Characterization of Listeria monocytogenes Strains Involved in Invasive and Noninvasive Listeriosis Outbreaks by PCR-Based Fingerprinting Techniques

A total of 32 Listeria monocytogenes strains (16 from a recent outbreak of invasive listeriosis and 16 from two outbreaks of noninvasive listeriosis, all three occurring in Italy) were characterized by PCR-ribotyping, arbitrarily primed PCR (AP-PCR), and the recently developed infrequent-restriction-site PCR (IRS-PCR). The discriminatory ability of the techniques, first evaluated on 29 unrelated L. monocytogenes food isolates using Simpson's index of diversity, was 0.714 for PCR-ribotyping, 0.690 for AP-PCR, and 0.919 for IRS-PCR. IRS-PCR was also more capable of distinguishing among strains from the invasive listeriosis outbreak: three different clusters were identified by IRS-PCR compared to two clusters identified by both PCR-ribotyping and AP-PCR. Within each of the two outbreaks of noninvasive listeriosis, the patterns were practically identical, as demonstrated by all three techniques. Only IRS-PCR succeeded in clearly discriminating the strains related to noninvasive listeriosis from all of the other strains included in this study, including those from the outbreak of invasive listeriosis. This finding may suggest the presence of unique differences in their DNA sequences.     

Genome Analysis of Listeria monocytogenes Sequence Type 8 Strains Persisting in Salmon and Poultry Processing Environments and Comparison with Related Strains

Listeria monocytogenes is an important foodborne pathogen responsible for the disease listeriosis, and can be found throughout the environment, in many foods and in food processing facilities. The main cause of listeriosis is consumption of food contaminated from sources in food processing environments. Persistence in food processing facilities has previously been shown for the L. monocytogenes sequence type (ST) 8 subtype. In the current study, five ST8 strains were subjected to whole-genome sequencing and compared with five additionally available ST8 genomes, allowing comparison of strains from salmon, poultry and cheese industry, in addition to a human clinical isolate. Genome-wide analysis of single-nucleotide polymorphisms (SNPs) confirmed that almost identical strains were detected in a Danish salmon processing plant in 1996 and in a Norwegian salmon processing plant in 2001 and 2011. Furthermore, we show that L. monocytogenes ST8 was likely to have been transferred between two poultry processing plants as a result of relocation of processing equipment. The SNP data were used to infer the phylogeny of the ST8 strains, separating them into two main genetic groups. Within each group, the plasmid and prophage content was almost entirely conserved, but between groups, these sequences showed strong divergence. The accessory genome of the ST8 strains harbored genetic elements which could be involved in rendering the ST8 strains resilient to incoming mobile genetic elements. These included two restriction-modification loci, one of which was predicted to show phase variable recognition sequence specificity through site-specific domain shuffling. Analysis indicated that the ST8 strains harbor all important known L. monocytogenes virulence factors, and ST8 strains are commonly identified as the causative agents of invasive listeriosis. Therefore, the persistence of this L. monocytogenes subtype in food processing facilities poses a significant concern for food safety.     

Characterization by Multilocus Enzyme Electrophoresis of Listeria monocytogenes Isolates Involved in Ovine Listeriosis Outbreaks in Scotland from 1989 to 1991

Initial results from a study of five small ovine listeriosis outbreaks in Scotland in 1989 to 1991 are presented. Forty-eight isolates including three from silage were typed at 10 polymorphic enzyme loci by using multilocus enzyme electrophoresis resulting in the identification of 12 electrophoretic types. Phylogenetic analysis partitioned the 12 electrophoretic types into two statistically distinct divisions distinguishing 1/2a serotypes from non-1/2a serotypes.     

The Connection between Persistent,Disinfectant-Resistant Listeria monocytogenes Strains from Two Geographically Separate Iberian Pork Processing Plants: Evidence from Comparative Genome Analysis

The aim of this study was to investigate the basis of the putative persistence of Listeria monocytogenes in a new industrial facility dedicated to the processing of ready-to-eat (RTE) Iberian pork products. Quaternary ammonium compounds, which included benzalkonium chloride (BAC), were repeatedly used as surface disinfectants in the processing plant. Clean and disinfected surfaces were sampled to evaluate if resistance to disinfectants was associated with persistence. Of the 14 isolates obtained from product contact and non-product contact surfaces, only five different pulsed-field gel electrophoresis (PFGE) types were identified during the 27-month study period. Two of these PFGE types (S1 and S10-1) were previously identified to be persistent and BAC-resistant (BAC^r) strains in a geographically separate slaughterhouse belonging to the same company. The remaining three PFGE types, which were first identified in this study, were also BAC^r. Whole-genome sequencing and in silico multilocus sequence typing (MLST) analysis of five BAC^r isolates of the different PFGE types identified in this study showed that the isolate of the S1 PFGE type belonged to MLST sequence type 31 (ST31), a low-virulence type characterized by mutations in the inlA and prfA genes. The isolates of the remaining four PFGE types were found to belong to MLST ST121, a persistent type that has been isolated in several countries. The ST121 strains contained the BAC resistance transposon Tn6188. The disinfection-resistant L. monocytogenes population in this RTE pork product plant comprised two distinct genotypes with different multidrug resistance phenotypes. This work offers insight into the L. monocytogenes subtypes associated with persistence in food processing environments.     

Examining Phylogenetic Relationships Among Gibbon Genera Using Whole Genome Sequence Data Using an Approximate Bayesian Computation Approach

Gibbons are believed to have diverged from the larger great apes ∼16.8 MYA and today reside in the rainforests of Southeast Asia. Based on their diploid chromosome number, the family Hylobatidae is divided into four genera, Nomascus, Symphalangus, Hoolock, and Hylobates. Genetic studies attempting to elucidate the phylogenetic relationships among gibbons using karyotypes, mitochondrial DNA (mtDNA), the Y chromosome, and short autosomal sequences have been inconclusive . To examine the relationships among gibbon genera in more depth, we performed second-generation whole genome sequencing (WGS) to a mean of ∼15× coverage in two individuals from each genus. We developed a coalescent-based approximate Bayesian computation (ABC) method incorporating a model of sequencing error generated by high coverage exome validation to infer the branching order, divergence times, and effective population sizes of gibbon taxa. Although Hoolock and Symphalangus are likely sister taxa, we could not confidently resolve a single bifurcating tree despite the large amount of data analyzed. Instead, our results support the hypothesis that all four gibbon genera diverged at approximately the same time. Assuming an autosomal mutation rate of 1 × 10⁻⁹/site/year this speciation process occurred ∼5 MYA during a period in the Early Pliocene characterized by climatic shifts and fragmentation of the Sunda shelf forests. Whole genome sequencing of additional individuals will be vital for inferring the extent of gene flow among species after the separation of the gibbon genera.     

PathogenFinder - Distinguishing Friend from Foe Using Bacterial Whole Genome Sequence Data

Although the majority of bacteria are harmless or even beneficial to their host, others are highly virulent and can cause serious diseases, and even death. Due to the constantly decreasing cost of high-throughput sequencing there are now many completely sequenced genomes available from both human pathogenic and innocuous strains. The data can be used to identify gene families that correlate with pathogenicity and to develop tools to predict the pathogenicity of newly sequenced strains, investigations that previously were mainly done by means of more expensive and time consuming experimental approaches. We describe PathogenFinder (http://cge.cbs.dtu.dk/services/PathogenFinder/), a web-server for the prediction of bacterial pathogenicity by analysing the input proteome, genome, or raw reads provided by the user. The method relies on groups of proteins, created without regard to their annotated function or known involvement in pathogenicity. The method has been built to work with all taxonomic groups of bacteria and using the entire training-set, achieved an accuracy of 88.6% on an independent test-set, by correctly classifying 398 out of 449 completely sequenced bacteria. The approach here proposed is not biased on sets of genes known to be associated with pathogenicity, thus the approach could aid the discovery of novel pathogenicity factors. Furthermore the pathogenicity prediction web-server could be used to isolate the potential pathogenic features of both known and unknown strains.     

Sequence Analysis of the actA Gene of Listeria monocytogenes Isolated from Human

The region encoding proline-rich units of actA genes was amplified from 24 strains of Listeria monocytogenes using polymerase chain reaction (PCR). PCR products of 13 strains showed the expected size of 623 bp, whereas those of 11 strains showed a short size of 518 bp. The shortening of these PCR products resulted from the deletion of one proline-rich unit. These results indicate that ActA proteins are divided into at least two different types which are unrelated to bacterial serotypes.     

TreeSeq,a Fast and Intuitive Tool for Analysis of Whole Genome and Metagenomic Sequence Data

Next-generation sequencing is not yet commonly used in clinical laboratories because of a lack of simple and intuitive tools. We developed a software tool (TreeSeq) with a quaternary tree search structure for the analysis of sequence data. This permits rapid searches for sequences of interest in large datasets. We used TreeSeq to screen a gut microbiota metagenomic dataset and a whole genome sequencing (WGS) dataset of a strain of Klebsiella pneumoniae for antibiotic resistance genes and compared the results with BLAST and phenotypic resistance determination. TreeSeq was more than thirty times faster than BLAST and accurately detected resistance gene sequences in complex metagenomic data and resistance genes corresponding with the phenotypic resistance pattern of the Klebsiella strain. Resistance genes found by TreeSeq were visualized as a gene coverage heat map, aiding in the interpretation of results. TreeSeq brings analysis of metagenomic and WGS data within reach of clinical diagnostics.     

Prevalence and Fingerprinting of Listeria monocytogenes Strains Isolated from Raw Whole Milk in Farm Bulk Tanks and in Dairy Plant Receiving Tanks

The incidence of Listeria species in raw whole milk from farm bulk tanks and from raw milk in storage at a Swedish dairy plant was studied. Listeria monocytogenes was found in 1.0% and Listeria innocua was found in 2.3% of the 294 farm bulk tank (farm tank) milk specimens. One farm tank specimen contained 60 CFU of L. monocytogenes ml⁻¹. L. monocytogenes was detected in 19.6% and L. innocua was detected in 8.5% of the milk specimens from the silo receiving tanks at the dairy (dairy silos). More dairy silo specimens were positive for both Listeria species during winter than during summer. Restriction enzyme analysis and pulsed-field gel electrophoresis were applied to 65 isolates of L. monocytogenes, resulting in 16 different clonal types. Two clonal types were shared by the farm tank milk and the dairy silo milk. All except one clonal type belonged to serovar 1/2a. In the dairy silo milk five clonal types were found more frequently and for a longer period than the others. No Listeria species were found in any other samples from the plant.     

Whole Genome Sequence of Treponema pallidum ssp. pallidum,Strain Mexico A,Suggests Recombination between Yaws and Syphilis Strains

Background

Treponema pallidum ssp. pallidum (TPA), the causative agent of syphilis, and Treponema pallidum ssp. pertenue (TPE), the causative agent of yaws, are closely related spirochetes causing diseases with distinct clinical manifestations. The TPA Mexico A strain was isolated in 1953 from male, with primary syphilis, living in Mexico. Attempts to cultivate TPA Mexico A strain under in vitro conditions have revealed lower growth potential compared to other tested TPA strains.

Methodology/Principal Findings

The complete genome sequence of the TPA Mexico A strain was determined using the Illumina sequencing technique. The genome sequence assembly was verified using the whole genome fingerprinting technique and the final sequence was annotated. The genome size of the Mexico A strain was determined to be 1,140,038 bp with 1,035 predicted ORFs. The Mexico A genome sequence was compared to the whole genome sequences of three TPA (Nichols, SS14 and Chicago) and three TPE (CDC-2, Samoa D and Gauthier) strains. No large rearrangements in the Mexico A genome were found and the identified nucleotide changes occurred most frequently in genes encoding putative virulence factors. Nevertheless, the genome of the Mexico A strain, revealed two genes (TPAMA_0326 (tp92) and TPAMA_0488 (mcp2-1)) which combine TPA- and TPE- specific nucleotide sequences. Both genes were found to be under positive selection within TPA strains and also between TPA and TPE strains.

Conclusions/Significance

The observed mosaic character of the TPAMA_0326 and TPAMA_0488 loci is likely a result of inter-strain recombination between TPA and TPE strains during simultaneous infection of a single host suggesting horizontal gene transfer between treponemal subspecies.     

Comparative Analysis of Extracellular and Intracellular Proteomes of Listeria monocytogenes Strains Reveals a Correlation between Protein Expression and Serovar

Listeria monocytogenes, the etiologic agent of listeriosis, remains a serious public health concern, with its frequent occurrence in food environments coupled with a high mortality rate. Among the 13 serovars, human listeriosis is mostly associated with the serovar 4b, 1/2b, and 1/2a strains. To investigate the diversity of L. monocytogenes, the intracellular and extracellular proteins of 12 strains were analyzed by two-dimensional gel electrophoresis. These strains had different origins, belonged to different serovars (4b, 1/2a, and 1/2b), and presented with different levels of virulence in chicken embryos. The clustering of the strains in two groups based on proteomic patterns is in agreement with the L. monocytogenes phylogenetic lineages. Statistical analysis did not allow for identification of proteins specific to the isolate origin or the virulence level of the strains, but 26 and 21 protein spots were shown to be significantly overexpressed and underexpressed, respectively, in the six strains of serovar 1/2a (lineage II) compared to strains of serovar 1/2b or 4b. Moreover, a penicillin-binding protein was specific for serovar 1/2b and two protein spots identified as a serine protease were specific to serovar 4b. These protein spots, identified through peptide mass fingerprinting using matrix-assisted laser desorption ionization-time of flight mass spectrometry, were essentially found in the extracellular proteome and may have uses as potential markers for serotyping and risk analysis.     

Multi-locus Sequence Typing and Whole Genome Sequence Analysis of Cryptococcus neoformans Isolated from Clinical Specimens in Vajira Hospital,Bangkok, Thailand

Mycopathologia - The basidiomycete yeast Cryptococcus neoformans causes disease in immunocompromized patients. Whole genome sequencing (WGS) technology provides insights into the molecular...     

Analysis of the Clonality of Candida tropicalis Strains from a General Hospital in Beijing Using Multilocus Sequence Typing

Multilocus sequence typing (MLST) based on six loci was used to analyze the relationship of 58 Candida tropicalis isolates from individual patients in a general hospital in Beijing, China. A total of 52 diploid sequence types (DSTs) were generated by the MLST, all of which were new to the central database. Unweighted Pair Group Method with Arithmetic Mean (UPGMA) dendrograms were constructed, which showed that the 58 isolates were distributed robustly and 6 main groups were clustered regardless of the specimen source and medical department. The minimum spanning tree (MST) of the 58 isolates (52 DSTs) and all 401 isolates (268 DSTs) in the C. tropicalis central database (http://pubmlst.org/ctropicalis/) indicated that the isolates in this study clustered in three relative pure clonal complexes, and 2 clustered with isolates from Taiwan, Belgium, Brazil, and the US. This study presents the first MLST analysis of C. tropicalis isolates from Mainland China, which may be useful for further studies on the similarity, genetic relationship, and molecular epidemiology of C. tropicalis strains worldwide.     

Subtyping of a Large Collection of Historical Listeria monocytogenes Strains from Ontario,Canada, by an Improved Multilocus Variable-Number Tandem-Repeat Analysis (MLVA)

Listeria monocytogenes is responsible for severe and often fatal food-borne infections in humans. A collection of 2,421 L. monocytogenes isolates originating from Ontario''s food chain between 1993 and 2010, along with Ontario clinical isolates collected from 2004 to 2010, was characterized using an improved multilocus variable-number tandem-repeat analysis (MLVA). The MLVA method was established based on eight primer pairs targeting seven variable-number tandem-repeat (VNTR) loci in two 4-plex fluorescent PCRs. Diversity indices and amplification rates of the individual VNTR loci ranged from 0.38 to 0.92 and from 0.64 to 0.99, respectively. MLVA types and pulsed-field gel electrophoresis (PFGE) patterns were compared using Comparative Partitions analysis involving 336 clinical and 99 food and environmental isolates. The analysis yielded Simpson''s diversity index values of 0.998 and 0.992 for MLVA and PFGE, respectively, and adjusted Wallace coefficients of 0.318 when MLVA was used as a primary subtyping method and 0.088 when PFGE was a primary typing method. Statistical data analysis using BioNumerics allowed for identification of at least 8 predominant and persistent L. monocytogenes MLVA types in Ontario''s food chain. The MLVA method correctly clustered epidemiologically related outbreak strains and separated unrelated strains in a subset analysis. An MLVA database was established for the 2,421 L. monocytogenes isolates, which allows for comparison of data among historical and new isolates of different sources. The subtyping method coupled with the MLVA database will help in effective monitoring/prevention approaches to identify environmental contamination by pathogenic strains of L. monocytogenes and investigation of outbreaks.     

Molecular Analysis of Vibrio cholerae O1, O139, non-O1, and non-O139 Strains: Clonal Relationships between Clinical and Environmental Isolates

A total of 26 strains of Vibrio cholerae, including members of the O1, O139, and non-O1, non-O139 serogroups from both clinical and environmental sources, were examined for the presence of genes encoding cholera toxin (ctxA), zonula occludens toxin (zot), accessory cholera enterotoxin (ace), hemolysin (hlyA), NAG-specific heat-stable toxin (st), toxin-coregulated pilus (tcpA), and outer membrane protein (ompU), for genomic organization, and for the presence of the regulatory protein genes tcpI and toxR in order to determine relationships between epidemic serotypes and sources of isolation. While 22 of the 26 strains were hemolytic on 5% sheep blood nutrient agar, all strains were PCR positive for hlyA, the hemolysin gene. When multiplex PCR was used, all serogroup O1 and O139 strains were positive for tcpA, ompU, and tcpI. All O1 and O139 strains except one O1 strain and one O139 strain were positive for the ctxA, zot, and ace genes. Also, O1 strain VO3 was negative for the zot gene. All of the non-O1, non-O139 strains were negative for the ctxA, zot, ace, tcpA, and tcpI genes, and all of the non-O1, non-O139 strains except strain VO26 were negative for ompU. All of the strains except non-O1, non-O139 strain VO22 were PCR positive for the gene encoding the central regulatory protein, toxR. All V. cholerae strains were negative for the NAG-specific st gene. Of the nine non-ctx-producing strains of V. cholerae, only one, non-O1, non-O139 strain VO24, caused fluid accumulation in the rabbit ileal loop assay. The other eight strains, including an O1 strain, an O139 strain, and six non-O1, non-O139 strains, regardless of the source of isolation, caused fluid accumulation after two to five serial passages through the rabbit gut. Culture filtrates of all non-cholera-toxigenic strains grown in AKI media also caused fluid accumulation, suggesting that a new toxin was produced in AKI medium by these strains. Studies of clonality performed by using enterobacterial repetitive intergenic consensus sequence PCR, Box element PCR, amplified fragment length polymorphism (AFLP), and pulsed-field gel electrophoresis (PFGE) collectively indicated that the V. cholerae O1 and O139 strains had a clonal origin, whereas the non-O1, non-O139 strains belonged to different clones. The clinical isolates closely resembled environmental isolates in their genomic patterns. Overall, there was an excellent correlation among the results of the PCR, AFLP, and PFGE analyses, and individual strains derived from clinical and environmental sources produced similar fingerprint patterns. From the results of this study, we concluded that the non-cholera-toxin-producing strains of V. cholerae, whether of clinical or environmental origin, possess the ability to produce a new secretogenic toxin that is entirely different from the toxin produced by toxigenic V. cholerae O1 and O139 strains. We also concluded that the aquatic environment is a reservoir for V. cholerae O1, O139, non-O1, and non-O139 serogroup strains.     

Genomic Relationships between Enterococcus faecium Strains from Different Sources and with Different Antibiotic Resistance Profiles Evaluated by Restriction Endonuclease Analysis of Total Chromosomal DNA Using EcoRI and PvuII

Forty-seven Enterococcus faecium strains from different sources were evaluated by restriction endonuclease analysis (REA) of total chromosomal DNA. Strains from chicken, pork, and humans were clearly divided into separate clusters, whereas strains from different countries, strains with different antibiotic resistance profiles, or clinical and healthy-subject strains were not.