Plasmid-borne Virulence Genes Research Articles

Genomic Characteristics of a Carbapenem-Resistant Klebsiella pneumoniae Co-Carrying bla NDM-5 and bla KPC-2 Capsular Type KL25 Recovered from a County Level Hospital in China.

Hypervirulent carbapenem-resistant K. pneumoniae (hv-CRKP) has been spreading rapidly worldwide. Here, we investigated the genomic characteristics of ST11 K. pneumoniae isolate SM117 with capsular serotype KL25, co-carrying bla NDM-5, two copies of bla KPC-2 and multiple plasmid-borne virulence genes from a county level hospital in China. Antimicrobial susceptibility of K. pneumoniae SM117 was evaluated. The Illumina NovaSeq 6000 and Oxford Nanopore MinION platforms were applied to sequence the genome and then de novo assembled. The genome sequence was annotated using the NCBI Prokaryotic Genome Annotation Pipeline and further subjected to identify the sequence type (ST), capsular type, antibiotic resistance genes, plasmid replicon types and virulence genes. The phylogenetic analysis was performed based on the core genome single nucleotide polymorphisms (cgSNPs) using CSI Phylogeny 1.4, and further visualized by Interactive Tree of Life (iTOL) V5 web server. The whole-genome sequence of K. pneumoniae SM117 is made up of eight contigs totaling 6,104,486 bp, contain a 5,612,620 bp single chromosome and seven plasmids. The isolate was assigned to ST11 with capsular serotype KL25, co-carrying including bla NDM-5, bla KPC-2 and multiple plasmid-borne virulence genes including rmpA2 and aerobactin genes iucABCD-iutA. The coexistence of bla KPC and bla NDM in K. pneumoniae strains exhibit a high degree of resistance to β-lactam antibiotics. The strain SM117 also carries multiple antibiotic resistance genes, making it resistant to all antibiotics except polymyxin. The closest relative of K. pneumoniae C793 was identified in 2023 from a hospital surface sample in Zhejiang, China, with just 52 SNPs difference. This study reported the genomic characteristics of a multidrug-resistant ST11 K. pneumoniae with capsular serotype KL25, co-carrying bla NDM-5, two copies of bla KPC-2 genes and multiple plasmid-borne virulence genes in China. These findings will provide important knowledge of the antibiotic resistance mechanisms, genomic epidemiological characteristics and transmission dynamics of multidrug-resistant K. pneumoniae.

Genotypic virulence profiles and associations in Salmonella isolated from meat samples in wet markets and abattoirs of Metro Manila, Philippines

BackgroundSalmonella are pathogenic foodborne bacteria with complex pathogenicity from numerous virulence genes housed in Salmonella pathogenicity islands (SPIs), plasmids, and other gene cassettes. However, Salmonella virulence gene distributions and mechanisms remain unestablished. In the Philippines, studies mainly report Salmonella incidences and antimicrobial resistance, but little to none on virulence profiles, their associations to animal sources, collection sites and Salmonella serogroups. Hence, a total of 799 Salmonella isolates, previously obtained from pig, cow, and chicken meat samples in wet markets and abattoirs (wet markets: 124 chicken, 151 cow, and 352 pig meat isolates; abattoirs: 172 pig tonsil and jejunum isolates) in Metro Manila, Philippines, were revived and confirmed as Salmonella through invA gene polymerase chain reaction (PCR). Isolates were then screened for eight virulence genes, namely avrA, hilA, sseC, mgtC, spi4R, pipB, spvC and spvR, by optimized multiplex PCR and significant pair associations between virulence genes were determined through Fisher’s exact test. Gene frequency patterns were also determined. Salmonella serogroups in addition to animal sources and location types were also used to predict virulence genes prevalence using binary logistic regression.ResultsHigh frequencies (64 to 98%) of SPI virulence genes were detected among 799 Salmonella isolates namely mgtC, pipB, avrA, hilA, spi4R and sseC, from most to least. However, only one isolate was positive for plasmid-borne virulence genes, spvC and spvR. Diversity in virulence genes across Salmonella serogroups for 587 Salmonella isolates (O:3 = 250, O:4 = 133, O:6,7 = 99, O:8 = 93, O:9 = 12) was also demonstrated through statistical predictions, particularly for avrA, hilA, sseC, and mgtC. mgtC, the most frequent virulence gene, was predicted by serogroup O:9, while sseC, the least frequent, was predicted by serogroup O:4 and chicken animal source. The highest virulence gene pattern involved SPIs 1-5 genes which suggests the wide distribution and high pathogenic potential of Salmonella. Statistical analyses showed five virulence gene pair associations, namely avrA and hilA, avrA and spi4R, hilA and spi4R, sseC and spi4R, and mgtC and pipB. The animal sources predicted the presence of virulence genes, sseC and pipB, whereas location type for hilA and spi4R, suggesting that these factors may contribute to the type and pathogenicity of Salmonella present.ConclusionThe high prevalence of virulence genes among Salmonella in the study suggests the high pathogenic potential of Salmonella from abattoirs and wet markets of Metro Manila, Philippines which poses food safety and public health concerns and threatens the Philippine food animal industry. Statistical associations between virulence genes and prediction analyses across Salmonella serogroups and external factors such as animal source and location type and presence of virulence genes suggest the diversity of Salmonella virulence and illustrate determining factors to Salmonella pathogenicity. This study recommends relevant agencies in the Philippines to improve standards in food animal industries and increase efforts in monitoring of foodborne pathogens.

Prevalence, virulence determinants, and genetic diversity in Yersinia enterocolitica isolated from slaughtered pigs and pig carcasses

Yersinia enterocolitica is an important zoonotic foodborne pathogen that could be transferred from infected pigs to their carcasses at slaughter, with subsequent introduction of the pathogen into the food chain. The aim of the present study was to study the prevalence, virulence characteristics, and genetic diversity of Y. enterocolitica isolates present in slaughtered pig tonsils and carcasses by using the WGS approach. A total of 200 slaughtered pig tonsils from 11 pig farms were collected in 2020–2021 at six slaughterhouses located in Latvia. Out of these samples, n = 190 were obtained from slaughtered pigs raised on Latvian farms while n = 10 were of Lithuanian origin, with the number of farms sampled being 10 and 1, respectively. Additionally, 30 pig carcasses were sampled at five slaughterhouses from pigs originating from five farms in 2021. Samples were investigated microbiologically, Y. enterocolitica isolates were biotyped and serotyped. Y. enterocolitica 4/O:3 was screened for antimicrobial resistance with the EUVSEC test panels. Whole genome sequence analysis (WGS) was performed in order to detect virulence genes and to assess the genetic diversity of Y. enterocolitica isolates. A total of 139 isolates, including one to three isolates from 84 Y. enterocolitica positive slaughtered pig tonsils and 13 pig carcass samples, were subjected to WGS analysis. The prevalence of Y. enterocolitica 4/O:3 in slaughtered pig tonsils and carcasses was 35% (70/200) and 13% (4/30), respectively. Antimicrobial resistance to ampicillin and tetracycline was detected in 97% (72/74) and 1% (1/74) of Y. enterocolitica 4/O:3 isolates. Y. enterocolitica 4/O:3 was represented only by ST18, while Y. enterocolitica 1A by ST3, ST147, ST304, ST307, and ST473. The ST18 isolates harbored the same main chromosomal (ail, inv, myfA, ystA) and majority shared plasmid-borne virulence genes (virF, yadA, yop virulon). The main virulence genes were not identified within the STs of Y. enterocolitica 1A and only minor differences were found between ST3, ST147, ST304, ST307, and ST473. Among ST18 isolates, cgMLST analysis revealed 43 cgMLST genotypes while 16 cgMLST genotypes were found among Y. enterocolitica 1A STs. The present study has shown the distribution of genetically distant cgMLST genotypes in slaughtered pigs from pig farms located in different geographical regions of Latvia, with one to 11 cgMLSTs identified within each sampled farm. The presence of undistinguishable cgMLST genotypes in slaughtered pig tonsils and the respective carcasses supported the link between the slaughter of Y. enterocolitica - positive pigs and carcass contamination with Y. enterocolitica 4/O:3.

Population Analysis of O26 Shiga Toxin-Producing Escherichia coli Causing Hemolytic Uremic Syndrome in Italy, 1989-2020, Through Whole Genome Sequencing.

Shiga toxin-producing Escherichia coli (STEC) belonging to the O26 serogroup represent an important cause of Hemolitic Uremic Syndrome (HUS) in children worldwide. The localization of STEC virulence genes on mobile genetic elements allowed the emergence of clones showing different assets of this accessory genomic fraction. A novel O26 STEC clone belonging to Sequence Type (ST) 29 and harboring stx2a, ehxA and etpD plasmid-borne genes has emerged and spread in Europe since the mid-1990s, while another ST29 clone positive for stx2d and lacking plasmid-borne virulence genes was recently described as emerging in France. In Italy, O26 has been the most frequently detected STEC serogroup from HUS cases since the late 1990s. In this study we describe the genomic characterization and population structure of 144 O26 STEC strains isolated from human sources in Italy in the period 1989-2020. A total of 89 strains belonged to ST21, 52 to ST29, two to ST396 and one to ST4944. ST29 strains started to be isolated from 1999. 24 strains were shown to harbour stx1a, alone (n=20) or in combination with stx2a (n=4). The majority of the strains (n=118) harbored stx2a genes only and the two ST396 strains harbored stx2d. A Hierarchical Clustering on Principal Components (HCPC) analysis, based on the detection of accessory virulence genes, antimicrobial resistance (AMR) genes and plasmid replicons, classified the strains in seven clusters identified with numbers from 1 to 7, containing two, 13, 39, 63, 16, 10 and one strain, respectively. The majority of the genetic features defining the clusters corresponded to plasmid-borne virulence genes, AMR genes and plasmid replicons, highlighting specific assets of plasmid-borne features associated with different clusters. Core genome Multi Locus Sequence Typing grouped ST21 and ST29 strains in three clades each, with each ST29 clade exactly corresponding to one HCPC cluster. Our results showed high conservation of either the core or the accessory genomic fraction in populations of ST29 O26 STEC, differently from what observed in ST21 strains, suggesting that a different selective pressure could drive the evolution of different populations of these pathogens possibly involving different ecological niches.

Characterization of Human Origin Salmonella Serovar 1,4,[5],12:i:- in Eastern China, 2014 to 2018.

The prevalence of Salmonella serovar 1,4,[5],12:i:- among diarrhea patients has increased considerably in many countries around the world, including China. However, the characterization of this serovar of human origin has been less reported from China. We characterized 76 isolates of Salmonella 1,4,[5],12:i:- gained from diarrhea patients from 2014 to 2018 in the Jiangsu Province of eastern China. These isolates fell into a single-sequence type (ST34) determined by multilocus sequence typing (MLST), and into 44 pulsed-field gel electrophoresis band patterns, with 1 pattern (JSSMM015) comprising 12 isolates (15.9%). By means of PCR-based assays, the seven prophage located virulence genes were detected in our Salmonella 1,4,[5],12:i:- isolates with a high rate of gipA, gtgB, sspH1, sspH2, sodC1, and gtgE (93.4-97.4%), and with a moderate rate of sopE (42.1%). In contrast, none of the five plasmid-borne virulence genes (spvC, pefA, mig5, rck, and srgA) was identified. We tested the isolates' susceptibility to 18 antibiotics of 9 categories using the VITEK 2 system. A high proportion (89.5%) of the isolates were multidrug resistant (MDR) strains with full resistance to cefazolin, cefotetan, amikacin, gentamycin, and tobramycin, followed by resistance to ampicillin (88.2%) and ampicillin/sulbactam (80.3%). The resistance to piperacillin/tazobactam, ceftazidime, cefepime, and levofloxacin was scarce (2.6-9.2%). Notably, an isolate from 2018 was resistant to carbapenems. blaTEM-1B and aac(6')-Ib-cr were the most common drug resistance genes presented in cephalosporin- and fluoroquinolone-resistant strains. All Salmonella 1,4,[5],12:i:- isolates were capable of forming biofilm, with 13.2% of them having strong ability. However, no association was indicated between the scale of biofilm formation ability and MDR. Our results indicate that the combination of these characteristics may together provide a selective and competitive advantage to those Salmonella 1,4,[5],12:i:- isolates, contributing to their increasing prevalence observed worldwide.

The Antiactivator of Type III Secretion, OspD1, Is Transcriptionally Regulated by VirB and H-NS from Remote Sequences in Shigella flexneri.

Shigella species, the causal agents of bacillary dysentery, use a type III secretion system (T3SS) to inject two waves of virulence proteins, known as effectors, into the colonic epithelium to subvert host cell machinery. Prior to host cell contact and secretion of the first wave of T3SS effectors, OspD1, an effector and antiactivator protein, prevents premature production of the second wave of effectors. Despite this important role, regulation of the ospD1 gene is not well understood. While ospD1 belongs to the large regulon of VirB, a transcriptional antisilencing protein that counters silencing mediated by the histone-like nucleoid structuring protein H-NS, it remains unclear if VirB directly or indirectly regulates ospD1 Additionally, it is not known if ospD1 is regulated by H-NS. Here, we identify the primary ospD1 transcription start site (+1) and show that the ospD1 promoter is remotely regulated by both VirB and H-NS. Our findings demonstrate that VirB regulation of ospD1 requires at least one of the two newly identified VirB regulatory sites, centered at -978 and -1270 relative to the ospD1 +1. Intriguingly, one of these sites lies on a 193-bp sequence found in three conserved locations on the large virulence plasmids of Shigella The region required for H-NS-dependent silencing of ospD1 lies between -1120 and -820 relative to the ospD1 +1. Thus, our study provides further evidence that cis-acting regulatory sequences for transcriptional antisilencers and silencers, such as VirB and H-NS, can lie far upstream of the canonical bacterial promoter region (i.e., -250 to +1).IMPORTANCE Transcriptional silencing and antisilencing mechanisms regulate virulence gene expression in many important bacterial pathogens. In Shigella species, plasmid-borne virulence genes, such as those encoding the type III secretion system (T3SS), are silenced by the histone-like nucleoid structuring protein H-NS and antisilenced by VirB. Previous work at the plasmid-borne icsP locus revealed that VirB binds to a remotely located cis-acting regulatory site to relieve transcriptional silencing mediated by H-NS. Here, we characterize a second example of remote VirB antisilencing at ospD1, which encodes a T3SS antiactivator and effector. Our study highlights that remote transcriptional silencing and antisilencing occur more frequently in Shigella than previously thought, and it raises the possibility that long-range transcriptional regulation in bacteria is commonplace.

DNA Microarray-Based Genomic Characterization of the Pathotypes of Escherichia coli O26, O45, O103, O111, and O145 Isolated from Feces of Feedlot Cattle

Shiga toxin-producing Escherichia coli (STEC) serogroups O26, O45, O103, O111, O121, and O145, referred to as the top six non-O157 serogroups, are responsible for more than 70% of human non-O157 STEC infections in North America. Cattle harbor non-O157 strains in the hindgut and shed them in the feces. The objective of this study was to use the U.S. Food and Drug Administration (FDA) E. coli identification (ECID) DNA microarray to identify the serotype, assess the virulence potential of each, and determine the phylogenetic relationships among five of the six non-O157 E. coli serogroups isolated from feedlot cattle feces. Forty-four strains of STEC, enterohemorrhagic E. coli (EHEC), enteropathogenic E. coli (EPEC), or putative nonpathotype E. coli (NPEC) of cattle origin and five human clinical strains of EHEC were assayed with the FDA-ECID DNA microarray. The cattle strains harbored diverse flagellar genes. The bovine and human strains belonging to serogroups O26, O45, and O103 carried stx1 only, O111 carried both stx1 and stx2, and O145 carried either stx1 or stx2. The strains were also positive for various subtypes of intimin and other adhesins (IrgA homologue adhesin, long polar fimbriae, mannose-specific adhesin, and curli). Both human and cattle strains were positive for LEE-encoded type III secretory system genes and non-LEE-encoded effector genes. SplitsTree4, a program used to determine the phylogenetic relationship among the strains, revealed that the strains within each serogroup clustered according to their pathotype. In addition to genes encoding Shiga toxins, bovine non-O157 E. coli strains possessed other major virulence genes, including those for adhesins, type III secretory system proteins, and plasmid-borne virulence genes, similar to human clinical strains. Because virulence factors encoded by these genes are involved in the pathogenesis of various pathotypes of E. coli, the bovine non-O157 strains could cause human illness. The FDA-ECID DNA microarray assay rapidly provided a profile of the virulence genes for assessment of the virulence potential of each strain.

Characterization of Salmonella Typhimurium and its monophasic variant 1,4, [5],12:i:- isolated from different sources.

In order to characterize the most commonly detected Salmonella serotypes, we tested 124 isolates of S. Typhimurium and 89 isolates of the monophasic variant of S. Typhimurium (S. 1,4, [5],12:i:-) for their antimicrobial susceptibility by means of the Kirby-Bauer disk-diffusion method, and for the detection of 19 genes (four Phage Markers (g13, Sieb, eat, g8), ten prophage-related virulence genes (gipA, gtgB, nanH, gogB, grvA, sopE, sspH1, sspH2, sodC1, gtgE), and five plasmid-borne virulence genes (spvC, pefA, mig5, rcK, srgA)) by means of PCR-based assays. A total of 213 strains were analyzed from, humans (n = 122), animals (n = 25), food (n = 46), and irrigation water (n = 20). S. Typhimurium isolates showed higher variability, in both their resistance profiles and molecular typing, than S. 1,4, [5],12:i:-. Strains from irrigation water displayed significantly higher susceptibility to antibiotics than those from the other sources. Resistance to ampicillin, streptomycin, sulfonamide, and tetracycline was the most commonly detected resistance profile (R-type), being in serovar S. 1,4, [5],12:i:-, frequently associated to resistance to other antimicrobials. Significant differences in genetic profiles in the two abovementioned Salmonella serotypes were found. None of the plasmid-borne virulence genes investigated were detected in S. 1,4, [5],12:i:- isolates, while those genes, characterized 37.9% of the S. Typhimurium strains. Differences in the prevalence of some molecular targets between the two Salmonella serotypes deserve further study. Importantly, the grvA gene was found exclusively in S. Typhimurium strains, whereas sopE, sodC, gtgB, and gipA were mainly detected, with a statistically significant difference, in S. 1,4, [5],12:i:- isolates.

Low prevalence of human enteropathogenic Yersinia spp. in brown rats (Rattus norvegicus) in Flanders.

Brown rats (Rattus norvegicus) have been identified as potential carriers of Yersinia enterocolitica and Y. pseudotuberculosis, the etiological agents of yersiniosis, the third most reported bacterial zoonosis in Europe. Enteropathogenic Yersinia spp. are most often isolated from rats during yersiniosis cases in animals and humans, and from rats inhabiting farms and slaughterhouses. Information is however lacking regarding the extent to which rats act as carriers of these Yersinia spp.. In 2013, 1088 brown rats across Flanders, Belgium, were tested for the presence of Yersinia species by isolation method. Identification was performed using MALDI-TOF MS, PCR on chromosomal- and plasmid-borne virulence genes, biotyping and serotyping. Yersinia spp. were isolated from 38.4% of the rats. Of these, 53.4% were designated Y. enterocolitica, 0.7% Y. pseudotuberculosis and 49.0% other Yersinia species. Two Y. enterocolitica possessing the virF-, ail- and ystA-gene were isolated. Additionally, the ystB-gene was identified in 94.1% of the other Y. enterocolitica isolates, suggestive for biotype 1A. Three of these latter isolates simultaneously possessed the ail-virulence gene. Significantly more Y. enterocolitica were isolated during winter and spring compared to summer. Based on our findings we can conclude that brown rats are frequent carriers for various Yersinia spp., including Y. pseudotuberculosis and (human pathogenic) Y. enterocolitica which are more often isolated during winter and spring.

Sheep carrying pathogenic Yersinia enterocolitica bioserotypes 2/O:9 and 5/O:3 in the feces at slaughter

Yersinia enterocolitica is a heterogeneous species including non-pathogenic strains belonging to biotype 1A and pathogenic strains belonging to biotypes 1B and 2–5. Pathogenic strains of biotypes 2–4 carrying the ail virulence gene have frequently been isolated from domestic pigs at slaughter. In sheep, mostly non-pathogenic biotype 1A strains have been reported. In our study, the prevalence of ail-positive Y. enterocolitica was studied by PCR and culturing in 406 young sheep (<1year of age) and 139 older sheep at slaughter in Finland. When using PCR, the detection rate was 11% (45/406) in young sheep originating from 11 (18%) farms. Surprisingly, Y. enterocolitica belonging to bioserotypes 2/O:9 and 5/O:3, carrying both chromosomal and plasmid-borne virulence genes, were isolated from the fecal samples of 10 (2%) and 23 (4%) sheep, respectively. All isolates of bioserotypes 2/O:9 (19 isolates) and 5/O:3 (53 isolates) carried the chromosomal virulence genes ail, inv, ystA, and myfA, and almost all isolates (71/72) also carried the virulence genes virF and yadA located on the virulence plasmid. The isolates showed high susceptibility to tested antimicrobials and low genetic diversity by PFGE. Y. enterocolitica bioserotype 5/O:3 is a very rare bioserotype, and has earlier only sporadically been reported in European wildlife and in sheep in Australia and New Zealand. Bioserotype 2/O:9 is a common bioserotype found in humans with yersiniosis, and has sporadically been isolated in wild and domestic animals.

Evolutionary study of Yersinia genomes deciphers emergence of human pathogenic species.

On record, there are 17 species in the Yersinia genus, of which three are known to be pathogenic to human. While the chromosomal and pYV (or pCD1) plasmid-borne virulence genes as well as pathogenesis of these three species are well studied, their genomic evolution is poorly understood. Our study aims to predict the key evolutionary events that led to the emergence of pathogenic Yersinia species by analyzing gene gain-and-loss, virulence genes, and “Clustered regularly-interspaced short palindromic repeats”. Our results suggest that the most recent ancestor shared by the human pathogenic Yersinia was most probably an environmental species that had adapted to the human body. This might have led to ecological specialization that diverged Yersinia into ecotypes and distinct lineages based on differential gene gain-and-loss in different niches. Our data also suggest that Y. pseudotuberculosis group might be the donor of the ail virulence gene to Y. enterocolitica. Hence, we postulate that evolution of human pathogenic Yersinia might not be totally in parallel, but instead, there were lateral gene transfer events. Furthermore, the presence of virulence genes seems to be important for the positive selection of virulence plasmid. Our studies provide better insights into the evolutionary biology of these bacteria.

Editorial: Ecology, Virulence, and Detection of Pathogenic and Pandemic Vibrio parahaemolyticus.

Vibrio parahaemolyticus is a very versatile halophilic organism that can adapt to a wide variety of environments and can cause infections in both humans and aquatic animals. This versatility in terms of host and habitat is attributable to the ability to acquire genes that improve fitness of the organism in different situations. Several genomic islands have been described in this organism. Human pathogenic strains are characterized by the presence of pathogenicity islands that encode certain specific Type Three Secretion Systems (TTSS) and hemolysins that are not present in most environmental strains (Chen et al., 2011). Recently characterized shrimp pathogenic V. parahaemolyticus strains have plasmid borne virulence genes (Sirikharin et al., 2015). V. parahaemolyticus is associated with zooplankton like copepods in off-shore waters and same genotype has been found over large areas (Martinez-Urtaza et al., 2012). The global spread of pandemic V. parahaemolyticus has been attributed to the El Nino phenomenon characterized by the arrival of equatorial warm waters to South American coast in a sequence of invasive waves lasting about 6 months in 1997 (Martinez-Urtaza et al., 2008). Studies conducted using molecular techniques such as realtime PCR and multilocus sequence typing have helped detecting pathogenic V. parahaemolyticus in environmental samples and in understanding their global spread. Thus, this organism has attracted attention of both seafood safety managers as well as aquatic animal health professionals. Being an autochthonous aquatic organism, V. parahaemolyticus has global distribution, occurring wherever environmental conditions are favorable. V. parahaemolyticus is a model organism for the “one health” concept, which recognizes that human health is connected to the health of animals and the environment. In order to better manage both public health and aquatic animal health, we need a better understanding of the factors effecting the ecology of this organism, the virulence factors present in human and animal pathogenic strains. The papers in this research topic cover the three major aspects of pathogenic and pandemic V. parahaemolyticus: ecology, virulence, and detection. Lopez-Joven et al. discuss the prevalence of pathogenic and non-pathogenic strains in association with molluscs while Zavala-Norzagaray et al. describe Vibrio spp. associated with sea turtles in Mexico. Host colonization depends on the ability of the organism to acquire difficult to get nutrients such as iron. Leon-Sicairos et al. describe strategies of V. parahaemolyticus to obtain iron. Improvements in the detection methods of pathogenic strains has been presented by Escalante-Maldonado et al. Genetic characterization of clinical and environmental strains has enabled Xu et al. to understand the emergence of indigenous and non-indegenous pathogen lineages. Genomic and molecular typing studies provide insights into the environmental reservoirs and genetic diversity of pathogenic and pandemic strains as described by Hazen et al., de Jesus Hernandez-Diaz et al., Ludeke et al., and Haendiges et al. Function of genes involved in Type IV secretion system of V. parahemolyticus has been investigated by Yu et al. and conditions leading to loss of plasmid in this organism has been described by Letchumanan et al. Raghunath presented insights into the role of virulence genes involved in human infections. Thus, the articles presented in this research topic contribute to a better understanding of the ecology, virulence, and detection of this important aquatic organism that impacts for both public health and aquaculture.

Yersinia enterocoliticaof Porcine Origin: Carriage of Virulence Genes and Genotypic Diversity

Yersinia enterocolitica is an important foodborne pathogen, and pigs are recognized as a major reservoir and potential source of pathogenic strains to humans. A total of 172 Y. enterocolitica recovered from conventional and antimicrobial-free pig production systems from different geographic regions (North Carolina, Ohio, Michigan, Wisconsin, and Iowa) were investigated to determine their pathogenic significance to humans. Phenotypic and genotypic diversity of the isolates was assessed using antibiogram, serogrouping, and amplified fragment length polymorphism (AFLP). Carriage of chromosomal and plasmid-borne virulence genes were investigated using polymerase chain reaction. A total of 12 antimicrobial resistance patterns were identified. More than two-thirds (67.4%) of Y. enterocolitica were pan-susceptible, and 27.9% were resistant against β-lactams. The most predominant serogroup was O:3 (43%), followed by O:5 (25.6%) and O:9 (4.1%). Twenty-two of 172 (12.8%) isolates were found to carry Yersinia adhesion A (yadA), a virulence gene encoded on the Yersinia virulence plasmid. Sixty-nine (40.1%) isolates were found to carry ail gene. The ystA and ystB genes were detected in 77% and 26.2% of the strains, respectively. AFLP genotyping of isolates showed wide genotypic diversity and were grouped into nine clades with an overall genotypic similarity of 66.8-99.3%. AFLP analysis revealed that isolates from the same production system showed clonal relatedness, while more than one genotype of Y. enterocolitica circulates within a farm.

Characterization of phenotypic variants of Clavibacter michiganensis subsp. michiganensis isolated from Capsicum annuum

Phenotypic variants of Clavibacter michiganensis subsp. michiganensis (Cmm) were isolated from pepper fields and from pepper seeds during quarantine inspections. All strains isolated from pepper (pepper isolates) produced orange-coloured colonies with lower mucoidy than typical Cmm strains isolated from tomato (tomato isolates). However, the results of ELISA, fatty acid analysis, 16S rDNA sequencing, and PCR analysis showed that all pepper isolates were similar enough to be identified as Cmm. In addition to phenotypic variations, the pepper isolates showed different pathogenic and genetic characteristics from tomato isolates from the USA, Europe, or other countries. They could be clearly distinguished in terms of pathogenicity, as they showed increased pathogenicity to pepper but reduced pathogenicity to tomato. Tomato isolates caused strong wilting and canker in tomato, but caused only canker and no wilting in pepper and bell pepper. However, pepper isolates caused no wilting, even in tomato, and only caused canker in the three host plants. In addition, compared to tomato isolates, pepper isolates showed increased colonization efficiency and caused a greater reduction in shoot dry weight in pepper. Pepper and tomato isolates could be separated into two groups according to host origin on the basis of 16S rDNA and ITS sequence analysis. They also showed different rep-PCR genomic fingerprints. All pepper isolates showed higher cellulase activity than tomato isolates on M9CMC plates. However, two plasmid-borne virulence genes of Cmm, pat-1, and celA, were not detected in any pepper isolates by PCR. Furthermore, PCR for pathogenicity-related genes located on a pathogenicity island (PAI) revealed that all tomato isolates were positive for these genes, whereas the pepper isolates did not show any PCR products for the chpC, chpG, ppaA, or tomA genes. Therefore, we suggest that the pepper isolates may represent a separate Cmm population that has evolved within the limits of this host.

Comparative Virulence Genotyping and Antimicrobial Susceptibility Profiling of Environmental and Clinical Salmonella enterica from Cochin, India

Salmonella enterica serotype Newport is an important cause of non-typhoidal salmonellosis, a clinically less severe infection than typhoid fever caused by S. enterica serotype Typhi. In this investigation, the virulence genotypes of S. enterica Newport isolated from a backwater environment were compared with Salmonella Typhi from clinical cases in the same region where salmonellosis is endemic. Genotyping was done by PCR screening for virulence markers associated with Salmonella pathogenicity islands (SPIs) and plasmids. The virulence genes associated with SPIs I-VI were detected in 95-100% of all the isolates, while the viaB locus representing SPI-7 was detectable in 66 and 73% of the environmental and clinical isolates, respectively. A significant number of Salmonella Newport lacked virulence genes shdA and sopE compared to S. Typhi. All S. Typhi and S. Newport isolates lacked large plasmid-borne virulence genes spvR and pefA. Further investigations into the antimicrobial resistance of S. Newport revealed multiple drug resistance to ampicillin, amoxicillin/clavulanic acid, trimethoprim-sulfamethoxazole, chloramphenicol, tetracycline, cephalothin, and cephalexin. In comparison, S. Typhi were susceptible to all clinically relevant antimicrobials. The results of this study will help in understanding the spread of virulence genotypes and antibiotic resistance in S. Newport in the region of study.

50 Years Ago in The Journal of Pediatrics: Enteritis Due to Enteropathogenic Escherichia Coli: Present-day Status and Unsolved Problems

Neter E. J Pediatr 1959;55:223-39 In the 1950s, if a child with diarrhea was not infected with Salmonella, Shigella, or Vibrio cholerae, the cause would remain unknown. In this context, Neter reviewed mid-century knowledge of enteropathogenic Escherichia coli (EPEC). Elegant studies by Varela, et al1Varela G.A. Aquirre A. Carrillo J. E coli-gomez, nueva especie aislada de un caso mortal de diarrea.Boletin Medico Hospital Infantil (Mexico). 1946; 3: 623-627Google Scholar and Bray and Beavan in the 1940s1Varela G.A. Aquirre A. Carrillo J. E coli-gomez, nueva especie aislada de un caso mortal de diarrea.Boletin Medico Hospital Infantil (Mexico). 1946; 3: 623-627Google Scholar (neither of which were cited in Neter's review), introduced a definable diarrheagenic subset of E. coli. termed EPEC,3Neter E. Westphal O. Luderitz O. Gino R.M. Gorzynski E.A. Demonstration of antibodies against enteropathogenic Escherichia coli in sera of children of various ages.Pediatrics. 1955; 16: 801-808PubMed Google Scholar which belonged to a limited number of serotypes. EPEC caused severe epidemics into the 1980's in this country. Typical EPEC are defined by their clustered adherence to epithelial cells, in and their ability to induce cytoskeletal rearrangements in host cells. EPEC are fascinating phylogenetically: many distantly related EPEC contain similar suites of chromosomal and plasmid-borne virulence genes. Atypical EPEC (aEPEC) are also pathogens; some aEPEC are enterohemorrhagic E coli (EHEC) that have lost Shiga toxin genes.4Bielaszewska M. Middendorf B. Köck R. Friedrich A.W. Fruth A. Karch H. et al.Shiga toxin-negative attaching and effacing Escherichia coli: distinct clinical associations with bacterial phylogeny and virulence traits and inferred in-host pathogen evolution.Clin Infect Dis. 2008; 47: 208-217Crossref PubMed Scopus (88) Google Scholar It is ironic that as our understanding of EPEC biology has blossomed, the epidemiological and clinical importance of EPEC has been overshadowed by other diarrheagenic E coli, including EHEC and aEPEC.2Bray's discovery of pathogenic Esch. coli as a cause of infantile gastroenteritis.Arch Dis Child. 1973; 48: 923-926Crossref PubMed Scopus (16) Google Scholar, 4Bielaszewska M. Middendorf B. Köck R. Friedrich A.W. Fruth A. Karch H. et al.Shiga toxin-negative attaching and effacing Escherichia coli: distinct clinical associations with bacterial phylogeny and virulence traits and inferred in-host pathogen evolution.Clin Infect Dis. 2008; 47: 208-217Crossref PubMed Scopus (88) Google Scholar, 5Ochoa T.J. Barletta F. Contreras C. Mercado E. New insights into the epidemiology of enteropathogenic Escherichia coli infection.Trans R Soc Trop Med Hyg. 2008; 102: 852-856Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar However, except for EHEC O157:H7 (which has aEPEC characteristics), clinicians cannot easily identify diarrheagenic E coli. Many diarrheal episodes remain undiagnosed even when stools are subjected to comprehensive evaluations. We are seeing genomic- and viromic-based nominations of many new candidate enteric pathogens. Investigators will be challenged to expeditiously confirm or refute the virulence of such agents with the rigor and curiosity of the investigators from the 1940's and 1950's.

Plasmid diversity in Vibrio vulnificus biotypes

Vibrio vulnificus is a heterogeneous bacterial species that can be virulent for humans and fish. Virulence in fish seems to rely on a recently described plasmid that can be transmitted between strains, aided by a conjugative plasmid. The main objective of this work was to analyse the plasmid content of a wide collection of strains from the three biotypes of the species, as well as to identify putative conjugative and virulence plasmids by means of Southern hybridization with specific probes and sequence analysis of selected gene markers. We found 28 different plasmid profiles in a total of 112 strains, which were relatively biotype- or serovar-specific. Biotype 1 lacked high-molecular-mass plasmids, with the exception of a putative conjugative plasmid of 48 kb that was present in 42.8% of clinical and environmental strains isolated worldwide. All biotype 2 strains possessed the virulence plasmid, whose molecular mass ranged between 68 and 70 kb, and 89.65% of these strains also had a putative conjugative plasmid with a molecular size of 52-56 kb. Finally, a 48 kb putative conjugative plasmid was present in all biotype 3 strains. Data from partial sequencing of traD, traI and the whole vep07 (a recently described plasmid-borne virulence gene) from a selection of strains suggest that the plasmids of 48-56 kb probably belong to the same family of F-plasmids as pYJ016 and that the gene vep07 is absolutely essential for fish virulence. Additional cryptic plasmids of low molecular mass were present in the three biotypes. In conclusion, plasmids are widespread among V. vulnificus species and could contribute substantially to genetic plasticity of the species.

Pathogenic Isolates of Rhodococcus fascians from New Hosts in the United States

Rhodococcus fascians is important to the nursery industry due to its broad host range (68 genera) (2) and potential for horizontal gene transfer of plasmid-borne virulence genes. Since 2001, many herbaceous ornamental plants with symptoms of leafy galls or basal or axillary shoot proliferation suggestive of infection by R. fascians have been submitted to the Oregon State University Plant Clinic for diagnosis. R. fascians was isolated from symptomatic plants by placing affected tissues into saline or liquid D2 medium (1) for 30 to 120 min and then dilution plating onto D2 agar. Orange colonies were purified by dilution streaking and identified as R. fascians by substrate utilization (Biolog, Hayward, CA) and fatty acid analysis (L. Barnes, Texas A & M University, College Station, TX). Pathogenicity was confirmed by inoculation of 10 newly germinated Pisum sativum 'Laxton Progress' and 'Sugar Pod' seedlings with bacteria from 2-day-old cultures (107 CFU/ml) or water (controls). Our isolates produced shoot proliferations typical of R. fascians infection of peas, confirming pathogenicity. Control plants remained healthy. Pathogenic R. fascians isolates were associated with and isolated from eight species not previously reported as hosts: Acanthus mollis, Campanula sarastro, Heliopsis helianthoides 'Loraine Sunshine', Nemesia × 'Natalie', Hosta × 'Blue Umbrella', Verbascum 'Sierra Sunset', Veronica spicata 'Minuet' and Viola × 'Purple Showers'.

PCR detection of virulence genes in Yersinia enterocolitica and Yersinia pseudotuberculosis and investigation of virulence gene distribution.

PCR-based assays were developed for the detection of plasmid- and chromosome-borne virulence genes in Yersinia enterocolitica and Yersinia pseudotuberculosis, to investigate the distribution of these genes in isolates from various sources. The results of PCR genotyping, based on 5 virulence-associated genes of 140 strains of Y. enterocolitica, were compared to phenotypic tests, such as biotyping and serotyping, and to virulence plasmid-associated properties such as calcium-dependent growth at 37 degrees C and Congo red uptake. The specificity of the PCR results was validated by hybridization. Genotyping data correlated well with biotype data, and most biotypes resulted in (nearly) homogeneous genotypes for the chromosomal virulence genes (ystA, ystB, and ail); however, plasmid-borne genes (yadA and virF) were detected with variable efficiency, due to heterogeneity within the bacterial population for the presence of the virulence plasmid. Of the virulence genes, only ystB was present in biotype 1A; however, within this biotype, pathogenic and apathogenic isolates could not be distinguished based on the detection of virulence genes. Forty Y. pseudotuberculosis isolates were tested by PCR for the presence of inv, yadA, and lcrF. All isolates were inv positive, and 88% of the isolates contained the virulence plasmid genes yadA and lcrF. In conclusion, this study shows that genotyping of Yersinia spp., based on both chromosome- and plasmid-borne virulence genes, is feasible and informative and can provide a rapid and reliable genotypic characterization of field isolates.

Detection of pathogenic Yersinia enterocolitica in enrichment media and pork by a multiplex PCR: a study of sample preparation and PCR-inhibitory components

A multiplex PCR assay including sample preparation was developed to detect viable pathogenic strains of Yersinia enterocolitica in PCR-inhibitory samples, such as pork and enrichment media. The method developed was used to simultaneously detect the plasmid-borne virulence gene yadA and a Yersinia-specific region of the 16S rRNA gene. According to an auto-agglutination test for virulence-plasmid-bearing strains of Y. enterocolitica, all potential pathogenic strains tested were detected by the assay. A DNA extraction procedure, an aqueous two-phase system composed of polyethylene glycol 4000 and dextran 40 and a buoyant density centrifugation method, based on Percoll, were compared with regard to their efficiency in separating Yersinia enterocolitica from PCR inhibitors originating from enrichment media and pork. Using the density gradient centrifugation method resulted in a detection level of 4.0·10 2 CFU Y. enterocolitica per ml enrichment media. To ensure detection of viable bacteria a short enrichment step was included in the sample preparation together with the density gradient centrifugation. When this sample treatment method was evaluated with a selective enrichment medium together with a background flora inoculated with approximately 1.0·10 1 CFU per ml of Y. enterocolitica and incubated at 25°C, a positive PCR result was obtained after 6 to 8 h. Our results indicate that selective enrichment followed by buoyant density gradient centrifugation provides a convenient and user-friendly sample preparation method prior to PCR.