attained higher quality scores. Moreover, a subsequent study concluded that sequencing technologies generating shorter sequence reads (i.e., the genome sequence is encompassed in many contiguous fragments) present major difficulties for bioinformatics algorithms seeking to analyze such data (Land et al., 2015). Taken together, it is perhaps not surprising that our study only identified the putative virulenceplasmid described here when complementary approaches for generating the complete genome sequence were used. Short-read second generation sequencing remains the most commonly used and cost-effective genome sequencing strategy for bacterial genomes (Land et al., 2015), but as our study indicates, the reduced financial cost can come at a price for biological data that may be of significance.
We find that pADEO16, a recombinant cosmid carrying the rck gene of the Salmonella typhimurium virulenceplasmid, when cloned into either rough or smooth Escherichia coli and Salmonella strains, confers high level resistance to the bactericidal activity of pooled normal human serum. The rck gene encodes a 17-kD outer membrane protein that is homologous to a family of virulence-associated outer membrane proteins, including pagC and Ail. Complement depletion, C3 and C5 binding, and membrane-bound C3 cleavage products are similar in strains with and without rck. Although a large difference in C9 binding was not seen, trypsin cleaved 55.7% of bound 125I-C9 counts from rough S. typhimurium with pADEO16, whereas only 26.4% were released from S. typhimurium with K2011, containing a mutation in rck. The majority of C9 extracted from rck strain membranes sediments at a lower molecular weight than in strains without rck, suggesting less C9
is expressed during infection and codes for SpvB, a toxin that ADP-ribosylates actin of the macrophage cell desta- bilizing its cytoskeleton (Heithoff et al. 1997; Lesnick et al. 2001; Tezcan-Merdol et al. 2001) and SpvC, a phos- phothreonine lyase that helps subvert natural inflamma- tion response to Salmonella enterica (Haneda et al. 2012; Mazurkiewicz et al. 2008). Intra-serovar diversity in plas- mid composition has also been reported, although, on a more limited scale. For example, insertions led to the acquisition of antibiotic resistance genes in the virulenceplasmid of the serovar Typhimurium, thus making it not only a virulence determinant, but also the source a multi- drug resistance phenotype (Herrero-Fresno et al. 2015).
The spv operon is common to all Salmonella virulence plasmids. DNA hybridization analysis indicates that the spv region is limited in distribution to serovars of Salmonella enterica subspecies I, II, IIIa, IV, and VII and is absent from Salmonella bongori isolates. Among strains of subspecies II, IIIa, and VII, all isolates examined contained sequences that hybridized with the spv region. However, among isolates of subspecies I, DNA sequences capable of hybridizing with the spv region were found in some isolates of certain serovars. Furthermore, in isolates of subspecies I, the virulenceplasmid was found in the same set of isolates as an F-related plasmid, as determined by the presence of the spv region of the virulenceplasmid and the finO, traD, and repA sequences of the F-plasmid. The concordance of the virulenceplasmid and all three F-plasmid sequences in subspecies I serovar Choleraesuis, Paratyphi, and Typhimurium is most easily explained if the spv region is carried in an F-related plasmid in these isolates. In contrast, among S. enterica subspecies II, IIIa, IV, and VII, the isolates that contain spv sequences did not hybridize with an F-related plasmid or any other identifiable plasmid. With the use of pulse-field gel electrophoresis, the spv region in subspecies II, IIIa, and VII was found to be encoded on the chromosome. Analysis of the phylogenetic distribution of spv among Salmonella isolates and comparative nucleotide sequence analysis of spvA and spvC suggests that the spv region was acquired very recently, after speciation of the salmonellae.
Different derivatives of pESI were recently found to be associated with emergent strains of S. Infantis (7, 9). Illuminating the ecol- ogy, regulation, and transmission properties of pESI will add to the current understanding of the global S. Infantis emergence. Screening of recent S. Infantis isolates demonstrated the fixation of pESI in the domestic S. Infantis population. This observation and the single copy number of pESI are consistent with the finding that at least in vitro, pESI does not impose a significant metabolic burden on its bacterial host. A low- or even single-copy-number plasmid is somewhat expected, as other large conjugative plasmids were also shown to be maintained in a very low copy number (25). Because they are energy-consuming processes and because conjugative pili are often utilized by bacteriophage as receptors for bacterial infection (26), pilus assembly and conjugation are usu- ally tightly regulated, ensuring the expression of this system in a spatially and timely appropriate manner (27). A fundamental as- pect of HGT is the assimilation of the newly acquired genes into the preexisting regulatory setup of the new carrier (28, 29). In- sights into this mechanism were obtained by the identification of two regulators controlling pil gene expression and pESI conjuga- tion. The gene coding for TraB is carried by pESI, and its homolog in the R64 plasmid was previously suggested to encode a putative positive regulator of T4SS genes (30). Here we established that TraB is a transcriptional regulator of the pil genes in pESI. The second identified regulator is FNR, a global oxygen-responsive transcriptional regulator required for the switch from aerobic to anaerobic metabolism (31). In S. Typhimurium, FNR was previ- ously shown to regulate more than 300 genes involving in aerobic metabolism, nitric oxide detoxification, anaerobic carbon, and ethanolamine utilization, as well as the transcription of numerous Salmonella pathogenicity island 1 flagellar chemotaxis and viru- lence genes (32). Under microaerobic conditions, the absence of FNR resulted in a downregulation of pilV, pilS, and pilT transcrip- tion and lower pESI conjugation, demonstrating the integration of the pilus gene expression with the global metabolic regulatory setup of Salmonella.
The virulenceplasmid of Escherichia coli O157 strain EDL933 carries a 10-kb putative virulence gene designated toxB. Little is known about the distribution of this gene among E. coli O157 strains or its presence in other enterohemorrhagic E. coli (EHEC) and enteropathogenic E. coli (EPEC) strains. We developed PCR and hybridization tools for the detection of the entire toxB sequence and investigated its presence in a collection of EHEC O157 strains and other EHEC and EPEC strains belonging to different serogroups and isolated from different sources. The EHEC O157 strains reacted with all of the PCR primers and probes used, thus indicating the presence of a complete toxB gene regardless of the human or bovine origin of the isolates. Similar positive reactions were observed for about 50% of the EHEC O26 strains tested and a few other EHEC and EPEC strains. However, the size of the DNA fragments hybridizing with the toxB probes differed from that of the positive fragments from EHEC O157, suggesting a polymorphism in the toxB genes present in the different E. coli serogroups. Moreover, several EHEC and EPEC strains belonging to different serogroups reacted with only some of the genetic tools used, suggesting either the existence of major variants of toxB or the presence of fragments of the gene. Southern blotting analysis showed that toxB sequences were located on large plasmids in EHEC and EPEC O26 as well.
coli (STEC) strains that usually lack the locus for enterocyte effacement (LEE). We report for the first time the production of SubAB by two Stx-negative E. coli strains, isolated from unrelated cases of childhood diarrhea. The characterization of the SubAB-coding genes showed a 90% nucleotide sequence similarity with that of the prototype subAB, located on the virulenceplasmid of the STEC O113 strain 98NK2 (pO113). In both strains, subAB was physically associated with tia, an invasion genetic determinant of enterotoxigenic E. coli. The strains were negative for the saa gene, encoding an adhesin located on pO113 and present in many of the SubAB- positive strains described so far. PCR screening of 61 STEC and 100 Stx-negative E. coli strains in our collection revealed the presence of subAB in five LEE-negative STEC strains but not in the Stx-negative strains. subAB was contiguous to tia in three of the positive strains, which were all negative for saa. These results indicate that SubAB production is not restricted to STEC and suggest that a subAB-tia putative pathogenicity island is involved in the dissemination of subAB genes, as an alternative to plasmid pO113.
To detect the pLVPK-like virulenceplasmid and carbapen- emase gene-encoding plasmid, whole chromosomal DNA of 15 strains positive for both rmpA2 and bla KPC-2 were subjected to S1 nuclease (Takara, Shiga, Japan) digestion. Digested fragments were subjected to PFGE. Then, the gels were blotted onto nylon membranes (Millipore, Burlington, MA, USA) according to standard technique. The membranes were hybridized with digoxigenin-labeled rmpA2 probe and bla KPC-2 probe, respectively. Consistent with the findings from Russo et al, 15 K. pneumoniae strains that were confirmed to
Salmonellosis is one of the most common infectious diseases in both humans and animals (1- 2). Salmonella entrica serovar typhimurium is the most frequently isolated serovar worldwide (1, 3). Therefore, it is necessary and important to discriminate Salmonella serovars from each other in order to ensure that each pathogen and epidemiology is correctly recognized (4-5). On the other hand, Salmonella control requires rapid and reliable methods (4, 6-8). Traditional Salmonella detection methods are based on cultures using selective media after overnight enrichment broth and characterization of suspicious colonies by chemical and serological tests. These methods are laborious and time- consuming (5-6, 9-10). The S. typhimurium often harbor a serovar – specific virulenceplasmid (90 kb) containing the spv operon (11-13). Only a 7.8 kb region of spv is necessary to confer the virulence phenotype. The spv region harbors five genes spvR, spvA, spvB, spvC, spvD (14-16). Studies showed that a major function of the spv operon is to potentiate the systemic spread of the pathogen (17). There are also studies describing the genetic contents of spv, its role in the virulence and multiplication of intracellular Salmonella (14, 18). Brain et al. demonstrated that virulenceplasmid of S. typhimurium which is self-transmissible, provides an example of horizontal gene transfer (19). This therefore, necessitates investigating the plasmid profile for the presence of virulence genes (spvA, spvB, spvC) in Salmonella isolates. Simple and multiplex PCR (M-PCR) assays were utilized to detect the presence of the sequence spvA, spvB, spvC genes in the study. We selected simple PCR with SpvA and SpvB primers that target spvA, spvB gene sequences in Salmonella serovar (20). M-PCR with two pairs of oligonucleotide primers were performed according to the sequences of the chromosomal invA and plasmid spvC genes (21). We selected M-PCR targeting four genes sequences namely invA, rfbj, fliC and fljB specific for detecting genus Salmonella and serovar
A bacterial plasmid is an important agent for introducing adaptive traits horizontally to bacterial hosts as well as contributing to bacterial pathogenesis and evolution. Plasmids that carry virulence and resistance genes besides genes essential for their own transmission and maintenance are termed virulence plasmids and resistance plasmids, respectively (Sengupta and Austin, 2011). Large virulence plasmids (>100 kb) belonging to the incompatibility group IncF are widely disseminated in clinically relevant Enterobacteriaceae (Villa et al., 2010) including extraintestinal pathogenic E. coli (Johnson and Nolan, 2009; Mellata et al., 2009; Peigne et al., 2009; Woodford et al., 2009; Johnson et al., 2010a; Brolund et al., 2013). An example of an IncF virulenceplasmid that is often associated with ExPEC is the colicin-producing plasmid. Colicins are antimicrobial substances produced by certain members of colicin-producing Enterobacteriaceae that can kill susceptible strains, contributing to the virulence of the bacterial host (Waters and Crosa, 1991). Colicin V (ColV) and colicin B and M (ColBM) are among the most common types of colicins described that are associated with IncF plasmids. These plasmids are also known to harbor a repertoire of virulence determinants (extensively reviewed by Johnson and Nolan, 2009) that are essential for ExPEC pathogenesis. Nonetheless, putative evolutionary intermediates of ColV/ColBM plasmids which harbor the incomplete classical ColV components have also been reported, albeit rarely (Johnson and Nolan, 2009). Notoriously, IncF plasmids are also responsible for the dissemination of CTX- M genes that confer resistance to the first-line antimicrobial therapy against Enterobacteriaceae (i.e., cephalosporins; Villa et al., 2010; Cantón et al., 2012; Brolund et al., 2013). In fact, the successful global dissemination of IncF plasmids may be attributed to its multi-replicon characteristic which
lance initiative at a Beijing hospital. Using WGS analyses, we show that these isolates had been in circulation for at least 1 year prior to the initiation of our sampling efforts and had accumulated extensive genetic and phenotypic diversity that cannot be captured by commonly used typing schemes which rely on variation on the chromosome. The application of WGS allowed us to gain deeper insight into the extensive transmission of these isolates between patients. For example, the patient initially suspected to be the index case (Patient X) was probably not involved in any subsequent transmission to other patients. Interestingly, the two CRKP samples isolated from Patient X were unique in their carriage of a bla OXA-1 virulenceplasmid, probably
The species Y. enterocolitica is divided into six biotypes. Strains of biotype 1A are generally regar- ded as nonpathogenic, whereas strains of biotypes 1B, 2, 3, 4, and 5 carry a virulenceplasmid pYV. This plasmid encodes type III secretion system and the outer membrane protein YadA (Yersinia adhesin A). YadA was found to play multiple functions in pathogenesis because it protects bacterial cells against antibacterial activity of complement and mediates specific binding of Y. enterocolitica to laminin, collagen and cellular fibronectin . The chromosomal Y. enterocolitica virulence markers are ail, ystA and myfA genes. The ail gene encodes a small outer membrane protein (Ail adhesin), which promotes adhesion of Y. enterocolitica and invasion of epithelial cells. The ystA gene encodes entero- toxin YstA, which activates the guanylate cyclase that leads to the increased cGMP level. High level of cGMP causes fluid accumulation in the intestine . The major subunit of antigen Myf is encoded by the myfA gene. This fibrillar structure promotes the colonization of the intestine by yersiniae . Biotyping is used for clinical and epidemiological classification of Y. enterocolitica, but the hetero- genous nature of Y. enterocolitica, including diffe- rences in virulence, requires genotyping methods and this may be a novel way of pathogenic characterization of this microorganism.
chromosome 1, an oriCI region similar to that found in other γ-proteobacteria was found and spans 481 nucleo- tides (3,119,582 - 367 nt) [36–38]. For chromosome 2, an oriCII region similar to that of V. cholerae and other Vibrio species [36, 39, 40] was found and spans 366 nu- cleotides (1-366 nt). In addition, an incII incompatibility region similar to that of V. cholerae was found upstream of the oriCII region (1,186,667 - 1,187,342 nt). In V. cho- lerae, incII negatively regulates chromosome II replica- tion [36, 39]. Of the 3,912 genes predicted, 3,783 encode proteins, 25 encode rRNAs, 91 encode tRNAs, and at least 13 encode ncRNAs. Fifty-five pseudogenes were found with 33 located on chromosome 1, 21 located on chromosome 2, and 1 located on p67-NB10. Of the predicted CDSs, a functional prediction was made for 84.8 % and 66.9 % were assigned a putative COG function with the remaining annotated as hypothetical proteins. The plasmid is a pJM1- like virulenceplasmid that contains 58 protein-coding genes. Four new insertion sequences, named ISVa3, ISVa4, ISVa5, and ISVa6, were identified in this strain and were
Atypical enteropathogenic Escherichia coli (aEPEC) strains are increasingly recognized as an emerging pathotype respon- sible for childhood diarrhea in many countries (2–4, 19, 23). Atypical EPEC strains together with typical EPEC (tEPEC) strains constitute two distinct groups of organisms that have in common the locus of enterocyte effacement (LEE), a patho- genicity island responsible for the development of attaching- effacing (A/E) lesions. This island encodes the type III secre- tion system with multiple secreted proteins and a bacterial adhesin called “intimin” encoded by the eae gene (12, 16–18). Unlike tEPEC, aEPEC strains do not possess the EPEC ad- herence factor (EAF) virulenceplasmid that contains the bun- dle-forming pili (BFP) responsible for a localized adherence (LA) pattern on cultured epithelial cells (5, 8, 28), but aEPEC strains display different adherence patterns. The typical EPEC strain exhibits only the LA pattern, while aEPEC strains dis- play LA-like (LAL), diffuse adherence (DA), or aggregative adherence (AA) patterns (1, 10, 26, 32, 33). In addition, aEPEC strains belong to serotypes other than those of tEPEC strains (1, 10, 32, 33).
Previous studies of outbreaks have suggested that they are primarily associated with cKp, with few reports demon- strating that outbreaks were triggered by hvKp. 18 To date, only ST11 CR-hvKp has been reported as associated with HAI outbreaks. One reason may be the original capsule, 17 while another possible explanation is a strong association with plasmid compatibility. 30 Interestingly, a fatal outbreak induced by hvKp reported by Gu et al also originated from the same clone of ST11; 18 however, in this study, various ST11-MDR-hvKp clones were involved inter-host evolution and transmission and distributed among different clades. It is alarming that hvKp, as a source of hospital infection, is tending toward polymorphism, indicating that an increase in ongoing surveillance is urgently required. Yang et al 30 reported a conjugative virulenceplasmid that can rapidly enhance dissemination of virulence-encoding elements among Gram-negative bacterial pathogens, particularly Kp. In our study, all hvKp strains in clade 2 carried the IncFIB, but not the IncHI1B, plasmid replicon and harbored the same virulence gene cluster (iucA+ p rmpA), similar to the pVir-CR-
In recent years increased attention has been focused on infections caused by isolates of verocytotoxin- producing Escherichia coli (VTEC) serotypes other than O157. These non-O157 VTEC isolates are commonly present in food and food production animals. Easy detection, isolation, and characterization of non-O157 VTEC isolates are essential for improving our knowledge of these organisms. In the present study, we detected VTEC isolates in bovine fecal samples by a duplex 5 ⴕ nuclease PCR assay (real-time PCR) that targets vtx1 and vtx2. VTEC isolates were obtained by colony replication by use of hydrophobic-grid membrane filters and DNA probe hybridization. Furthermore, we have developed 5 ⴕ nuclease PCR assays for the detection of virulence factors typically present in VTEC isolates, including subtypes of three genes of the locus of enterocyte effacement (LEE) pathogenicity island. The 22 assays included assays for the detection of verocytotoxin genes (vtx1, vtx2), pO157-associated genes (ehxA, katP, espP, and etpD), a recently identified adhesin (saa), intimin (eae, all variants), seven subtypes of eae, four subtypes of tir, and three subtypes of espD. A number of reference strains (VTEC and enteropathogenic E. coli strains) and VTEC strains isolated from calves were tested to validate the PCR assays. The expected virulence profiles were detected for all reference strains. In addition, new information on the subtypes of LEE genes was obtained. For reference strains as well as bovine isolates, a consistent relationship between subtypes of the LEE genes was found, so that a total of seven different combinations of these were recognized (corresponding to the seven subtypes of eae). Isolates with 15 different serogroup-virulence profiles were isolated from 16 calves. Among these, 53% harbored LEE and 73% harbored factors carried by the large virulenceplasmid. One LEE-negative isolate had the gene for the adhesin Saa. The most common virulence profile among the bovine isolates was vtx1, eae- , tir- ␣ , ehxA, and espP. This panel of assays offers an easy method for the extensive characterization of VTEC isolates.