytogenes isolates obtained as part of routine surveillance and outbreak investigations. Many of these entities submit the sequences to the National Center for Biotechnology Information Pathogen Detection (NCBI PD) database, which groups the L. monocytogenes isolates into single nucleotide polymorphism (SNP) clusters based on a pairwise SNP difference threshold of 50 SNPs. Our goal was to assess whether isolates with metadata that suggest different sources or locations could show evidence for close genetic relatedness indicating a recent common ancestor and a possible unknown common source. We compared the whole genome sequencing (WGS) data of 249 L. monocytogenes isolates sequenced here, which have detailed metadata, with WGS data of nonclinical isolates on NCBI PD. The 249 L. monocytogenes isolates originated from natural environments (n = 91) as well as from smoked fish (n = 62), dairy (n = 56), and deli meat (n = 40) operations in the United States. Using a combination of subtyping by core genome multilocus sequence typing and high-quality SNP, we observed five SNP clusters in which study isolates and SNP cluster isolates seemed to be closely related and either (i) shared the same geolocation but showed different source types (one SNP cluster); (ii) shared the same source type but showed different geolocations (two SNP clusters); or (iii) shared neither source type nor geolocation (two SNP clusters). For one of the two clusters under (iii), there was, however, no strong bootstrap support for a common ancestor shared between the study isolates and SNP cluster isolates, indicating the value of in-depth evolutionary analyses when WGS data are used for traceback and epidemiological investigations. Overall, our results demonstrate that some L. monocytogenes subtypes may be associated with specific locations or commodities; these associations can help in investigations involving multi-ingredient foods such as sandwiches. However, at least some L. monocytogenes subtypes can be widespread geographically and can be associated with different sources, which may present a challenge to traceback investigations involving these subtypes. Closely related L. monocytogenes strains may have no apparent common source. Some L. monocytogenes strains are widespread geographically and source-wise. Traceback and epidemiological evidence is important for interpretation of WGS data. [ABSTRACT FROM AUTHOR]

Brucellosis is a zoonotic disease caused by Brucella spp. and a major concern for livestock. Most human cases are caused by B. melitensis and clinical presentation is usually a mild febrile illness. However, treatment failure is frequent and more severe complications can occur. In Austria, every human brucellosis is investigated to determine whether it was imported from endemic areas or is the sign of an undetected autochthonous transmission. For this study, 21 B. melitensis strains isolated in Austria between 2005 and 2019 were collected, 17 strains from 15 different patients and four strains from cattle. Whole genome sequencing combined with core-genome MLST analysis was used to characterize these strains. A cluster of seven isolates from 2018 (three human and four cattle isolates) was identified, with fewer than two allelic differences. They corresponded to the only Austrian B. melitensis outbreak that happened over the past 15 years. The other 12 Austrian brucellosis cases were single cases, and geographical origins were available for 8/12. Genomic data was used to locate probable geographical origins and compared with the results of the epidemiological investigations. Austrian strains were compared with 67 published B. melitensis sequences available on NCBI. The result of genomic analysis matched for 7/8 cases with documented conclusion of the epidemiological investigation. Genome analysis also pointed to the geographical origin for three of the four cases with missing epidemiological data. Strains from six cases were grouped together (

Pseudomonas aeruginosa is a ubiquitous microorganism and an important opportunistic pathogen responsible for a broad spectrum of infections mainly in immunosuppressed and critically ill patients. Molecular investigations traditionally rely on pulsed field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). In this work we propose a core genome multilocus sequence typing (cgMLST) scheme for P. aeruginosa, a methodology that combines traditional MLST principles with whole genome sequencing data. All publicly available complete P. aeruginosa genomes, representing the diversity of this species, were used to establish a cgMLST scheme targeting 2,653 genes. The scheme was then tested using genomes available at contig, chromosome and scaffold levels. The proposed cgMLST scheme for P. aeruginosa typed over 99% (2,314/2,325) of the genomes available for this study considering at least 95% of the cgMLST target genes present. The absence of a certain number gene targets at the threshold considered for both the creation and validation steps due to low genome sequence quality is possibly the main reason for this result. The cgMLST scheme was compared with previously published whole genome single nucleotide polymorphism analysis for the characterization of the population structure of the epidemic clone ST235 and results were highly similar. In order to evaluate the typing resolution of the proposed scheme, collections of isolates belonging to two important STs associated with cystic fibrosis, ST146 and ST274, were typed using this scheme, and ST235 isolates associated with an outbreak were evaluated. Besides confirming the relatedness of all the isolates, earlier determined by MLST, the higher resolution of cgMLST denotes that it may be suitable for surveillance programs, overcoming possible shortcomings of classical MLST. The proposed scheme is publicly available at: https://github.com/BioinformaticsHIAEMolecularMicrobiology/cgMLST-Pseudomonas-aeruginosa.

Pseudomonas cichorii is divided into two subclades based on the 16S ribosomal RNA gene sequence and core genome multilocus sequence typing. It was shown that subclade 2 strains utilize d -tartrate as a sole carbon source, whereas subclade 1 strains do not. Draft genome sequencing was performed with P. cichorii strains to identify d -tartrate utilization genes. By genome comparative and homology search studies, an ∼7.1-kb region was identified to be involved in d -tartrate utilization. The region is subclade 2 specific, and contains tarD and dctA genes, which encode a putative enzyme and transporter of d -tartrate, respectively. When the region was introduced into subclade 1 strains, the transformants were able to utilize d -tartrate. Partial fragments of tarD and dctA were amplified from all subclade 2 strains tested in this study by PCR using gene-specific primers, but not from subclade 1 strains. This is the first report on the genetic analysis of biochemical characteristics corresponding to a specific phylogenetic group in P. cichorii. [ABSTRACT FROM AUTHOR]

Pseudomonas aeruginosa is a ubiquitous microorganism and an important opportunistic pathogen responsible for a broad spectrum of infections mainly in immunosuppressed and critically ill patients. Molecular investigations traditionally rely on pulsed field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). In this work we propose a core genome multilocus sequence typing (cgMLST) scheme for P. aeruginosa , a methodology that combines traditional MLST principles with whole genome sequencing data. All publicly available complete P. aeruginosa genomes, representing the diversity of this species, were used to establish a cgMLST scheme targeting 2,653 genes. The scheme was then tested using genomes available at contig, chromosome and scaffold levels. The proposed cgMLST scheme for P. aeruginosa typed over 99% (2,314/2,325) of the genomes available for this study considering at least 95% of the cgMLST target genes present. The absence of a certain number gene targets at the threshold considered for both the creation and validation steps due to low genome sequence quality is possibly the main reason for this result. The cgMLST scheme was compared with previously published whole genome single nucleotide polymorphism analysis for the characterization of the population structure of the epidemic clone ST235 and results were highly similar. In order to evaluate the typing resolution of the proposed scheme, collections of isolates belonging to two important STs associated with cystic fibrosis, ST146 and ST274, were typed using this scheme, and ST235 isolates associated with an outbreak were evaluated. Besides confirming the relatedness of all the isolates, earlier determined by MLST, the higher resolution of cgMLST denotes that it may be suitable for surveillance programs, overcoming possible shortcomings of classical MLST. The proposed scheme is publicly available at: https://github.com/BioinformaticsHIAEMolecularMicrobiology/cgMLST-Pseudomonas-aeruginosa. [ABSTRACT FROM AUTHOR]

Abstract Background In 2015, in addition to a United States multistate outbreak linked to contaminated ice cream, another outbreak linked to ice cream was reported in the Pacific Northwest of the United States. It was a hospital-acquired outbreak linked to milkshakes, made from contaminated ice cream mixes and milkshake maker, served to patients. Here we performed multiple analyses on isolates associated with this outbreak: pulsed-field gel electrophoresis (PFGE), whole genome single nucleotide polymorphism (SNP) analysis, species-specific core genome multilocus sequence typing (cgMLST), lineage-specific cgMLST and whole genome-specific MLST (wgsMLST)/outbreak-specific cgMLST. We also analyzed the prophages and virulence genes. Results The outbreak isolates belonged to sequence type 1038, clonal complex 101, genetic lineage II. There were no pre-mature stop codons in inlA. Isolates contained Listeria Pathogenicity Island 1 and multiple internalins. PFGE and multiple whole genome sequencing (WGS) analyses all clustered together food, environmental and clinical isolates when compared to outgroup from the same clonal complex, which supported the finding that L. monocytogenes likely persisted in the soft serve ice cream/milkshake maker from November 2014 to November 2015 and caused 3 illnesses, and that the outbreak strain was transmitted between two ice cream production facilities. The whole genome SNP analysis, one of the two species-specific cgMLST, the lineage II-specific cgMLST and the wgsMLST/outbreak-specific cgMLST showed that L. monocytogenes cells persistent in the milkshake maker for a year formed a unique clade inside the outbreak cluster. This clustering was consistent with the cleaning practice after the outbreak was initially recognized in late 2014 and early 2015. Putative prophages were conserved among prophage-containing isolates. The loss of a putative prophage in two isolates resulted in the loss of the AscI restriction site in the prophage, which contributed to their AscI-PFGE banding pattern differences from other isolates. Conclusions The high resolution of WGS analyses allowed the differentiation of epidemiologically unrelated isolates, as well as the elucidation of the microevolution and persistence of isolates within the scope of one outbreak. We applied a wgsMLST scheme which is essentially the outbreak-specific cgMLST. This scheme can be combined with lineage-specific cgMLST and species-specific cgMLST to maximize the resolution of WGS.

Prior to the identification of Xanthomonas perforans associated with bacterial spot of tomato in 1991, X. euvesicatoria was the only known species in Florida. Currently, X. perforans is the Xanthomonas sp. associated with tomato in Florida. Changes in pathogenic race and sequence alleles over time signify shifts in the dominant X. perforans genotype in Florida. We previously reported recombination of X. perforans strains with closely related Xanthomonas species as a potential driving factor for X. perforans evolution. However, the extent of recombination across the X. perforans genomes was unknown. We used a core genome multilocus sequence analysis approach to identify conserved genes and evaluated recombination-associated evolution of these genes in X. perforans. A total of 1,356 genes were determined to be “core” genes conserved among the 58 X. perforans genomes used in the study. Our approach identified three genetic groups of X. perforans in Florida based on the principal component analysis (PCA) using core genes. Nucleotide variation in 241 genes defined these groups, that are referred as Phylogenetic-group Defining (PgD) genes. Furthermore, alleles of many of these PgD genes showed 100% sequence identity with X. euvesicatoria, suggesting that variation likely has been introduced by recombination at multiple locations throughout the bacterial chromosome. Site-specific recombinase genes along with plasmid mobilization and phage associated genes were observed at different frequencies in the three phylogenetic groups and were associated with clusters of recombinant genes. Our analysis of core genes revealed the extent, source, and mechanisms of recombination events that shaped the current population and genomic structure of X. perforans in Florida.

Prior to the identification of Xanthomonas perforans associated with bacterial spot of tomato in 1991, X. euvesicatoria was the only known species in Florida. Currently, X. perforans is the Xanthomonas sp. associated with tomato in Florida. Changes in pathogenic race and sequence alleles over time signify shifts in the dominant X. perforans genotype in Florida. We previously reported recombination of X. perforans strains with closely related Xanthomonas species as a potential driving factor for X. perforans evolution. However, the extent of recombination across the X. perforans genomes was unknown. We used a core genome multilocus sequence analysis approach to identify conserved genes and evaluated recombination-associated evolution of these genes in X. perforans. A total of 1,356 genes were determined to be "core" genes conserved among the 58 X. perforans genomes used in the study. Our approach identified three genetic groups of X. perforans in Florida based on the principal component analysis (PCA) using core genes. Nucleotide variation in 241 genes defined these groups, that are referred as Phylogenetic-group Defining (PgD) genes. Furthermore, alleles of many of these PgD genes showed 100% sequence identity with X. euvesicatoria , suggesting that variation likely has been introduced by recombination at multiple locations throughout the bacterial chromosome. Site-specific recombinase genes along with plasmid mobilization and phage associated genes were observed at different frequencies in the three phylogenetic groups and were associated with clusters of recombinant genes. Our analysis of core genes revealed the extent, source, and mechanisms of recombination events that shaped the current population and genomic structure of X. perforans in Florida. [ABSTRACT FROM AUTHOR]

Dichelobacter nodosus (D. nodosus) is the causative pathogen of ovine footrot, a disease that has a significant welfare and financial impact on the global sheep industry. Previous studies into the phylogenetics of D. nodosus have focused on Australia and Scandinavia, meaning the current diversity in the United Kingdom (U.K.) population and its relationship globally, is poorly understood. Numerous epidemiological methods are available for bacterial typing; however, few account for whole genome diversity or provide the opportunity for future application of new computational techniques. Multilocus sequence typing (MLST) measures nucleotide variations within several loci with slow accumulation of variation to enable the designation of allele numbers to determine a sequence type. The usage of whole genome sequence data enables the application of MLST, but also core and whole genome MLST for higher levels of strain discrimination with a negligible increase in experimental cost. An MLST database was developed alongside a seven loci scheme using publically available whole genome data from the sequence read archive. Sequence type designation and strain discrimination was compared to previously published data to ensure reproducibility. Multiple D. nodosus isolates from U.K. farms were directly compared to populations from other countries. The U.K. isolates define new clades within the global population of D. nodosus and predominantly consist of serogroups A, B and H, however serogroups C, D, E, and I were also found. The scheme is publically available at https://pubmlst.org/dnodosus/.

Dichelobacter nodosus (D. nodosus) is the causative pathogen of ovine footrot, a disease that has a significant welfare and financial impact on the global sheep industry. Previous studies into the phylogenetics of D. nodosus have focused on Australia and Scandinavia, meaning the current diversity in the United Kingdom (U.K.) population and its relationship globally, is poorly understood. Numerous epidemiological methods are available for bacterial typing; however, few account for whole genome diversity or provide the opportunity for future application of new computational techniques. Multilocus sequence typing (MLST) measures nucleotide variations within several loci with slow accumulation of variation to enable the designation of allele numbers to determine a sequence type. The usage of whole genome sequence data enables the application of MLST, but also core and whole genome MLST for higher levels of strain discrimination with a negligible increase in experimental cost. An MLST database was developed alongside a seven loci scheme using publically available whole genome data from the sequence read archive. Sequence type designation and strain discrimination was compared to previously published data to ensure reproducibility. Multiple D. nodosus isolates from U.K. farms were directly compared to populations from other countries. The U.K. isolates define new clades within the global population of D. nodosus and predominantly consist of serogroups A, B and H, however serogroups C, D, E, and I were also found. The scheme is publically available at https://pubmlst.org/dnodosus/. [ABSTRACT FROM AUTHOR]

During 2015-2016, we evaluated the performance of whole-genome sequencing (WGS) as a routine typing tool. Its added value for microbiological and epidemiologic surveillance of listeriosis was compared with that for pulsed-field gel electrophoresis (PFGE), the current standard method. A total of 2,743 Listeria monocytogenes isolates collected as part of routine surveillance were characterized in parallel by PFGE and core genome multilocus sequence typing (cgMLST) extracted from WGS. We investigated PFGE and cgMLST clusters containing human isolates. Discrimination of isolates was significantly higher by cgMLST than by PFGE (p<0.001). cgMLST discriminated unrelated isolates that shared identical PFGE profiles and phylogenetically closely related isolates with distinct PFGE profiles. This procedure also refined epidemiologic investigations to include only phylogenetically closely related isolates, improved source identification, and facilitated epidemiologic investigations, enabling identification of more outbreaks at earlier stages. WGS-based typing should replace PFGE as the primary typing method for L. monocytogenes. [ABSTRACT FROM AUTHOR]

At present, the most used methods for Klebsiella pneumoniae subtyping are multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE). However, the discriminatory power of MLST could not meet the need for distinguishing outbreak and non-outbreak isolates and the PFGE is time-consuming and labor-intensive. A core genome multilocus sequence typing (cgMLST) scheme for whole-genome sequence-based typing of K. pneumoniae was developed for solving the disadvantages of these traditional molecular subtyping methods. Firstly, we used the complete genome of K. pneumoniae strain HKUOPLC as the reference genome and 907 genomes of K. pneumoniae download from NCBI database as original genome dataset to determine cgMLST target genes. A total of 1,143 genes were retained as cgMLST target genes. Secondly, we used 26 K. pneumoniae strains from a nosocomial infection outbreak to evaluate the cgMLST scheme. cgMLST enabled clustering of outbreak strains with <10 alleles difference and unambiguous separation from unrelated outgroup strains. Moreover, cgMLST revealed that there may be several sub-clones of epidemic ST11 clone. In conclusion, the novel cgMLST scheme not only showed higher discriminatory power compared with PFGE and MLST in outbreak investigations but also showed ability to reveal more population structure characteristics than MLST. [ABSTRACT FROM AUTHOR]

This study investigated the prevalence of Salmonella in 210 retail meat samples (105 raw chicken and 105 raw pork) collected from supermarkets and wet markets in 13 areas of Hebei Province, China, from June to October 2018. Whole-genome sequencing was performed on all 125 Salmonella isolates to investigate their genetic relationship. Core genome multilocus sequence typing of 77 representative isolates was used to further elucidate the genetic relatedness among the Salmonella isolated from retail meat. The mean detection rate of Salmonella in all samples was 59.5% (125/210). The prevalence of Salmonella was 53.3% (56/105) in chicken and 65.7% (69/105) in pork. Chicken and pork samples collected in July had the highest detection rate of Salmonella among the sampling months. The isolates were assigned to 19 serotypes, with S. Derby, S. London, and S. Thompson being the most frequent serotypes. Resistance to tetracycline (primarily used for the treatment of bacterial infections) was observed in 89.6% of the isolates, and 84.0% were resistant to doxycycline (also a tetracycline antibiotic) or gemifloxacin (commonly used for clinical treatment of human acute bronchitis). More than 80% of the isolates were multidrug resistant. A total of 21 sequence types were identified. Sequence type 40 (ST-40), the predominant genotype among all isolates, was found only in pork; the sequence types of chicken isolates were more diverse. A total of 58 different antibiotic resistance genes (ARGs) were detected in the 125 isolates. Most types of ARGs were associated with aminoglycoside and β-lactam resistance. Nevertheless, the tetracycline resistance gene tet(A) was the most frequently occurring ARG in all isolates at 78.4%. Multiple isolates of ST-26 contained 20 ARGs. All isolates of ST-40 were divided into two clusters, with at least 160 allelic differences between them. The findings highlight the need to continually monitor ARGs in foodborne Salmonella with particular emphasis on ST-40 and ST-26; the monitoring should include as many retail meat types as possible in the study area. • Salmonella was detected in 53.3% of retail chicken and 65.7% of retail pork. • Most Salmonella isolates (89.6%) were resistant to tetracycline. • Among the Salmonella isolates, 83.2% were multidrug resistant strains. • Sequence type 40, found only in pork samples, appeared the most in Salmonella isolates. • In Salmonella isolates, 58 different antibiotic resistance genes were detected. [ABSTRACT FROM AUTHOR]

Whole genome sequencing (WGS) is replacing traditional microbiological typing methods for investigation of outbreaks in clinical settings. Here, we used a clinical microbiology laboratory core genome multilocus sequence typing (cgMLST) workflow to analyze 40 isolates of K. pneumoniae which are part of the Antimicrobial Resistance Leadership Group (ARLG) isolate collection, alongside 10 Mayo Clinic K. pneumoniae isolates, comparing results to those of pulsed-field gel electrophoresis (PFGE). Additionally, we used the WGS data to predict phenotypic antimicrobial susceptibility (AST). Thirty-one of 40 ARLG K. pneumoniae isolates belonged to the same PFGE type, all of which, alongside 3 isolates of different PFGE types, formed a large cluster by cgMLST. PFGE and cgMLST were completely concordant for the 10 Mayo Clinic K. pneumoniae isolates. For AST prediction, the overall agreement between phenotypic AST and genotypic prediction was 95.6%. [ABSTRACT FROM AUTHOR]

Burkholderia pseudomallei causes the severe disease melioidosis. Whole-genome sequencing (WGS)-based typing methods currently offer the highest resolution for molecular investigations of this genetically diverse pathogen. Still, its routine application in diagnostic laboratories is limited by the need for high computing power, bioinformatic skills, and variable bioinformatic approaches, with the latter affecting the results. We therefore aimed to establish and validate a WGS-based core genome multilocus sequence typing (cgMLST) scheme, applicable in routine diagnostic settings. A soft defined core genome was obtained by challenging the B. pseudomallei reference genome K96243 with 469 environmental and clinical genomes, resulting in 4,221 core and 1,351 accessory targets. The scheme was validated with 320 WGS data sets. We compared our novel typing scheme with single nucleotide polymorphism-based approaches investigating closely and distantly related strains. Finally, we applied our scheme for tracking the environmental source of a recent infection. The validation of the scheme detected >95% good cgMLST target genes in 98.4% of the genomes. Comparison with existing typing methods revealed very good concordance. Our scheme proved to be applicable to investigating not only closely related strains but also the global B. pseudomallei population structure. We successfully utilized our scheme to identify a sugarcane field as the presumable source of a recent melioidosis case. In summary, we developed a robust cgMLST scheme that integrates high resolution, maximized standardization, and fast analysis for the nonbioinformatician. Our typing scheme has the potential to serve as a routinely applicable classification system in B. pseudomallei molecular epidemiology.

Objective: To analyze the molecular characteristics of Listeria monocytogenes strains from ready-to eat food in China. Methods: A total of 239 Listeria monocytogenes strains isolated from ready-to-eat food in 2017, all strains underwent whole-genome sequencing (WGS) , and comparisons uncovered population structure derived from lineages, clonal complex, serogroups, antimicrobial susceptibility and virulence, which were inferred in silico from the WGS data. Core genome multilocus sequence typing was used to subtype isolates. Results: All strains were categorized into three different lineages, lineage Ⅱ was the predominant types in food, and IIa was the main serogroups. CC8, CC101 and CC87 were the first three prevalent CCs among 23 detected CCs, accounting for 49.4%. Only 4.6% (11 isolates) of tested strains harbored antibiotic resistance genes, which were mostly trimethoprim genes (7 isolates, 2.9%). All strains were positive for LIPI-1, and only a part of strains harbored LIPI-3 and LIPI-4, accounting for 13.8% (33 isolates) and 14.2% (34 isolates), respectively. ST619 carried both LIPI-3 and LIPI-4. 51.5% (123 isolates) of strains carried SSI-1, and all CC121 strains harbored SSI-2. Different lineages, serogroups and CCs can be separated obviously through cgMLST analysis, and 24 sublineages were highly concordant with CCs. Conclusion: Ⅱa was the main serogroups in ready-to-eat food isolates in China; CC8, CC101 and CC87 were the prevalent CCs, and CC87 isolates was hypervirulent isolates, cgMLST method can be adopted for prospective foodborne disease surveillance and outbreaks detection.

Due to the potential of enterohemorrhagic Escherichia coli (EHEC) serogroup O157 to cause large food borne outbreaks, national and international surveillance is necessary. For developing an effective method of molecular surveillance, a conventional method, multilocus variable-number tandem-repeat analysis (MLVA), and whole-genome sequencing (WGS) analysis were compared. WGS of 369 isolates of EHEC O157 belonging to 7 major MLVA types and their relatives were subjected to comprehensive in silico typing, core genome single nucleotide polymorphism (cgSNP), and core genome multilocus sequence typing (cgMLST) analyses. The typing resolution was the highest in cgSNP analysis. However, determination of the sequence of the mismatch repair protein gene mutS is necessary because spontaneous deletion of the gene could lead to a hypermutator phenotype. MLVA had sufficient typing resolution for a short-term outbreak investigation and had advantages in rapidity and high throughput. cgMLST showed less typing resolution than cgSNP, but it is less time-consuming and does not require as much computer power. Therefore, cgMLST is suitable for comparisons using large data sets (e.g., international comparison using public databases). In conclusion, screening using MLVA followed by cgMLST and cgSNP analyses would provide the highest typing resolution and improve the accuracy and cost-effectiveness of EHEC O157 surveillance. IMPORTANCE Intensive surveillance for enterohemorrhagic Escherichia coli (EHEC) serogroup O157 is important to detect outbreaks and to prevent the spread of the bacterium. Recent advances in sequencing technology made molecular surveillance using whole-genome sequence (WGS) realistic. To develop rapid, high-throughput, and cost-effective typing methods for real-time surveillance, typing resolution of WGS and a conventional typing method, multilocus variable-number tandem-repeat analysis (MLVA), was evaluated. Nation-level systematic comparison of MLVA, core genome single nucleotide polymorphism (cgSNP), and core genome multilocus sequence typing (cgMLST) indicated that a combination of WGS and MLVA is a realistic approach to improve EHEC O157 surveillance., (Copyright © 2019 American Society for Microbiology.)

Epidemiological findings of a listeriosis outbreak in 2013 implicated Hispanic-style cheese produced by company A, and pulsed-field gel electrophoresis (PFGE) and whole genome sequencing (WGS) were performed on clinical isolates and representative isolates collected from company A cheese and environmental samples during the investigation. The results strengthened the evidence for cheese as the vehicle. Surveillance sampling and WGS 3 months later revealed that the equipment purchased by company B from company A yielded an environmental isolate highly similar to all outbreak isolates. The whole genome and core genome multilocus sequence typing and single nucleotide polymorphism (SNP) analyses results were compared to demonstrate the maximum discriminatory power obtained by using multiple analyses, which were needed to differentiate outbreak-associated isolates from a PFGE-indistinguishable isolate collected in a nonimplicated food source in 2012. This unrelated isolate differed from the outbreak isolates by only 7 to 14 SNPs, and as a result, the minimum spanning tree from the whole genome analyses and certain variant calling approach and phylogenetic algorithm for core genome-based analyses could not provide differentiation between unrelated isolates. Our data also suggest that SNP/allele counts should always be combined with WGS clustering analysis generated by phylogenetically meaningful algorithms on a sufficient number of isolates, and the SNP/allele threshold alone does not provide sufficient evidence to delineate an outbreak. The putative prophages were conserved across all the outbreak isolates. All outbreak isolates belonged to clonal complex 5 and serotype 1/2b and had an identical inlA sequence which did not have premature stop codons. IMPORTANCE In this outbreak, multiple analytical approaches were used for maximum discriminatory power. A PFGE-matched, epidemiologically unrelated isolate had high genetic similarity to the outbreak-associated isolates, with as few as 7 SNP differences. Therefore, the SNP/allele threshold should not be used as the only evidence to define the scope of an outbreak. It is critical that the SNP/allele counts be complemented by WGS clustering analysis generated by phylogenetically meaningful algorithms to distinguish outbreak-associated isolates from epidemiologically unrelated isolates. Careful selection of a variant calling approach and phylogenetic algorithm is critical for core-genome-based analyses. The whole-genome-based analyses were able to construct the highly resolved phylogeny needed to support the findings of the outbreak investigation. Ultimately, epidemiologic evidence and multiple WGS analyses should be combined to increase confidence levels during outbreak investigations., (Copyright © 2017 Chen et al.)

Vibrio parahaemolyticus is an important human foodborne pathogen whose transmission is associated with the consumption of contaminated seafood, with a growing number of infections reported over recent years worldwide. A multilocus sequence typing (MLST) database for V. parahaemolyticus was created in 2008, and a large number of clones have been identified, causing severe outbreaks worldwide (sequence type 3 [ST3]), recurrent outbreaks in certain regions (e.g., ST36), or spreading to other regions where they are nonendemic (e.g., ST88 or ST189). The current MLST scheme uses sequences of 7 genes to generate an ST, which results in a powerful tool for inferring the population structure of this pathogen, although with limited resolution, especially compared to pulsed-field gel electrophoresis (PFGE). The application of whole-genome sequencing (WGS) has become routine for trace back investigations, with core genome MLST (cgMLST) analysis as one of the most straightforward ways to explore complex genomic data in an epidemiological context. Therefore, there is a need to generate a new, portable, standardized, and more advanced system that provides higher resolution and discriminatory power among V. parahaemolyticus strains using WGS data. We sequenced 92 V. parahaemolyticus genomes and used the genome of strain RIMD 2210633 as a reference (with a total of 4,832 genes) to determine which genes were suitable for establishing a V. parahaemolyticus cgMLST scheme. This analysis resulted in the identification of 2,254 suitable core genes for use in the cgMLST scheme. To evaluate the performance of this scheme, we performed a cgMLST analysis of 92 newly sequenced genomes, plus an additional 142 strains with genomes available at NCBI. cgMLST analysis was able to distinguish related and unrelated strains, including those with the same ST, clearly showing its enhanced resolution over conventional MLST analysis. It also distinguished outbreak-related from non-outbreak-related strains within the same ST. The sequences obtained from this work were deposited and are available in the public database (http://pubmlst.org/vparahaemolyticus). The application of this cgMLST scheme to the characterization of V. parahaemolyticus strains provided by different laboratories from around the world will reveal the global picture of the epidemiology, spread, and evolution of this pathogen and will become a powerful tool for outbreak investigations, allowing for the unambiguous comparison of strains with global coverage., (Copyright © 2017 Gonzalez-Escalona et al.)