Nationwide genome surveillance of carbapenem-resistant Pseudomonas aeruginosa in Japan.

Japan is a country with an approximate 10% prevalence rate of carbapenem-resistant Pseudomonas aeruginosa (CRPA). Currently, a comprehensive overview of the genotype and phenotype patterns of CRPA in Japan is lacking. Herein, we conducted genome sequencing and quantitative antimicrobial susceptibility testing for 382 meropenem-resistant CRPA isolates that were collected from 78 hospitals across Japan from 2019 to 2020. CRPA exhibited susceptibility rates of 52.9%, 26.4%, and 88.0% against piperacillin-tazobactam, ciprofloxacin, and amikacin, respectively, whereas 27.7% of CRPA isolates was classified as difficult-to-treat resistance P. aeruginosa. Of the 148 sequence types detected, ST274 (9.7%) was predominant, followed by ST235 (7.6%). The proportion of urine isolates in ST235 was higher than that in other STs (P = 0.0056, χ2 test). Only 4.1% of CRPA isolates carried the carbapenemase genes: blaGES (2) and blaIMP (13). One ST235 isolate carried the novel blaIMP variant blaIMP-98 in the chromosome. Regarding chromosomal mutations, 87.1% of CRPA isolates possessed inactivating or other resistance mutations in oprD, and 28.8% showed mutations in the regulatory genes (mexR, nalC, and nalD) for the MexAB-OprM efflux pump. Additionally, 4.7% of CRPA isolates carried a resistance mutation in the PBP3-encoding gene ftsI. The findings from this study and other surveillance studies collectively demonstrate that CRPA exhibits marked genetic diversity and that its multidrug resistance in Japan is less prevailed than in other regions. This study contributes a valuable data set that addresses a gap in genotype/phenotype information regarding CRPA in the Asia-Pacific region, where the epidemiological background markedly differs between regions.

Evolving Populations in Biofilms Contain More Persistent Plasmids.

Bacterial plasmids substantially contribute to the rapid spread of antibiotic resistance, which is a crisis in healthcare today. Coevolution of plasmids and their hosts promotes this spread of resistance by ameliorating the cost of plasmid carriage. However, our knowledge of plasmid-bacteria coevolution is solely based on studies done in well-mixed liquid cultures, even though biofilms represent the main way of bacterial life on Earth and are responsible for most infections. The spatial structure and the heterogeneity provided by biofilms are known to lead to increased genetic diversity as compared with well-mixed liquids. Therefore, we expect that growth in this complex environment could affect the evolutionary trajectories of plasmid-host dyads. We experimentally evolved Shewanella oneidensis MR-1 with plasmid pBP136Gm in biofilms and chemostats and sequenced the genomes of clones and populations. Biofilm populations not only maintained a higher diversity of mutations than chemostat populations but contained a few clones with markedly more persistent plasmids that evolved via multiple distinct trajectories. These included the acquisition of a putative toxin-antitoxin transposon by the plasmid and chromosomal mutations. Some of these genetic changes resulted in loss of plasmid transferability or decrease in plasmid cost. Growth in chemostats led to a higher proportion of variants with decreased plasmid persistence, a phenomenon not detected in biofilms. We suggest that the presence of more stable plasmid-host dyads in biofilms reflects higher genetic diversity and possibly unknown selection pressures. Overall, this study underscores the importance of the mode of growth in the evolution of antibiotic-resistant bacteria.

Expression of an alcohol dehydrogenase gene in a heterotrophic bacterium induces carbon dioxide-dependent high-yield growth under oligotrophic conditions.

Sphingobium japonicum strain UT26, whose γ-hexachlorocyclohexane-degrading ability has been studied in detail, is a typical aerobic and heterotrophic bacterium that needs organic carbon sources for its growth, and cannot grow on a minimal salt agar medium prepared without adding any organic carbon sources. Here, we isolated a mutant of UT26 with the ability to grow to visible state on such an oligotrophic medium from a transposon-induced mutant library. This high-yield growth under oligotrophic conditions (HYGO) phenotype was CO2-dependent and accompanied with CO2 incorporation. In the HYGO mutant, a transposon was inserted just upstream of the putative Zn-dependent alcohol dehydrogenase (ADH) gene (adhX) so that the adhX gene was constitutively expressed, probably by the transposon-derived promoter. The adhX-deletion mutant (UT26DAX) harbouring a plasmid carrying the adhX gene under the control of a constitutive promoter exhibited the HYGO phenotype. Moreover, the HYGO mutants spontaneously emerged among the UT26-derived hypermutator strain cells, and adhX was highly expressed in these HYGO mutants, while no HYGO mutant appeared among UT26DAX-derived hypermutator strain cells, indicating the necessity of adhX for the HYGO phenotype. His-tagged AdhX that was expressed in Escherichia coli and purified to homogeneity showed ADH activity towards methanol and other alcohols. Mutagenesis analysis of the adhX gene indicated a correlation between the ADH activity and the HYGO phenotype. These results demonstrated that the constitutive expression of an adhX-encoding protein with ADH activity in UT26 leads to the CO2-dependent HYGO phenotype. Identical or nearly identical adhX orthologues were found in other sphingomonad strains, and most of them were located on plasmids, suggesting that the adhX-mediated HYGO phenotype may be an important adaptation strategy to oligotrophic environments among sphingomonads.

The recombination-cold region as an epidemiological marker of recombinogenic opportunistic pathogen Mycobacterium avium.

BACKGROUND: The rapid identification of lineage remains a challenge in the genotyping of clinical isolates of recombinogenic pathogens. The chromosome of Mycobacterium avium subsp. hominissuis (MAH), an agent of Mycobacterium avium complex (MAC) lung disease, is often mosaic and is composed of chromosomal segments originating from different lineages. This makes it difficult to infer the MAH lineage in a simple experimental set-up. To overcome this difficulty, we sought to identify chromosomal marker genes containing lineage-specific alleles by genome data mining. RESULTS: We conducted genetic population structure analysis, phylogenetic analysis, and a survey of historical recombination using data from 125 global MAH isolates. Six MAH lineages (EA1, EA2, SC1, SC2, SC3, and SC4) were identified in the current dataset. One P-450 gene (locus_tag MAH_0788/MAV_0940) in the recombination-cold region was found to have multiple alleles that could discriminate five lineages. By combining the information about allele type from one additional gene, the six MAH lineages as well as other M. avium subspecies were distinguishable. A recombination-cold region of 116 kb contains an insertion hotspot and is flanked by a mammalian cell-entry protein operon where allelic variants have previously been reported to occur. Hence, we speculate that the acquisition of lineage- or strain-specific insertions has introduced homology breaks in the chromosome, thereby reducing the chance of interlineage recombination. CONCLUSIONS: The allele types of the newly identified marker genes can be used to predict major lineages of M. avium. The single nucleotide polymorphism typing approach targeting multiallelic loci in recombination-cold regions will facilitate the epidemiological study of MAC, and may also be useful for equivalent studies of other nontuberculous mycobacteria potentially carrying mosaic genomes.

Restriction glycosylases: involvement of endonuclease activities in the restriction process.

All restriction enzymes examined are phosphodiesterases generating 3΄-OH and 5΄-P ends, but one restriction enzyme (restriction glycosylase) excises unmethylated bases from its recognition sequence. Whether its restriction activity involves endonucleolytic cleavage remains unclear. One report on this enzyme, R.PabI from a hyperthermophile, ascribed the breakage to high temperature while another showed its weak AP lyase activity generates atypical ends. Here, we addressed this issue in mesophiles. We purified R.PabI homologs from Campylobacter coli (R.CcoLI) and Helicobacter pylori (R.HpyAXII) and demonstrated their DNA cleavage, DNA glycosylase and AP lyase activities in vitro at 37°C. The AP lyase activity is more coupled with glycosylase activity in R.CcoLI than in R.PabI. R.CcoLI/R.PabI expression caused restriction of incoming bacteriophage/plasmid DNA and endogenous chromosomal DNA within Escherichia coli at 37°C. The R.PabI-mediated restriction was promoted by AP endonuclease action in vivo or in vitro. These results reveal the role of endonucleolytic DNA cleavage in restriction and yet point to diversity among the endonucleases. The cleaved ends are difficult to repair in vivo, which may indicate their biological significance. These results support generalization of the concept of restriction­modification system to the concept of self-recognizing epigenetic system, which combines any epigenetic labeling and any DNA damaging.

Evolutionary Paths That Expand Plasmid Host-Range: Implications for Spread of Antibiotic Resistance.

The World Health Organization has declared the emergence of antibiotic resistance to be a global threat to human health. Broad-host-range plasmids have a key role in causing this health crisis because they transfer multiple resistance genes to a wide range of bacteria. To limit the spread of antibiotic resistance, we need to gain insight into the mechanisms by which the host range of plasmids evolves. Although initially unstable plasmids have been shown to improve their persistence through evolution of the plasmid, the host, or both, the means by which this occurs are poorly understood. Here, we sought to identify the underlying genetic basis of expanded plasmid host-range and increased persistence of an antibiotic resistance plasmid using a combined experimental-modeling approach that included whole-genome resequencing, molecular genetics and a plasmid population dynamics model. In nine of the ten previously evolved clones, changes in host and plasmid each slightly improved plasmid persistence, but their combination resulted in a much larger improvement, which indicated positive epistasis. The only genetic change in the plasmid was the acquisition of a transposable element from a plasmid native to the Pseudomonas host used in these studies. The analysis of genetic deletions showed that the critical genes on this transposon encode a putative toxin-antitoxin (TA) and a cointegrate resolution system. As evolved plasmids were able to persist longer in multiple naïve hosts, acquisition of this transposon also expanded the plasmid's host range, which has important implications for the spread of antibiotic resistance.

Evolved plasmid-host interactions reduce plasmid interference cost.

Antibiotic selection drives adaptation of antibiotic resistance plasmids to new bacterial hosts, but the molecular mechanisms are still poorly understood. We previously showed that a broad-host-range plasmid was poorly maintained in Shewanella oneidensis, but rapidly adapted through mutations in the replication initiation gene trfA1. Here we examined if these mutations reduced the fitness cost of TrfA1, and whether this was due to changes in interaction with the host's DNA helicase DnaB. The strains expressing evolved TrfA1 variants showed a higher growth rate than those expressing ancestral TrfA1. The evolved TrfA1 variants showed a lower affinity to the helicase than ancestral TrfA1 and were no longer able to activate the helicase at the oriV without host DnaA. Moreover, persistence of the ancestral plasmid was increased upon overexpression of DnaB. Finally, the evolved TrfA1 variants generated higher plasmid copy numbers than ancestral TrfA1. The findings suggest that ancestral plasmid instability can at least partly be explained by titration of DnaB by TrfA1. Thus under antibiotic selection resistance plasmids can adapt to a novel bacterial host through partial loss of function mutations that simultaneously increase plasmid copy number and decrease unfavorably high affinity to one of the hosts' essential proteins.

Restriction-modification system with methyl-inhibited base excision and abasic-site cleavage activities.

The restriction-modification systems use epigenetic modification to distinguish between self and nonself DNA. A modification enzyme transfers a methyl group to a base in a specific DNA sequence while its cognate restriction enzyme introduces breaks in DNA lacking this methyl group. So far, all the restriction enzymes hydrolyze phosphodiester bonds linking the monomer units of DNA. We recently reported that a restriction enzyme (R.PabI) of the PabI superfamily with half-pipe fold has DNA glycosylase activity that excises an adenine base in the recognition sequence (5'-GTAC). We now found a second activity in this enzyme: at the resulting apurinic/apyrimidinic (AP) (abasic) site (5'-GT#C, # = AP), its AP lyase activity generates an atypical strand break. Although the lyase activity is weak and lacks sequence specificity, its covalent DNA-R.PabI reaction intermediates can be trapped by NaBH4 reduction. The base excision is not coupled with the strand breakage and yet causes restriction because the restriction enzyme action can impair transformation ability of unmethylated DNA even in the absence of strand breaks in vitro. The base excision of R.PabI is inhibited by methylation of the target adenine base. These findings expand our understanding of genetic and epigenetic processes linking those in prokaryotes and eukaryotes.

Mobile class A ß-lactamase gene bla GMA-1.

IMPORTANCE: Despite increasing reports, class A ß-lactamases of environmental bacteria remain very poorly characterized, with limited understanding of their transmission patterns. To address this knowledge gap, we focused on a recently designated GMA family ß-lactamase gene, bla GMA-1, found in marine bacterial genera such as Vibrio. This study shows that gammaproteobacterial mobile class A ß-lactamase is specialized for penicillin degradation, and bla GMA-1 is frequently linked to strand-biased circularizing integrative elements (SEs) in sequences in the RefSeq/GenBank database. Evidence for the implication of SEs in ß-lactamase environmental transmission provides insights for future surveillance studies of antimicrobial resistance genes in human clinical settings.

Exploring the effects of antimicrobial treatment on the gut and oral microbiomes and resistomes from elderly long-term care facility residents via shotgun DNA sequencing.

Monitoring antibiotic-resistant bacteria (ARB) and understanding the effects of antimicrobial drugs on the human microbiome and resistome are crucial for public health. However, no study has investigated the association between antimicrobial treatment and the microbiome-resistome relationship in long-term care facilities, where residents act as reservoirs of ARB but are not included in the national surveillance for ARB. We conducted shotgun metagenome sequencing of oral and stool samples from long-term care facility residents and explored the effects of antimicrobial treatment on the human microbiome and resistome using two types of comparisons: cross-sectional comparisons based on antimicrobial treatment history in the past 6 months and within-subject comparisons between stool samples before, during and 2-4 weeks after treatment using a single antimicrobial drug. Cross-sectional analysis revealed two characteristics in the group with a history of antimicrobial treatment: the archaeon Methanobrevibacter was the only taxon that significantly increased in abundance, and the total abundance of antimicrobial resistance genes (ARGs) was also significantly higher. Within-subject comparisons showed that taxonomic diversity did not decrease during treatment, suggesting that the effect of the prescription of a single antimicrobial drug in usual clinical treatment on the gut microbiota is likely to be smaller than previously thought, even among very elderly people. Additional analysis of the detection limit of ARGs revealed that they could not be detected when contig coverage was <2.0. This study is the first to report the effects of usual antimicrobial treatments on the microbiome and resistome of long-term care facility residents.

Host range diversification within the IncP-1 plasmid group.

Broad-host-range plasmids play a critical role in the spread of antibiotic resistance and other traits. In spite of increasing information about the genomic diversity of closely related plasmids, the relationship between sequence divergence and host range remains unclear. IncP-1 plasmids are currently classified into six subgroups based on the genetic distance of backbone genes. We investigated whether plasmids from two subgroups exhibit a different host range, using two IncP-1γ plasmids, an IncP-1ß plasmid and their minireplicons. Efficiencies of plasmid establishment and maintenance were compared using five species that belong to the Alphaproteobacteria, Betaproteobacteria and Gammaproteobacteria. The IncP-1ß plasmid replicated and persisted in all five hosts in the absence of selection. Of the two IncP-1γ plasmids, both were unable to replicate in alphaproteobacterial host Sphingobium japonicum, and one established itself in Agrobacterium tumefaciens but was very unstable. In contrast, both IncP-1γ minireplicons, which produced higher levels of replication initiation protein than the wild-type plasmids, replicated in all strains, suggesting that poor establishment of the native plasmids is in part due to suboptimal replication initiation gene regulation. The findings suggest that host ranges of distinct IncP-1 plasmids only partially overlap, which may limit plasmid recombination and thus result in further genome divergence.

Diverse broad-host-range plasmids from freshwater carry few accessory genes.

Broad-host-range self-transferable plasmids are known to facilitate bacterial adaptation by spreading genes between phylogenetically distinct hosts. These plasmids typically have a conserved backbone region and a variable accessory region that encodes host-beneficial traits. We do not know, however, how well plasmids that do not encode accessory functions can survive in nature. The goal of this study was to characterize the backbone and accessory gene content of plasmids that were captured from freshwater sources without selecting for a particular phenotype or cultivating their host. To do this, triparental matings were used such that the only required phenotype was the plasmid's ability to mobilize a nonconjugative plasmid. Based on complete genome sequences of 10 plasmids, only 5 carried identifiable accessory gene regions, and none carried antibiotic resistance genes. The plasmids belong to four known incompatibility groups (IncN, IncP-1, IncU, and IncW) and two potentially new groups. Eight of the plasmids were shown to have a broad host range, being able to transfer into alpha-, beta-, and gammaproteobacteria. Because of the absence of antibiotic resistance genes, we resampled one of the sites and compared the proportion of captured plasmids that conferred antibiotic resistance to their hosts with the proportion of such plasmids captured from the effluent of a local wastewater treatment plant. Few of the captured plasmids from either site encoded antibiotic resistance. A high diversity of plasmids that encode no or unknown accessory functions is thus readily found in freshwater habitats. The question remains how the plasmids persist in these microbial communities.

Cointegrate-resolution of toluene-catabolic transposon Tn4651: determination of crossover site and the segment required for full resolution activity.

Tn3-family transposon Tn4651 from Pseudomonas putida mt-2 plasmid pWW0 carries two divergently transcribed genes, tnpS and tnpT, for cointegrate-resolution. While tnpS encodes a tyrosine recombinase, tnpT encodes a protein that shows no homology to any other characterized protein. The Tn4651 resolution site was previously mapped within the 203-bp fragment that covered the tnpS and tnpT promoter region. To better understand the molecular mechanisms underlying the Tn4651 cointegrate-resolution, we determined the extent of the functional resolution site (designated the rst site) of Tn4651 and the location of the crossover site for the cointegrate-resolution. Deletion analysis of the rst region localized the fully functional rst site to a 136-bp segment. The analysis of the site-specific recombination between Tn4651 rst and a rst variant from the Tn4651-related transposon, Tn4661, indicated that the crossover occurs in the 33-bp inverted repeat region, which separates the 136-bp functional rst site into the tnpS- and tnpT-proximal segments. Electrophoretic mobility shift assays demonstrated specific binding of TnpT to the 20-bp inverted repeat region in the tnpT-proximal segment. The requirement for accessory sequences on both sides of the crossover site and the involvement of the unique DNA-binding protein TnpT suggest that the Tn4651-specified resolution system uses a different mechanism than other known resolution systems. Furthermore, comparative sequence analysis for Tn4651-related transposons revealed the occurrence of DNA exchange at the rst site among different transposons, suggesting an additional role of the TnpS-TnpT-rst system in the evolution of Tn4651-related transposons.

Host range of strand-biased circularizing integrative elements: a new class of mobile DNA elements nesting in Gammaproteobacteria.

BACKGROUND: The strand-biased circularizing integrative elements (SEs) are putatively non-mobilizable integrative elements for transmitting antimicrobial resistance genes. The transposition mode and the prevalence of SEs in prokaryotes remain vague. RESULTS: To corroborate the transposition mode and the prevalence of SEs, hypothetical transposition intermediates of an SE were searched for in genomic DNA fractions of an SE host. Then, the SE core genes were defined based on gene knockout experiments, and the synteny blocks of their distant homologs were searched for in the RefSeq complete genome sequence database using PSI-BLAST. A genomic DNA fractionation experiment revealed that SE copies are present in a double-stranded nicked circular form in vivo. Operonic structure of three conserved coding sequences (intA, tfp, intB) and srap located at the left end of SEs were identified as essential for attL × attR recombination. The synteny blocks of tfp and srap homologs were detected in 3.6% of the replicons of Gammaproteobacteria but not in other taxa, implying that SE movement is host-dependent. SEs have been discovered most frequently in the orders Vibrionales (19% of replicons), Pseudomonadales (18%), Alteromonadales (17%), and Aeromonadales (12%). Genomic comparisons revealed 35 new SE members with identifiable termini. SEs are present at 1 to 2 copies per replicon and have a median length of 15.7 kb. Three newly identified SE members carry antimicrobial resistance genes, like tmexCD-toprJ, mcr-9, and blaGMA-1. Further experiments validated that three new SE members possess the strand-biased attL × attR recombination activity. CONCLUSIONS: This study suggested that transposition intermediates of SEs are double-stranded circular DNA. The main hosts of SEs are a subset of free-living Gammaproteobacteria; this represents a rather narrow host range compared to those of mobile DNA element groups discovered to date. As the host range, genetic organization, and movements are unique among the mobile DNA elements, SEs provide a new model system for host-mobile DNA element coevolution studies.

Unique genomic sequences in a novel Mycobacterium avium subsp. hominissuis lineage enable fine scale transmission route tracing during pig movement.

Mycobacterium avium subsp. hominissuis (MAH) is one of the most prevalent mycobacteria causing non-tuberculous mycobacterial disease in humans and animals. Of note, MAH is a major cause of mycobacterial granulomatous mesenteric lymphadenitis outbreaks in pig populations. To determine the precise source of infection of MAH in a pig farm and to clarify the epidemiological relationship among pig, human and environmental MAH lineages, we collected 50 MAH isolates from pigs reared in Japan and determined draft genome sequences of 30 isolates. A variable number of tandem repeat analysis revealed that most pig MAH isolates in Japan were closely related to North American, European and Russian human isolates but not to those from East Asian human and their residential environments. Historical recombination analysis revealed that most pig isolates could be classified into SC2/4 and SC3, which contain MAH isolated from pig, European human and environmental isolates. Half of the isolates in SC2/4 had many recombination events with MAH lineages isolated from humans in East Asia. To our surprise, four isolates belonged to a new lineage (SC5) in the global MAH population. Members of SC5 had few footprints of inter-lineage recombination in the genome, and carried 80 unique genes, most of which were located on lineage specific-genomic islands. Using unique genetic features, we were able to trace the putative transmission route via their host pigs. Together, we clarify the possibility of species-specificity of MAH in addition to local adaptation. Our results highlight two transmission routes of MAH, one exposure on pig farms from the environment and the other via pig movement. Moreover, our study also warns that the evolution of MAH in pigs is influenced by MAH from patients and their residential environments, even if the MAH are genetically distinct.

Roles of long and short replication initiation proteins in the fate of IncP-1 plasmids.

Broad-host-range IncP-1 plasmids generally encode two replication initiation proteins, TrfA1 and TrfA2. TrfA2 is produced from an internal translational start site within trfA1. While TrfA1 was previously shown to be essential for replication in Pseudomonas aeruginosa, its role in other bacteria within its broad host range has not been established. To address the role of TrfA1 and TrfA2 in other hosts, efficiency of transformation, plasmid copy number (PCN), and plasmid stability were first compared between a mini-IncP-1ß plasmid and its trfA1 frameshift variant in four phylogenetically distant hosts: Escherichia coli, Pseudomonas putida, Sphingobium japonicum, and Cupriavidus necator. TrfA2 was sufficient for replication in these hosts, but the presence of TrfA1 enhanced transformation efficiency and PCN. However, TrfA1 did not contribute to, and even negatively affected, long-term plasmid persistence. When trfA genes were cloned under a constitutive promoter in the chromosomes of the four hosts, strains expressing either both TrfA1 and TrfA2 or TrfA1 alone, again, generally elicited a higher PCN of an IncP1-ß replicon than strains expressing TrfA2 alone. When a single species of TrfA was produced at different concentrations in E. coli cells, TrfA1 maintained a 3- to 4-fold higher PCN than TrfA2 at the same TrfA concentrations, indicating that replication mediated by TrfA1 is more efficient than that by TrfA2. These results suggest that the broad-host-range properties of IncP-1 plasmids are essentially conferred by TrfA2 and the intact replication origin alone but that TrfA1 is nonetheless important to efficiently establish plasmid replication upon transfer into a broad range of hosts.

Atypical integrative element with strand-biased circularization activity assists interspecies antimicrobial resistance gene transfer from Vibrio alfacsensis.

The exchange of antimicrobial resistance (AMR) genes between aquaculture and terrestrial microbial populations has emerged as a serious public health concern. However, the nature of the mobile genetic elements in marine bacteria is poorly documented. To gain insight into the genetic mechanisms underlying AMR gene transfer from marine bacteria, we mated a multidrug-resistant Vibrio alfacsensis strain with an Escherichia coli strain, and then determined the complete genome sequences of the donor and the transconjugant strains. Sequence analysis revealed a conjugative multidrug resistance plasmid in the donor strain, which was integrated into the chromosome of the recipient. The plasmid backbone in the transconjugant chromosome was flanked by two copies of a 7.1 kb unclassifiable integrative element harboring a ß-lactamase gene. The 7.1 kb element and the previously reported element Tn6283 share four coding sequences, two of which encode the catalytic R-H-R-Y motif of tyrosine recombinases. Polymerase chain reaction and sequencing experiments revealed that these elements generate a circular copy of one specific strand without leaving an empty site on the donor molecule, in contrast to the movement of integron gene cassettes or ICE/IMEs discovered to date. These elements are termed SEs (strand-biased circularizing integrative elements): SE-6945 (the 7.1 kb element) and SE-6283 (Tn6283). The copy number and location of SE-6945 in the chromosome affected the antibiotic resistance levels of the transconjugants. SEs were identified in the genomes of other Vibrio species. Overall, these results suggest that SEs are involved in the spread of AMR genes among marine bacteria.

Genomic features of Mycobacterium avium subsp. hominissuis isolated from pigs in Japan.

Mycobacterium avium subsp. hominissuis (MAH) is one of the most important agents causing non-tuberculosis mycobacterial infection in humans and pigs. There have been advances in genome analysis of MAH from human isolates, but studies of isolates from pigs are limited despite its potential source of infection to human. Here, we obtained 30 draft genome sequences of MAH from pigs reared in Japan. The 30 draft genomes were 4,848,678-5,620,788 bp in length, comprising 4652-5388 coding genes and 46-75 (median: 47) tRNAs. All isolates had restriction modification-associated genes and 185-222 predicted virulence genes. Two isolates had tRNA arrays and one isolate had a clustered regularly interspaced short palindromic repeat (CRISPR) region. Our results will be useful for evaluation of the ecology of MAH by providing a foundation for genome-based epidemiological studies.

Genome-wide association study of gastric cancer- and duodenal ulcer-derived Helicobacter pylori strains reveals discriminatory genetic variations and novel oncoprotein candidates.

Genome-wide association studies (GWASs) can reveal genetic variations associated with a phenotype in the absence of any hypothesis of candidate genes. The problem of false-positive sites linked with the responsible site might be bypassed in bacteria with a high homologous recombination rate, such as Helicobacter pylori, which causes gastric cancer. We conducted a small-sample GWAS (125 gastric cancer cases and 115 controls) followed by prediction of gastric cancer and control (duodenal ulcer) H. pylori strains. We identified 11 single nucleotide polymorphisms (eight amino acid changes) and three DNA motifs that, combined, allowed effective disease discrimination. They were often informative of the underlying molecular mechanisms, such as electric charge alteration at the ligand-binding pocket, alteration in subunit interaction, and mode-switching of DNA methylation. We also identified three novel virulence factors/oncoprotein candidates. These results provide both defined targets for further informatic and experimental analyses to gain insights into gastric cancer pathogenesis and a basis for identifying a set of biomarkers for distinguishing these H. pylori-related diseases.

Predicting plasmid promiscuity based on genomic signature.

Despite the important contribution of self-transmissible plasmids to bacterial evolution, little is understood about the range of hosts in which these plasmids have evolved. Our goal was to infer this so-called evolutionary host range. The nucleotide composition, or genomic signature, of plasmids is often similar to that of the chromosome of their current host, suggesting that plasmids acquire their hosts' signature over time. Therefore, we examined whether the evolutionary host range of plasmids could be inferred by comparing their trinucleotide composition to that of all completely sequenced bacterial chromosomes. The diversity of candidate hosts was determined using taxonomic classification and genetic distance. The method was first tested using plasmids from six incompatibility (Inc) groups whose host ranges are generally thought to be narrow (IncF, IncH, and IncI) or broad (IncN, IncP, and IncW) and then applied to other plasmid groups. The evolutionary host range was found to be broad for IncP plasmids, narrow for IncF and IncI plasmids, and intermediate for IncH and IncN plasmids, which corresponds with their known host range. The IncW plasmids as well as several plasmids from the IncA/C, IncP, IncQ, IncU, and PromA groups have signatures that were not similar to any of the chromosomal signatures, raising the hypothesis that these plasmids have not been ameliorated in any host due to their promiscuous nature. The inferred evolutionary host range of IncA/C, IncP-9, and IncL/M plasmids requires further investigation. In this era of high-throughput sequencing, this genomic signature method is a useful tool for predicting the host range of novel mobile elements.