September 22, 2019  |  

In situ analyses directly in diarrheal stool reveal large variations in bacterial load and active toxin expression of enterotoxigenic Escherichia coli and Vibrio cholerae.

The bacterial pathogens enterotoxigenicEscherichia coli(ETEC) andVibrio choleraeare major causes of diarrhea. ETEC causes diarrhea by production of the heat-labile toxin (LT) and heat-stable toxins (STh and STp), whileV. choleraeproduces cholera toxin (CT). In this study, we determined the occurrence and bacterial doses of the two pathogens and their respective toxin expression levels directly in liquid diarrheal stools of patients in Dhaka, Bangladesh. By quantitative culture and real-time quantitative PCR (qPCR) detection of the toxin genes, the two pathogens were found to coexist in several of the patients, at concentrations between 102and 108bacterial gene copies per ml. Even in culture-negative samples, gene copy numbers of 102to 104of either ETEC orV. choleraetoxin genes were detected by qPCR. RNA was extracted directly from stool, and gene expression levels, quantified by reverse transcriptase qPCR (RT-qPCR), of the genes encoding CT, LT, STh, and STp showed expression of toxin genes. Toxin enzyme-linked immunosorbent assay (ELISA) confirmed active toxin secretion directly in the liquid diarrhea. Analysis of ETEC isolates by multiplex PCR, dot blot analysis, and genome sequencing suggested that there are genetic ETEC profiles that are more commonly found as dominating single pathogens and others that are coinfectants with lower bacterial loads. The ETEC genomes, including assembled genomes of dominating ETEC isolates expressing LT/STh/CS5/CS6 and LT/CS7, are provided. In addition, this study highlights an emerging important ETEC strain expressing LT/STp and the novel colonization factor CS27b. These findings have implications for investigations of pathogenesis as well as for vaccine development. IMPORTANCEThe cause of diarrheal disease is usually determined by screening for several microorganisms by various methods, and sole detection is used to assign the agent as the cause of disease. However, it has become increasingly clear that many infections are caused by coinfections with several pathogens and that the dose of the infecting pathogen is important. We quantified the absolute numbers of enterotoxigenicE. coli(ETEC) andVibrio choleraedirectly in diarrheal fluid. We noted several events where both pathogens were found but also a large dose dependency. In three samples, we found ETEC as the only pathogen sought for. These isolates belonged to globally distributed ETEC clones and were the dominating species in stool with active toxin expression. This suggests that certain superior virulent ETEC lineages are able to outcompete the gut microbiota and be the sole cause of disease and hence need to be specifically monitored.


September 22, 2019  |  

Comparative genomic analysis reveals the evolution and environmental adaptation strategies of vibrios.

Vibrios are among the most diverse and ecologically important marine bacteria, which have evolved many characteristics and lifestyles to occupy various niches. The relationship between genome features and environmental adaptation strategies is an essential part for understanding the ecological functions of vibrios in the marine system. The advent of complete genome sequencing technology has provided an important method of examining the genetic characteristics of vibrios on the genomic level.Two Vibrio genomes were sequenced and found to occupy many unique orthologues families which absent from the previously genes pool of the complete genomes of vibrios. Comparative genomics analysis found vibrios encompass a steady core-genome and tremendous pan-genome with substantial gene gain and horizontal gene transfer events in the evolutionary history. Evolutionary analysis based on the core-genome tree suggested that V. fischeri emerged ~?385 million years ago, along with the occurrence of cephalopods and the flourish of fish. The relatively large genomes, the high number of 16S rRNA gene copies, and the presence of R-M systems and CRISPR system help vibrios live in various marine environments. Chitin-degrading related genes are carried in nearly all the Vibrio genomes. The number of chitinase genes in vibrios has been extremely expanded compared to which in the most recent ancestor of the genus. The chitinase A genes were estimated to have evolved along with the genus, and have undergone significant purifying selective force to conserve the ancestral state.Vibrios have experienced extremely genome expansion events during their evolutionary history, allowing them to develop various functions to spread globally. Despite their close phylogenetic relationships, vibrios were found to have a tremendous pan-genome with a steady core-genome, which indicates the highly plastic genome of the genus. Additionally, the existence of various chitin-degrading related genes and the expansion of chitinase A in the genus demonstrate the importance of the chitin utilization for vibrios. Defensive systems in the Vibrio genomes may protect them from the invasion of external DNA. These genomic features investigated here provide a better knowledge of how the evolutionary process has forged Vibrio genomes to occupy various niches.


September 22, 2019  |  

Basic characterization of natural transformation in a highly transformable Haemophilus parasuis strain SC1401.

Haemophilus parasuis causes Glässer’s disease and pneumonia, incurring serious economic losses in the porcine industry. In this study, natural competence was investigated in H. parasuis. We found competence genes in H. parasuis homologous to ones in Haemophilus influenzae and a high consensus battery of Sxy-dependent cyclic AMP (cAMP) receptor protein (CRP-S) regulons using bioinformatics. High rates of natural competence were found from the onset of stationary-phase growth condition to mid-stationary phase (OD600 from 0.29 to 1.735); this rapidly dropped off as cells reached mid-stationary phase (OD600 from 1.735 to 1.625). As a whole, bacteria cultured in liquid media were observed to have lower competence levels than those grown on solid media plates. We also revealed that natural transformation in this species is stable after 200 passages and is largely dependent on DNA concentration. Transformation competition experiments showed that heterogeneous DNA cannot outcompete intraspecific natural transformation, suggesting an endogenous uptake sequence or other molecular markers may be important in differentiating heterogeneous DNA. We performed qRT-PCR targeting multiple putative competence genes in an effort to compare bacteria pre-cultured in TSB++ vs. TSA++ and SC1401 vs. SH0165 to determine expression profiles of the homologs of competence-genes in H. influenzae. Taken together, this study is the first to investigate natural transformation in H. parasuis based on a highly naturally transformable strain SC1401.


September 22, 2019  |  

Emergence and genomic analysis of MDR Laribacter hongkongensis strain HLGZ1 from Guangzhou, China.

Laribacter hongkongensis is a facultative anaerobic, non-fermentative, Gram-negative bacillus associated with community-acquired gastroenteritis and traveller’s diarrhoea. No clinical MDR L. hongkongensis isolate has been reported yet.We performed WGS (PacBio and Illumina) on a clinical L. hongkongensis strain HLGZ1 with an MDR phenotype.HLGZ1 was resistant to eight classes of commonly used antibiotics. Its complete genome was a single circular chromosome of 3?424?272?bp with a G?+?C content of 62.29%. In comparison with the reference strain HLHK9, HLGZ1 had a higher abundance of genes associated with DNA metabolism and recombination. Several inserts including two acquired resistance gene clusters (RC1 and RC2) were also identified. RC1 carried two resistance gene cassette arrays, aac(6′)-Ib-cr-aadA2-?qac-?sul1-floR-tetR-tetG and arr-3-dfrA32-ereA2-?qac-sul1, which shared significant nucleotide sequence identities with the MDR region of Salmonella Genomic Island 1 from Salmonella enterica serovar Typhimurium DT104. There was also an integron-like structure, intl1-arr3-dfrA27-?qac-sul1-aph(3′)-Ic, and a tetR-tetA operon located on RC2. MLST analysis identified HLGZ1 as ST167, a novel ST clustered with two strains previously isolated from frogs.This study provides insight into the genomic characteristics of MDR L. hongkongensis and highlights the possibilities of horizontal resistance gene transfer in this bacterium with other pathogens.


September 22, 2019  |  

Synchronous termination of replication of the two chromosomes is an evolutionary selected feature in Vibrionaceae.

Vibrio cholerae, the causative agent of the cholera disease, is commonly used as a model organism for the study of bacteria with multipartite genomes. Its two chromosomes of different sizes initiate their DNA replication at distinct time points in the cell cycle and terminate in synchrony. In this study, the time-delayed start of Chr2 was verified in a synchronized cell population. This replication pattern suggests two possible regulation mechanisms for other Vibrio species with different sized secondary chromosomes: Either all Chr2 start DNA replication with a fixed delay after Chr1 initiation, or the timepoint at which Chr2 initiates varies such that termination of chromosomal replication occurs in synchrony. We investigated these two models and revealed that the two chromosomes of various Vibrionaceae species terminate in synchrony while Chr2-initiation timing relative to Chr1 is variable. Moreover, the sequence and function of the Chr2-triggering crtS site recently discovered in V. cholerae were found to be conserved, explaining the observed timing mechanism. Our results suggest that it is beneficial for bacterial cells with multiple chromosomes to synchronize their replication termination, potentially to optimize chromosome related processes as dimer resolution or segregation.


September 22, 2019  |  

Plasmids carrying DHA-1 ß-lactamases.

The aim of this review is to provide an update on the plasmids mediating DHA-1 cephalosporinase in Klebsiella pneumoniae. These plasmids have been mainly found in this bacterium but not only. The first was isolated from Salmonella sp. in France in the early 1990s. They are currently reported worldwide. BlaDHA-1 beta-lactamase gene is usually co-expressed with many other antibiotic resistance genes such as extended-spectrum ß-lactamases (blaCTX-M-, bla SHV -types), oxacillinases (blaOXA-1, blaOXA-30), penicillinases (bla TEM -type), carbapenemases (bla OXA48 , blaKPC-2), aminoglycosides (aacA, aadA, armA), fluoroquinolones (qnrB4, aac6′-1b-cr), and sulfonamide (sul1) resistance genes. Plasmids carrying DHA-1 cephalosporinase have different sizes (22 to 313 kb), belong to diverse groups of incompatibility (R, L/M, FII(k), FIB, A/C2, HI2, HIB), and are self-transferable or not. The multidrug resistance region consists of a mosaic structure composed of resistance genes, insertion sequences, composite transposon, and integrons.


September 22, 2019  |  

Redefinition and unification of the SXT/R391 family of integrative and conjugative elements.

Integrative and conjugative elements (ICEs) of the SXT/R391 family are key drivers of the spread of antibiotic resistance in Vibrio cholerae, the infectious agent of cholera, and other pathogenic bacteria. The SXT/R391 family of ICEs was defined based on the conservation of a core set of 52 genes and site-specific integration into the 5′ end of the chromosomal gene prfC Hence, the integrase gene int has been intensively used as a marker to detect SXT/R391 ICEs in clinical isolates. ICEs sharing most core genes but differing by their integration site and integrase gene have been recently reported and excluded from the SXT/R391 family. Here we explored the prevalence and diversity of atypical ICEs in GenBank databases and their relationship with typical SXT/R391 ICEs. We found atypical ICEs in V. cholerae isolates that predate the emergence and expansion of typical SXT/R391 ICEs in the mid-1980s in seventh-pandemic toxigenic V. cholerae strains O1 and O139. Our analyses revealed that while atypical ICEs are not associated with antibiotic resistance genes, they often carry cation efflux pumps, suggesting heavy metal resistance. Atypical ICEs constitute a polyphyletic group likely because of occasional recombination events with typical ICEs. Furthermore, we show that the alternative integration and excision genes of atypical ICEs remain under the control of SetCD, the main activator of the conjugative functions of SXT/R391 ICEs. Together, these observations indicate that substitution of the integration/excision module and change of specificity of integration do not preclude atypical ICEs from inclusion into the SXT/R391 family.IMPORTANCEVibrio cholerae is the causative agent of cholera, an acute intestinal infection that remains to this day a world public health threat. Integrative and conjugative elements (ICEs) of the SXT/R391 family have played a major role in spreading antimicrobial resistance in seventh-pandemic V. cholerae but also in several species of Enterobacteriaceae Most epidemiological surveys use the integrase gene as a marker to screen for SXT/R391 ICEs in clinical or environmental strains. With the recent reports of closely related elements that carry an alternative integrase gene, it became urgent to investigate whether ICEs that have been left out of the family are a liability for the accuracy of such screenings. In this study, based on comparative genomics, we broaden the SXT/R391 family of ICEs to include atypical ICEs that are often associated with heavy metal resistance. Copyright © 2018 American Society for Microbiology.


September 22, 2019  |  

Potential survival and pathogenesis of a novel strain, Vibrio parahaemolyticus FORC_022, isolated from a soy sauce marinated crab by genome and transcriptome analyses.

Vibrio parahaemolyticus can cause gastrointestinal illness through consumption of seafood. Despite frequent food-borne outbreaks of V. parahaemolyticus, only 19 strains have subjected to complete whole-genome analysis. In this study, a novel strain of V. parahaemolyticus, designated FORC_022 (Food-borne pathogen Omics Research Center_022), was isolated from soy sauce marinated crabs, and its genome and transcriptome were analyzed to elucidate the pathogenic mechanisms. FORC_022 did not include major virulence factors of thermostable direct hemolysin (tdh) and TDH-related hemolysin (trh). However, FORC_022 showed high cytotoxicity and had several V. parahaemolyticus islands (VPaIs) and other virulence factors, such as various secretion systems (types I, II, III, IV, and VI), in comparative genome analysis with CDC_K4557 (the most similar strain) and RIMD2210633 (genome island marker strain). FORC_022 harbored additional virulence genes, including accessory cholera enterotoxin, zona occludens toxin, and tight adhesion (tad) locus, compared with CDC_K4557. In addition, O3 serotype specific gene and the marker gene of pandemic O3:K6 serotype (toxRS) were detected in FORC_022. The expressions levels of genes involved in adherence and carbohydrate transporter were high, whereas those of genes involved in motility, arginine biosynthesis, and proline metabolism were low after exposure to crabs. Moreover, the virulence factors of the type III secretion system, tad locus, and thermolabile hemolysin were overexpressed. Therefore, the risk of foodborne-illness may be high following consumption of FORC_022 contaminated crab. These results provided molecular information regarding the survival and pathogenesis of V. parahaemolyticus FORC_022 strain in contaminated crab and may have applications in food safety.


September 22, 2019  |  

Periodic variation of mutation rates in bacterial genomes associated with replication timing

The causes and consequences of spatiotemporal variation in mutation rates remain to be explored in nearly all organisms. Here we examine relationships between local mutation rates and replication timing in three bacterial species whose genomes have multiple chromosomes: Vibrio fischeri, Vibrio cholerae, and Burkholderia cenocepacia Following five mutation accumulation experiments with these bacteria conducted in the near absence of natural selection, the genomes of clones from each lineage were sequenced and analyzed to identify variation in mutation rates and spectra. In lineages lacking mismatch repair, base substitution mutation rates vary in a mirrored wave-like pattern on opposing replichores of the large chromosomes of V. fischeri and V. cholerae, where concurrently replicated regions experience similar base substitution mutation rates. The base substitution mutation rates on the small chromosome are less variable in both species but occur at similar rates to those in the concurrently replicated regions of the large chromosome. Neither nucleotide composition nor frequency of nucleotide motifs differed among regions experiencing high and low base substitution rates, which along with the inferred ~800-kb wave period suggests that the source of the periodicity is not sequence specific but rather a systematic process related to the cell cycle. These results support the notion that base substitution mutation rates are likely to vary systematically across many bacterial genomes, which exposes certain genes to elevated deleterious mutational load.IMPORTANCE That mutation rates vary within bacterial genomes is well known, but the detailed study of these biases has been made possible only recently with contemporary sequencing methods. We applied these methods to understand how bacterial genomes with multiple chromosomes, like those of Vibrio and Burkholderia, might experience heterogeneous mutation rates because of their unusual replication and the greater genetic diversity found on smaller chromosomes. This study captured thousands of mutations and revealed wave-like rate variation that is synchronized with replication timing and not explained by sequence context. The scale of this rate variation over hundreds of kilobases of DNA strongly suggests that a temporally regulated cellular process may generate wave-like variation in mutation risk. These findings add to our understanding of how mutation risk is distributed across bacterial and likely also eukaryotic genomes, owing to their highly conserved replication and repair machinery. Copyright © 2018 Dillon et al.


September 22, 2019  |  

Identification of the DNA methyltransferases establishing the methylome of the cyanobacterium Synechocystis sp. PCC 6803.

DNA methylation in bacteria is important for defense against foreign DNA, but is also involved in DNA repair, replication, chromosome partitioning, and regulatory processes. Thus, characterization of the underlying DNA methyltransferases in genetically tractable bacteria is of paramount importance. Here, we characterized the methylome and orphan methyltransferases in the model cyanobacterium Synechocystis sp. PCC 6803. Single molecule real-time (SMRT) sequencing revealed four DNA methylation recognition sequences in addition to the previously known motif m5CGATCG, which is recognized by M.Ssp6803I. For three of the new recognition sequences, we identified the responsible methyltransferases. M.Ssp6803II, encoded by the sll0729 gene, modifies GGm4CC, M.Ssp6803III, encoded by slr1803, represents the cyanobacterial dam-like methyltransferase modifying Gm6ATC, and M.Ssp6803V, encoded by slr6095 on plasmid pSYSX, transfers methyl groups to the bipartite motif GGm6AN7TTGG/CCAm6AN7TCC. The remaining methylation recognition sequence GAm6AGGC is probably recognized by methyltransferase M.Ssp6803IV encoded by slr6050. M.Ssp6803III and M.Ssp6803IV were essential for the viability of Synechocystis, while the strains lacking M.Ssp6803I and M.Ssp6803V showed growth similar to the wild type. In contrast, growth was strongly diminished of the ?sll0729 mutant lacking M.Ssp6803II. These data provide the basis for systematic studies on the molecular mechanisms impacted by these methyltransferases.


September 22, 2019  |  

Tracing genomic divergence of Vibrio bacteria in the Harveyi clade.

The mechanism of bacterial speciation remains a topic of tremendous interest. To understand the ecological and evolutionary mechanisms of speciation in Vibrio bacteria, we analyzed the genomic dissimilarities between three closely related species in the so-called Harveyi clade of the genus Vibrio, V. campbellii, V. jasicida, and V. hyugaensis The analysis focused on strains isolated from diverse geographic locations over a long period of time. The results of phylogenetic analyses and calculations of average nucleotide identity (ANI) supported the classification of V. jasicida and V. hyugaensis into two species. These analyses also identified two well-supported clades in V. campbellii; however, strains from both clades were classified as members of the same species. Comparative analyses of the complete genome sequences of representative strains from the three species identified higher syntenic coverage between genomes of V. jasicida and V. hyugaensis than that between the genomes from the two V. campbellii clades. The results from comparative analyses of gene content between bacteria from the three species did not support the hypothesis that gene gain and/or loss contributed to their speciation. We also did not find support for the hypothesis that ecological diversification toward associations with marine animals contributed to the speciation of V. jasicida and V. hyugaensis Overall, based on the results obtained in this study, we propose that speciation in Harveyi clade species is a result of stochastic diversification of local populations, which was influenced by multiple evolutionary processes, followed by extinction events.IMPORTANCE To investigate the mechanisms underlying speciation in the genus Vibrio, we provided a well-assembled reference of genomes and performed systematic genomic comparisons among three evolutionarily closely related species. We resolved taxonomic ambiguities and identified genomic features separating the three species. Based on the study results, we propose a hypothesis explaining how species in the Harveyi clade of Vibrio bacteria diversified. Copyright © 2018 American Society for Microbiology.


September 22, 2019  |  

Large scale changes in host methylation patterns induced by IncA/C plasmid transformation in Vibrio cholerae

DNA methylation is a central epigenetic modification and has diverse biological functions in eukaryotic and prokaryotic organisms alike. The IncA/C plasmid genomes are approximately 150kb in length and harbour three methylase genes, two of which demonstrate cytosine specificity. Transformation of the Vibrio cholerae strain C6706 with the IncA/C plasmid pVC211 resulted in a significant relabelling of the methylation patterns on the host chromosomes. The new methylation patterns induced by transformation with IncA/C plasmid were accepted by the restriction enzymes of the hosttextquoterights restriction modification (RM) system. These data uncover a novel mechanism by which plasmids can be compatible with a hosttextquoterights RM system and suggest a possible reason that plasmids of the IncA/C family are broad-host-range.


September 22, 2019  |  

Comparative analyses of CTX prophage region of Vibrio cholerae seventh pandemic wave 1 strains isolated in Asia.

Vibrio cholerae O1 causes cholera, and cholera toxin, the principal mediator of massive diarrhea, is encoded by ctxAB in the cholera toxin (CTX) prophage. In this study, the structures of the CTX prophage region of V. cholerae strains isolated during the seventh pandemic wave 1 in Asian countries were determined and compared. Eighteen strains were categorized into eight groups by CTX prophage region-specific restriction fragment length polymorphism and PCR profiles and the structure of the region of a representative strain from each group was determined by DNA sequencing. Eight representative strains revealed eight distinct CTX prophage regions with various combinations of CTX-1, RS1 and a novel genomic island on chromosome I. CTX prophage regions carried by the wave 1 strains were diverse in structure. V. cholerae strains with an area specific CTX prophage region are believed to circulate in South-East Asian countries; additionally, multiple strains with distinct types of CTX prophage region are co-circulating in the area. Analysis of a phylogenetic tree generated by single nucleotide polymorphism differences across 2483 core genes revealed that V. cholerae strains categorized in the same group based on CTX prophage region structure were segregated in closer clusters. CTX prophage region-specific recombination events or gain and loss of genomic elements within the region may have occurred at much higher frequencies and contributed to producing a panel of CTX prophage regions with distinct structures among V. cholerae pathogenic strains in lineages with close genetic backgrounds in the early wave 1 period of the seventh cholera pandemic.© 2018 The Authors. Microbiology and Immunology published by The Societies and John Wiley & Sons Australia, Ltd.


September 22, 2019  |  

Functionality of two origins of replication in Vibrio cholerae strains with a single chromosome.

Chromosomal inheritance in bacteria usually entails bidirectional replication of a single chromosome from a single origin into two copies and subsequent partitioning of one copy each into daughter cells upon cell division. However, the human pathogen Vibrio cholerae and other Vibrionaceae harbor two chromosomes, a large Chr1 and a small Chr2. Chr1 and Chr2 have different origins, an oriC-type origin and a P1 plasmid-type origin, respectively, driving the replication of respective chromosomes. Recently, we described naturally occurring exceptions to the two-chromosome rule of Vibrionaceae: i.e., Chr1 and Chr2 fused single chromosome V. cholerae strains, NSCV1 and NSCV2, in which both origins of replication are present. Using NSCV1 and NSCV2, here we tested whether two types of origins of replication can function simultaneously on the same chromosome or one or the other origin is silenced. We found that in NSCV1, both origins are active whereas in NSCV2 ori2 is silenced despite the fact that it is functional in an isolated context. The ori2 activity appears to be primarily determined by the copy number of the triggering site, crtS, which in turn is determined by its location with respect to ori1 and ori2 on the fused chromosome.


September 22, 2019  |  

Role of phage ?1 in two strains of Salmonella Rissen, sensitive and resistant to phage ?1.

The study describes the Salmonella Rissen phage ?1 isolated from the ?1-sensitive Salmonella Rissen strain RW. The same phage was then used to select the resistant strain RR?1+, which can harbour or not ?1.Following this approach, we found that ?1, upon excision from RW cells with mitomycin, behaves as a temperate phage: lyses host cells and generates phage particles; instead, upon spontaneous excision from RR?1+ cells, it does not generate phage particles; causes loss of phage resistance; switches the O-antigen from the smooth to the rough phenotype, and favors the transition of Salmonella Rissen from the planktonic to the biofilm growth. The RW and RR?1+ strains differ by 10 genes; of these, only two (phosphomannomutase_1 and phosphomannomutase_2; both involved in the mannose synthesis pathway) display significant differences at the expression levels. This result suggests that phage resistance is associated with these two genes.Phage ?1 displays the unusual property of behaving as template as well as lytic phage. This feature was used by the phage to modulate several phases of Salmonella Rissen lifestyle.


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