From Investigating a Case of Cellulitis to Exploring Nosocomial Infection Control of ST1 Legionella pneumophila Using Genomic Approaches.

Legionella pneumophila can cause a large panel of symptoms besides the classic pneumonia presentation. Here we present a case of fatal nosocomial cellulitis in an immunocompromised patient followed, a year later, by a second case of Legionnaires' disease in the same ward. While the first case was easily assumed as nosocomial based on the date of symptom onset, the second case required clear typing results to be assigned either as nosocomial and related to the same environmental source as the first case, or community acquired. To untangle this specific question, we applied core-genome multilocus typing (MLST), whole-genome single nucleotide polymorphism and whole-genome MLST methods to a collection of 36 Belgian and 41 international sequence-type 1 (ST1) isolates using both thresholds recommended in the literature and tailored threshold based on local epidemiological data. Based on the thresholds applied to cluster isolates together, the three methods gave different results and no firm conclusion about the nosocomial setting of the second case could been drawn. Our data highlight that despite promising results in the study of outbreaks and for large-scale epidemiological investigations, next-generation sequencing typing methods applied to ST1 outbreak investigation still need standardization regarding both wet-lab protocols and bioinformatics. A deeper evaluation of the L. pneumophila evolutionary clock is also required to increase our understanding of genomic differences between isolates sampled during a clinical infection and in the environment.

Comparative genomics reveals high genetic similarity among strains of Salmonella enterica serovar Infantis isolated from multiple sources in Brazil.

Background: Salmonella enterica serovar Infantis (Salmonella Infantis) is a zoonotic, ubiquitous and foodborne pathogen of worldwide distribution. Despite Brazil's relevance as a major meat exporter, few studies were conducted to characterize strains of this serovar by genomic analyses in this country. Therefore, this study aimed to assess the diversity of 80 Salmonella Infantis strains isolated from veterinary, food and human sources in Brazil between 2013 and 2018 by comparative genomic analyses. Additional genomes of non-Brazilian countries (n = 18) were included for comparison purposes in some analyses. Methods: Analyses of whole-genome multi-locus sequence typing (wgMLST), using PGAdb-builder, and of fragmented genomes, using Gegenees, were conducted to compare the 80 Brazilian strains to the 18 non-Brazilian genomes. Pangenome analyses and calculations were performed for all Salmonella Infantis genomes analyzed. The presence of prophages was determined using PHASTER for the 80 Brazilian strains. The genome plasticity using BLAST Ring Image Generator (BRIG) and gene synteny using Mauve were evaluated for 20 selected Salmonella Infantis genomes from Brazil and ten from non-Brazilian countries. Unique orthologous protein clusters were searched in ten selected Salmonella Infantis genomes from Brazil and ten from non-Brazilian countries. Results: wgMLST and Gegenees showed a high genomic similarity among some Brazilian Salmonella Infantis genomes, and also the correlation of some clusters with non-Brazilian genomes. Gegenees also showed an overall similarity >91% among all Salmonella Infantis genomes. Pangenome calculations revealed an open pangenome for all Salmonella Infantis subsets analyzed and a high gene content in the core genomes. Fifteen types of prophages were detected among 97.5% of the Brazilian strains. BRIG and Mauve demonstrated a high structural similarity among the Brazilian and non-Brazilian isolates. Unique orthologous protein clusters related to biological processes, molecular functions, and cellular components were detected among Brazilian and non-Brazilian genomes. Conclusion: The results presented using different genomic approaches emphasized the significant genomic similarity among Brazilian Salmonella Infantis genomes analyzed, suggesting wide distribution of closely related genotypes among diverse sources in Brazil. The data generated contributed to novel information regarding the genomic diversity of Brazilian and non-Brazilian Salmonella Infantis in comparison. The different genetically related subtypes of Salmonella Infantis from Brazil can either occur exclusively within the country, or also in other countries, suggesting that some exportation of the Brazilian genotypes may have already occurred.

Flagella Phenotypic Variations of ST34 Type Salmonella Typhimurium and Variants.

Salmonella enterica serovar Typhimurium and its variants are the most common serotypes of human salmonellosis cases. Serotyping Salmonella Typhimurium and its variants has always been challenging. Our previous work found that among 14 Salmonella Typhimurium and variant strains, some different antigenic formulas had 100% pulsed-field gel electrophoresis (PFGE) similarity. The 14 strains were sorted into 3 groups; in each group, the different antigenic formulas had the same PFGE patterns. This phenomenon suggested that different antigenic formula identification might originate from a common ancestor subtyped by PFGE. To assess whether the serotyping method on Salmonella Typhimurium and variant strains reflected the genetic relationship, we improved the discrimination for the phylogenetic relationship among the 14 Salmonella Typhimurium and variant strains using Fourier-transform infrared spectroscopy (FTIR) and whole-genome multilocus sequence typing (wgMLST). We compared the wgMLST assay of 14 Salmonella Typhimurium and variant strains from this study with 50 public ST34 strain data of Salmonella Typhimurium and variant strains. We also compared flagella (H antigen)-related genes based on the whole genome of 14 strains and the other 293 ST34 public database for further understanding of this question. The phylogenetic results (PFGE) showed no regularity between the antigenic formulas and genotypes. The results of the higher discrimination power assays (FTIR and whole-genome multilocus sequence typing) also showed no regularity between the antigenic formulas and genotypes (or phenotypes). The 58 flagella encoding genes of different antigenic formulas were sorted into 13 patterns. However, a similar phenomenon was found: the same flagella encoding gene patterns could express different antigenic formulas. In conclusion, there is no consistency between the antigenic formulas and phylogenetic relationships among ST34 Salmonella Typhimurium and variant strains, even in flagella antigenic formula and flagella encoding genes.

Whole genome sequence-based molecular characterization of blood isolates of carbapenem-resistant Enterobacter cloacae complex from ICU patients in Kolkata, India, during 2017-2022: emergence of phylogenetically heterogeneous Enterobacter hormaechei subsp. xiangfangensis.

Blood-borne infections caused by the carbapenem-resistant Enterobacter cloacae complex (CR-ECC) are major public threats with respect to the challenges encountered during treatment. This study describes the whole genome sequencing-based molecular characteristics of blood isolates (n = 70) of CR-ECC from patients admitted to the intensive care unit of tertiary care hospitals in Kolkata, India, during 2017-2022 with respect to species identification, antimicrobial resistance (AMR) profiling, mechanism of drug resistance, and molecular subtypes. Vitek2 MALDI and species-specific PCR identified Enterobacter hormaechei subsp. xiangfangensis (47.14%) as the emerging CR-ECC subspecies in Kolkata. The predominating carbapenemase and extended-spectrum ß-lactamase genes found were blaNDM-1 (51.42%) and blaCTX-M-15 (27%), respectively. Besides, blaNDM-4, blaNDM-5, blaNDM-7, blaCMH-3, blaSFO-1, blaOXA-181, blaOXA-232, blaKPC-3, and blaDHA-7 genes were also detected, which were not previously reported from India. A multitude of Class 1 integrons (including In180, In4874, In4887, and In4888, which were novel) and plasmid replicon types (IncFIB, IncFII, IncX3, IncHI1-HI2, IncC, and IncR) involved in AMR dissemination were identified. Reverse transcription-PCR and western blot revealed that carbapenem resistance in non-carbapenemase-producing CR-ECC isolates was contributed by elevated levels of ampC, overexpression of acrAB, and loss of ompF. A total of 30 distinct sequence types (STs) were ascertained by multi-locus sequence typing; of which, ST2011, ST2018, ST2055, ST2721, and ST2722 were novel STs. Pulsed-field gel electrophoresis analysis showed heterogeneity (69 pulsotypes with a similarity coefficient of 48.40%) among the circulating isolates, suggesting multiple reservoirs of infections in humans. Phylogenetically and genetically diverse CR-ECC with multiple AMR mechanisms mandates close monitoring of nosocomial infections caused by these isolates to forestall the transmission and dissemination of AMR.IMPORTANCEThe emergence and extensive dissemination of the carbapenem-resistant Enterobacter cloacae complex (CR-ECC) have positioned it as a critical nosocomial global pathogen. The dearth of a comprehensive molecular study pertaining to CR-ECC necessitated this study, which is the first of its kind from India. Characterization of blood isolates of CR-ECC over the last 6 years revealed Enterobacter hormaechei subsp. xiangfangensis as the most prevalent subsp., exhibiting resistance to almost all antibiotics currently in use and harboring diverse transmissible carbapenemase genes. Besides the predominating blaNDM-1 and blaCTX-M-15, we document diverse carbapenemase and AmpC genes, such as blaNDM-4, blaNDM-7, blaOXA-181, blaOXA-232, blaKPC-3, blaCMH-3, blaSFO-1, and blaDHA-7, in CR-ECC, which were not previously reported from India. Furthermore, novel integrons and sequence types were identified. Our findings emphasize the need for strengthened vigilance for molecular epidemiological surveillance of CR-ECC due to the presence of epidemic clones with a phylogenetically diverse and wide array of antimicrobial resistance genes in vulnerable populations.

Epidemiology and molecular typing of multidrug-resistant bacteria in tertiary hospitals and nursing homes in Flanders, Belgium.

This study aimed to map MDRO carriage and potential transmission within and between three Flemish tertiary care hospitals and their neighbouring nursing homes. A cross-sectional MDRO prevalence survey was organized between October 2017 and February 2019. Perianal swabs were cultured for detection of MDRO. Determination of clonal relatedness based on wgMLST allelic profiles was performed. The prevalence of MDRO in Belgian hospitals and NHs is on the rise, compared to previous studies, and transmission in and between institutions is observed. These results re-emphasize the need for a healthcare network-wide infection prevention strategy in which WGS of MDRO strains can be supportive.

Core-genome multilocus sequence typing and core-SNP analysis of Clostridium neonatale strains isolated in different spatio-temporal settings.

IMPORTANCE: Clostridium neonatale has been isolated from the fecal samples of asymptomatic neonates and cases of necrotizing enterocolitis (NEC). Taking advantage of a large collection of independent strains isolated from different spatio-temporal settings, we developed and established a cgMLST scheme for the molecular typing of C. neonatale. Both the cgMLST and cgSNP methods demonstrate comparable discrimination power. Results indicate geographic- and temporal- independent clustering of C. neonatale NEC-associated strains. No specific cgMLST clade of C. neonatale was genetically associated with NEC.

Genome-Scale Characterization of Mycobacterium abscessus Complex Isolates from Portugal.

The Mycobacterium abscessus complex (MABC) is an emerging, difficult to treat, multidrug-resistant nontuberculous mycobacteria responsible for a wide spectrum of infections and associated with an increasing number of cases worldwide. Dominant circulating clones (DCCs) of MABC have been genetically identified as groups of strains associated with higher prevalence, higher levels of antimicrobial resistance, and worse clinical outcomes. To date, little is known about the genomic characteristics of MABC species circulating in Portugal. Here, we examined the genetic diversity and antimicrobial resistance profiles of 30 MABC strains isolated between 2014 and 2022 in Portugal. The genetic diversity of circulating MABC strains was assessed through a gene-by-gene approach (wgMLST), allowing their subspecies differentiation and the classification of isolates into DCCs. Antimicrobial resistance profiles were defined using phenotypic, molecular, and genomic approaches. The majority of isolates were resistant to at least two antimicrobials, although a poor correlation between phenotype and genotype data was observed. Portuguese genomes were highly diverse, and data suggest the existence of MABC lineages with potential international circulation or cross-border transmission. This study highlights the genetic diversity and antimicrobial resistance profile of circulating MABC isolates in Portugal while representing the first step towards the implementation of a genomic-based surveillance system for MABC at the Portuguese NIH.

Evaluation of whole and core genome multilocus sequence typing allele schemes for Salmonella enterica outbreak detection in a national surveillance network, PulseNet USA.

Salmonella enterica is a leading cause of bacterial foodborne and zoonotic illnesses in the United States. For this study, we applied four different whole genome sequencing (WGS)-based subtyping methods: high quality single-nucleotide polymorphism (hqSNP) analysis, whole genome multilocus sequence typing using either all loci [wgMLST (all loci)] and only chromosome-associated loci [wgMLST (chrom)], and core genome multilocus sequence typing (cgMLST) to a dataset of isolate sequences from 9 well-characterized Salmonella outbreaks. For each outbreak, we evaluated the genomic and epidemiologic concordance between hqSNP and allele-based methods. We first compared pairwise genomic differences using all four methods. We observed discrepancies in allele difference ranges when using wgMLST (all loci), likely caused by inflated genetic variation due to loci found on plasmids and/or other mobile genetic elements in the accessory genome. Therefore, we excluded wgMLST (all loci) results from any further comparisons in the study. Then, we created linear regression models and phylogenetic tanglegrams using the remaining three methods. K-means analysis using the silhouette method was applied to compare the ability of the three methods to partition outbreak and sporadic isolate sequences. Our results showed that pairwise hqSNP differences had high concordance with cgMLST and wgMLST (chrom) allele differences. The slopes of the regressions for hqSNP vs. allele pairwise differences were 0.58 (cgMLST) and 0.74 [wgMLST (chrom)], and the slope of the regression was 0.77 for cgMLST vs. wgMLST (chrom) pairwise differences. Tanglegrams showed high clustering concordance between methods using two statistical measures, the Baker's gamma index (BGI) and cophenetic correlation coefficient (CCC), where 9/9 (100%) of outbreaks yielded BGI values ≥ 0.60 and CCCs were ≥ 0.97 across all nine outbreaks and all three methods. K-means analysis showed separation of outbreak and sporadic isolate groups with average silhouette widths ≥ 0.87 for outbreak groups and ≥ 0.16 for sporadic groups. This study demonstrates that Salmonella isolates clustered in concordance with epidemiologic data using three WGS-based subtyping methods and supports using cgMLST as the primary method for national surveillance of Salmonella outbreak clusters.

Epidemiology and molecular typing of multidrug-resistant bacteria in day care centres in Flanders, Belgium.

The global prevalence and spread of multidrug-resistant organisms (MDROs) represent an emerging public health threat. Day care centre (DCC) attendance is a risk factor for MDRO carriage in children and their environment. This study aimed to map the epidemiology of carriage and potential transmission of these organisms within 18 Flemish DDCs (Belgium). An MDRO prevalence survey was organised between November 2018 and February 2019 among children attending the centres. Selective chromogenic culture media were used for the detection of extended-spectrum beta-lactamase-producing Enterobacterales (ESBL-E), carbapenemase-producing Enterobacterales (CPE), and vancomycin-resistant Enterococci (VRE) in faecal swabs obtained from diapers or jars (n = 448). All isolated MDROs were subjected to resistance gene sequencing. A total of 71 of 448 samples (15.8%) yielded isolates of ESBL-E with a predominance of Escherichia coli (92.2% of ESBL-E) and ESBL resistance gene blaCTX-M-15 (50.7% of ESBL coding genes in E. coli). ESBL-E prevalence varied between DCCs, ranging from 0 to 50%. Transmission, based on the clonal relatedness of ESBL-E strains, was observed. CPE was identified in only one child carrying an E. coli with an OXA-244 gene. VRE was absent from all samples. The observed prevalence of ESBL-E in Flemish DCCs is high compared with previous studies, and our findings re-emphasise the need for rigorous hygiene measures within such centres to control the further spread of MDROs in the community.

Etiological Survey and Traceability Analysis of a Foodborne Disease Outbreak of Salmonella Senftenberg in Guizhou Province.

To conduct a study that examined the molecular epidemiology and pathogenesis of Salmonella Senftenberg isolates associated with an outbreak of foodborne disease in Guizhou Province and to provide a reference basis for the traceability of foodborne salmonellosis outbreaks and clinical diagnosis and treatment in the province. Fourteen strains of suspected Salmonella isolated from patient stool and food samples were used for pathogenic identification and serotyping by biochemical and mass spectrometry methods. Fourteen types of antibiotics were tested for drug sensitivity by the microbroth dilution method, and molecular typing was performed by pulsed-field gel electrophoresis (PFGE) and whole genome sequencing (WGS). After the sequencing data were spliced by SPAdes, the gene protein sequences were compared with the Comprehensive Antibiotic Research Database and Virulence Factor Database, drug resistance and virulence genes were predicted, and whole genome multilocus sequence typing (wgMLST) was performed. The results were compared with those for Salmonella strains of the same serotype from the past 5 years in China detailed on the TraNet website. All 14 strains were identified as Salmonella Senftenberg (with the antigenic formula 1,3,19:g,s,t:-), and in the PFGE cluster tree, the strains were divided into two band types, with a similarity of 88.9%. The 14 strains were sensitive to the 14 antibiotics. WGS analysis showed that the 14 strains carried the same drug resistance and virulence genes and that all strains carried 3 aminoglycoside and lipopeptide drug resistance genes, including 114 virulence genes. The wgMLST results showed that the strains were distributed on the same small branch as those obtained from previous outbreaks of infection in Tianjin and Jilin. Salmonella Senftenberg, which caused the outbreak, carries a variety of virulence genes, which suggests that the strain is highly pathogenic. These pathogenic bacteria may be associated with the Salmonella strain in Tianjin, Jilin, and other places and have caused foodborne disease outbreaks as a result of imported contamination.

Evaluation of core genome and whole genome multilocus sequence typing schemes for Campylobacter jejuni and Campylobacter coli outbreak detection in the USA.

Campylobacter is a leading causing of bacterial foodborne and zoonotic illnesses in the USA. Pulsed-field gene electrophoresis (PFGE) and 7-gene multilocus sequence typing (MLST) have been historically used to differentiate sporadic from outbreak Campylobacter isolates. Whole genome sequencing (WGS) has been shown to provide superior resolution and concordance with epidemiological data when compared with PFGE and 7-gene MLST during outbreak investigations. In this study, we evaluated epidemiological concordance for high-quality SNP (hqSNP), core genome (cg)MLST and whole genome (wg)MLST to cluster or differentiate outbreak-associated and sporadic Campylobacter jejuni and Campylobacter coli isolates. Phylogenetic hqSNP, cgMLST and wgMLST analyses were also compared using Baker's gamma index (BGI) and cophenetic correlation coefficients. Pairwise distances comparing all three analysis methods were compared using linear regression models. Our results showed that 68/73 sporadic C. jejuni and C. coli isolates were differentiated from outbreak-associated isolates using all three methods. There was a high correlation between cgMLST and wgMLST analyses of the isolates; the BGI, cophenetic correlation coefficient, linear regression model R 2 and Pearson correlation coefficients were >0.90. The correlation was sometimes lower comparing hqSNP analysis to the MLST-based methods; the linear regression model R 2 and Pearson correlation coefficients were between 0.60 and 0.86, and the BGI and cophenetic correlation coefficient were between 0.63 and 0.86 for some outbreak isolates. We demonstrated that C. jejuni and C. coli isolates clustered in concordance with epidemiological data using WGS-based analysis methods. Discrepancies between allele and SNP-based approaches may reflect the differences between how genomic variation (SNPs and indels) are captured between the two methods. Since cgMLST examines allele differences in genes that are common in most isolates being compared, it is well suited to surveillance: searching large genomic databases for similar isolates is easily and efficiently done using allelic profiles. On the other hand, use of an hqSNP approach is much more computer intensive and not scalable to large sets of genomes. If further resolution between potential outbreak isolates is needed, wgMLST or hqSNP analysis can be used.

Genomes-based MLST, cgMLST, wgMLST and SNP analysis of Salmonella Typhimurium from animals and humans.

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.

A pseudo-outbreak of MRSA due to laboratory contamination related to MRSA carriage of a laboratory staff member.

BACKGROUND: Methicillin resistant Staphylococcus aureus (MRSA) is a major burden for hospitals globally. However, in the Netherlands, the MRSA prevalence is relatively low due to the 'search and destroy' policy. Routine multiple-locus variable-number of tandem repeat analysis (MLVA) of MRSA isolates supports outbreak detection. However, whole genome multiple locus sequence typing (wgMLST) is superior to MLVA in identifying (pseudo-)outbreaks with MRSA. The present study describes a pseudo-outbreak of MRSA at the bacteriology laboratory of a large Dutch teaching hospital. METHODS: All staff members of the bacteriology laboratory of the Elisabeth-TweeSteden hospital were screened for MRSA carriage, after a laboratory contamination with MRSA was suspected. Clonal relatedness between the index isolate and the MRSA isolates from laboratory staff members and all previous MRSA isolates from the Elisabeth-TweeSteden hospital with the same MLVA-type as the index case was examined based on wgMLST using whole genome sequencing. RESULTS: One of the staff members was identified as the probable source of the laboratory contamination, because of carriage of a MRSA possessing the same MLVA-type as the index case. Eleven other isolates with the same molecular characteristics were found in the database, of which seven were retrospectively suspected of contamination. Clonal relatedness was found between ten isolates, including the isolate found in the staff member and the MRSA found in the index patient with a maximum of eleven alleles difference. All isolates were epidemiologically linked through the laboratory staff member, who had worked on all these cultures. CONCLUSIONS: The present study describes a MRSA pseudo-outbreak over a 2.5-year period due to laboratory contamination caused by a MRSA carrying laboratory staff member involving nine patients. In case of unexpected bacteriological findings, the possibility of a laboratory contamination should be considered.

Whole Genome Multi-Locus Sequence Typing and Genomic Single Nucleotide Polymorphism Analysis for Epidemiological Typing of Pseudomonas aeruginosa From Indonesian Intensive Care Units.

We have previously studied carbapenem non-susceptible Pseudomonas aeruginosa (CNPA) strains from intensive care units (ICUs) in a referral hospital in Jakarta, Indonesia (Pelegrin et al., 2019). We documented that CNPA transmissions and acquisitions among patients were variable over time and that these were not significantly reduced by a set of infection control measures. Three high risk international CNPA clones (sequence type (ST)235, ST823, ST357) dominated, and carbapenem resistance was due to carbapenemase-encoding genes and mutations in the porin OprD. Pelegrin et al. (2019) reported core genome analysis of these strains. We present a more refined and detailed whole genome-based analysis of major clones represented in the same dataset. As per our knowledge, this is the first study reporting Single Nucleotide Polymorphisms (wgSNP) analysis of Pseudomonas strains. With whole genome-based Multi Locus Sequence Typing (wgMLST) of the 3 CNPA clones (ST235, ST357 and ST823), three to eleven subgroups with up to 200 allelic variants were observed for each of the CNPA clones. Furthermore, we analyzed these CNPA clone clusters for the presence of wgSNP to redefine CNPA transmission events during hospitalization. A maximum number 35350 SNPs (including non-informative wgSNPs) and 398 SNPs (ST-specific_informative-wgSNPs) were found in ST235, 34,570 SNPs (including non-informative wgSNPs) and 111 SNPs (ST-specific_informative-wgSNPs) in ST357 and 26,443 SNPs (including non-informative SNPs) and 61 SNPs (ST-specific_informative-wgSNPs) in ST823. ST-specific_Informative-wgSNPs were commonly noticed in sensor-response regulator genes. However, the majority of non-informative wgSNPs was found in conserved hypothetical proteins or in uncharacterized proteins. Of note, antibiotic resistance and virulence genes segregated according to the wgSNP analyses. A total of 8 transmission chains for ST235 strains followed by 9 and 4 possible transmission chains for ST357 and ST823 were traceable on the basis of pairwise distances of informative-wgSNPs (0 to 4 SNPs) among the strains. The present study demonstrates the value of detailed whole genome sequence analysis for highly refined epidemiological analysis of P. aeruginosa.

Genomic Analysis Reveals That Isolation Temperature on Selective Media Introduces Genetic Variation in Campylobacter jejuni from Bovine Feces.

Campylobacter jejuni is commonly isolated on selective media following incubation at 37 °C or 42 °C, but the impact of these temperatures on genome variation remains unclear. Previously, Campylobacter selective enrichments from the feces of steers before and after ceftiofur treatment were plated on selective agar media and incubated at either 37 °C or 42 °C. Here, we analyzed the whole genome sequence of C. jejuni strains of the same multilocus sequence typing (MLST)-based sequence type (ST) and isolated from the same sample upon incubation at both temperatures. Four such strain pairs (one ST8221 and three ST8567) were analyzed using core genome and whole genome MLST (cgMLST, wgMLST). Among the 1970 wgMLST loci, 7-25 varied within each pair. In all but one of the pairs more (1.7-8.5 fold) new alleles were found at 42 °C. Most frameshift, nonsense, or start-loss mutations were also found at 42 °C. Variable loci CAMP0575, CAMP0912, and CAMP0913 in both STs may regularly respond to different temperatures. Furthermore, frameshifts in four variable loci in ST8567 occurred at multiple time points, suggesting a persistent impact of temperature. These findings suggest that the temperature of isolation may impact the sequence of several loci in C. jejuni from cattle.

Dissecting Listeria monocytogenes Persistent Contamination in a Retail Market Using Whole-Genome Sequencing.

Listeria monocytogenes is a foodborne pathogen that can cause invasive disease with high mortality in immunocompromised individuals and can survive in a variety of food-associated environments for a long time. L. monocytogenes clonal complex (CC) 87 is composed of ST87 and three other STs and has been identified as the most common subgroup associated with both foods and human clinical infections in China. Therefore, the persistence of CC87 L. monocytogenes in food-associated environments poses a significant concern for food safety. In this study, 83 draft genomes of CC87 L. monocytogenes, including 60 newly sequenced genomes, were analyzed with all isolates from our previous surveillance in Zigong, Sichuang, China. Sixty-eight of the studied isolates were isolated from one retail market (M1 market), while the others were from seven other markets (M2-M8 markets) in the same city. Whole-genome multilocus sequence typing (wg-MLST) and the whole-genome single nucleotide polymorphism (wg-SNP) analysis were performed. Three persistent contamination routes were identified in the M1 market, caused by 2 clusters (A and B) and a wgST31 type. Cluster A isolates were associated with the persistent contamination in a raw meat stall (M1-S77), while Cluster B isolates caused a persistent contamination in aquatic foods stalls. Five wgST31 isolates caused persistent contamination in a single aquatic stall (M1-S65). A pLM1686-like plasmid was found in all Cluster A isolates. A novel plasmid, pLM1692, a truncated pLM1686 plasmid without the cadmium, and other heavy metal resistance genes were conserved in all wgST31 isolates. By comparing persistent and putative non-persistent isolates, four genes that were all located in the prophage comK might be associated with persistence. These findings enhanced our understanding of the underlying mechanisms of contamination and assist in formulating targeted strategies for the prevention and control of L. monocytogenes transmission from the food processing chain to humans. IMPORTANCE Contamination of food by Listeria monocytogenes at retail level leads to potential consumption of contaminated food with high risk of human infection. Our previous study found persistent contamination of CC87 L. monocytogenes from a retail market in China through pulsed-field gel electrophoresis and multilocus sequence typing. In this study, whole-genome sequencing was used to obtain the highest resolution inference of the source and reasons for persistent contamination; meat grinders and minced meat were the major reservoir of persistent contamination in meat stalls, whereas fishponds were the major reservoir in seafood stalls, with different L. monocytogenes isolates involved. These isolates carried different properties such as plasmids and prophages, which may have contributed to their ability to survive or adapt to the different environments. Our findings suggest that whole-genome sequencing will be an effective surveillance tool to detect persistent L. monocytogenes contamination in retail food markets and to design new control strategies to improve food safety.

Molecular Identification and Selection of Probiotic Strains Able to Reduce the Serum TMAO Level in Mice Challenged with Choline.

Trimethylamine oxide (TMAO) originates from trimethylamine (TMA), which is oxidized in the liver by hepatic flavin-containing monooxygenases (FMO3). TMA is produced by its dietary precursors such as choline, carnitine, and phosphatidylcholine by gut microbiota. TMAO attracts attention, identified as a novel and independent risk factor for promoting obesity, atherosclerosis and cardiovascular disease (CVD), chronic kidney disease (CKD), insulin tolerance, and colon cancer. Probiotics have been considered as live microorganisms, providing benefits to their host when they are given in sufficient quantities and administered continuously. The objective of this study is to suggest a method to select potential probiotic strains to reduce the serum concentration of TMAO in mice fed with choline. In this work, we chose three lactobacilli with strong adherence capability, and fed multistrain formula (MF) to the mice challenged with choline. On days 7, 14, and day 28, it was found that the MF-containing L. amylovorus LAM1345, Lpb. plantarum LP1145, and Lim. fermentum LF33 showed a significant reduction in serum TMAO and TMA levels. For the single strains, LP1145 reduced TMAO on days 14 and 28, and strain LAM1345 reduced TMAO significantly on days 7 and day 14. For strain LF1143 from strain LF33, it showed no significant effect on TMAO and TMA. Thus, MF showed the best effect, which may be due to the additive and synergetic effect and the contribution of strain LP1145 and LAM1345. Finally, for the LAM1345 and LP1145 strains, we used molecular identification and typing methods to assure that these two strains are unique strains. The methods used for LAM 1345 were leader peptidase A (lepA) gene analysis and phylogenetic analysis, while for strain LP 1145and other strains of Lpb. plantarum subsp. plantarum sequences were compared using the whole-genome multilocus sequence typing (wgMLST) method.

The global population structure and beta-lactamase repertoire of the opportunistic pathogen Serratia marcescens.

Serratia marcescens is a global spread nosocomial pathogen. This rod-shaped bacterium displays a broad host range and worldwide geographical distribution. Here we analyze an international collection of this multidrug-resistant, opportunistic pathogen from 35 countries to infer its population structure. We show that S. marcescens comprises 12 lineages; Sm1, Sm4, and Sm10 harbor 78.3% of the known environmental strains. Sm5, Sm6, and Sm7 comprise only human-associated strains which harbor smallest pangenomes, genomic fluidity and lowest levels of core recombination, indicating niche specialization. Sm7 and Sm9 lineages exhibit the most concerning resistome; blaKPC-2 plasmid is widespread in Sm7, whereas Sm9, also an anthropogenic-exclusive lineage, presents highest plasmid/lineage size ratio and plasmid-diversity encoding metallo-beta-lactamases comprising blaNDM-1. The heterogeneity of resistance patterns of S. marcescens lineages elucidated herein highlights the relevance of surveillance programs, using whole-genome sequencing, to provide insights into the molecular epidemiology of carbapenemase producing strains of this species.