Escherichia cryptic clade I is an emerging source of human intestinal pathogens.

BACKGROUND: Within the genus Escherichia, several monophyletic clades other than the traditionally defined species have been identified. Of these, cryptic clade I (C-I) appears to represent a subspecies of E. coli, but due to the difficulty in distinguishing it from E. coli sensu stricto, the population structure and virulence potential of C-I are unclear. RESULTS: We defined a set of true C-I strains (n = 465), including a Shiga toxin 2a (Stx2a)-producing isolate from a patient with bloody diarrhoea identified by the retrospective analyses using a C-I-specific detection system. Through genomic analysis of 804 isolates from the cryptic clades, including these C-I strains, we revealed their global population structures and the marked accumulation of virulence genes and antimicrobial resistance genes in C-I. In particular, half of the C-I strains contained hallmark virulence genes of Stx-producing E. coli (STEC) and/or enterotoxigenic E. coli (ETEC). We also found the host-specific distributions of virulence genes, which suggests bovines as the potential source of human infections caused by STEC- and STEC/ETEC hybrid-type C-I strains, as is known in STEC. CONCLUSIONS: Our findings demonstrate the emergence of human intestinal pathogens in C-I lineage. To better understand the features of C-I strains and their infections, extensive surveillance and larger population studies of C-I strains are needed. The C-I-specific detection system developed in this study will be a powerful tool for screening and identifying C-I strains.

Prophages integrating into prophages: A mechanism to accumulate type III secretion effector genes and duplicate Shiga toxin-encoding prophages in Escherichia coli.

Bacteriophages (or phages) play major roles in the evolution of bacterial pathogens via horizontal gene transfer. Multiple phages are often integrated in a host chromosome as prophages, not only carrying various novel virulence-related genetic determinants into host bacteria but also providing various possibilities for prophage-prophage interactions in bacterial cells. In particular, Escherichia coli strains such as Shiga toxin (Stx)-producing E. coli (STEC) and enteropathogenic E. coli (EPEC) strains have acquired more than 10 prophages (up to 21 prophages), many of which encode type III secretion system (T3SS) effector gene clusters. In these strains, some prophages are present at a single locus in tandem, which is usually interpreted as the integration of phages that use the same attachment (att) sequence. Here, we present phages integrating into T3SS effector gene cluster-associated loci in prophages, which are widely distributed in STEC and EPEC. Some of the phages integrated into prophages are Stx-encoding phages (Stx phages) and have induced the duplication of Stx phages in a single cell. The identified attB sequences in prophage genomes are apparently derived from host chromosomes. In addition, two or three different attB sequences are present in some prophages, which results in the generation of prophage clusters in various complex configurations. These phages integrating into prophages represent a medically and biologically important type of inter-phage interaction that promotes the accumulation of T3SS effector genes in STEC and EPEC, the duplication of Stx phages in STEC, and the conversion of EPEC to STEC and that may be distributed in other types of E. coli strains as well as other prophage-rich bacterial species.

Phylogenic position and low genomic diversity of "Candidatus Rickettsia kotlanii" inferred by complete genome sequences of two Japanese isolates.

Many Rickettsia species of the spotted fever group (SFG) cause tick-borne diseases known as "spotted fever." One of the candidate SFG Rickettsia species is "Candidatus Rickettsia kotlanii," which was first detected in Haemaphysalis concinna in Hungary in 2006. However, its precise phylogenetic position in the SFG is not clear because only single-gene sequence-based phylogenetic analyses were performed using very limited genes. Here, we present the complete genome sequences of two Japanese "Ca. R. kotlanii" isolates, which differed only by a 135 bp insertion/deletion (InDel). Using these genomes and publicly available whole genome sequences of other Rickettsia species, the precise phylogenetic position of "Ca. R. kotlanii" in Rickettsia was determined to be in a clade of the SFG. The phylogenetic relationships and average nucleotide identity of "Ca. R. kotlanii" relative to the other species indicated that "Ca. R. kotlanii" is an independent taxon in the SFG. Notably, although the genomes of the two isolates were almost identical, the isolates were obtained from different tick species in different regions and years, suggesting extremely low genomic diversity in "Ca. R. kotlanii." While the genome of "Ca. R. kotlanii" is the smallest in the transitional group and SFG Rickettsia sequenced to date, we identified genes uniquely present or absent in "Ca. R. kotlanii," but most were apparently degraded. Therefore, analyses of differences at the sequence (single nucleotide polymorphisms and small InDels) or gene expression level will be required to understand the functional or physiological features unique to "Ca. R. kotlanii."

Genomic and phylogenetic characterization of Elizabethkingia anophelis strains: The first two cases of life-threatening infection in Japan.

OBJECTIVE: Elizabethkingia anophelis causes meningitis, bloodstream infections, and respiratory infections in immunocompromised individuals. We examined two E. anophelis strains isolated from the first life-threatening cases caused by this species in Japan to determine the phylogenetic origin and genomic features of them. METHODS: We performed whole genome-based analysis to clarify the genetic relationship for the two strains (EK0004 and EK0079) and Elizabethkingia sp. strains isolated from worldwide and to characterize the genomic features such as the prevalence of virulence- and antimicrobial resistance (AMR)-related genes. PATIENTS: A 29-year-old man with hepatosplenic T-cell lymphoma and a 52-year-old man with systemic lupus erythematosus developed fatal bacteremia and meningitis due to E. anophelis, respectively. RESULTS: Two strains, EK0004 and EK0079, were genetically different but most closely related to the strains isolated from the largest outbreak in Wisconsin, USA from 2015 to 2016, and the strain isolated from cerebrospinal fluid of a patient in Florida, USA in 1982, respectively. The two strains contained AMR-related genes such as those encoding for an extended-spectrum ß-lactamase and multiple metallo-ß-lactamases and several virulence-related genes such as capsular polysaccharide synthesis gene clusters. CONCLUSIONS: Although further functional analyses are required to understand the virulence of these clones, these finding suggests that enough caution of E. anophelis infection in immunocompromised patients is required since the number of infections by this species is increasing outside Japan.

MetaPlatanus: a metagenome assembler that combines long-range sequence links and species-specific features.

De novo metagenome assembly is effective in assembling multiple draft genomes, including those of uncultured organisms. However, heterogeneity in the metagenome hinders assembly and introduces interspecies misassembly deleterious for downstream analysis. For this purpose, we developed a hybrid metagenome assembler, MetaPlatanus. First, as a characteristic function, it assembles the basic contigs from accurate short reads and then iteratively utilizes long-range sequence links, species-specific sequence compositions, and coverage depth. The binning information was also used to improve contiguity. Benchmarking using mock datasets consisting of known bacteria with long reads or mate pairs revealed the high contiguity MetaPlatanus with a few interspecies misassemblies. For published human gut data with nanopore reads from potable sequencers, MetaPlatanus assembled many biologically important elements, such as coding genes, gene clusters, viral sequences, and over-half bacterial genomes. In the benchmark with published human saliva data with high-throughput nanopore reads, the superiority of MetaPlatanus was considerably more evident. We found that some high-abundance bacterial genomes were assembled only by MetaPlatanus as near-complete. Furthermore, MetaPlatanus can circumvent the limitations of highly fragmented assemblies and frequent interspecies misassembles obtained by the other tools. Overall, the study demonstrates that MetaPlatanus could be an effective approach for exploring large-scale structures in metagenomes.

Large-scale genome analysis of bovine commensal Escherichia coli reveals that bovine-adapted E. coli lineages are serving as evolutionary sources of the emergence of human intestinal pathogenic strains.

How pathogens evolve their virulence to humans in nature is a scientific issue of great medical and biological importance. Shiga toxin (Stx)-producing Escherichia coli (STEC) and enteropathogenic E. coli (EPEC) are the major foodborne pathogens that can cause hemolytic uremic syndrome and infantile diarrhea, respectively. The locus of enterocyte effacement (LEE)-encoded type 3 secretion system (T3SS) is the major virulence determinant of EPEC and is also possessed by major STEC lineages. Cattle are thought to be the primary reservoir of STEC and EPEC. However, genome sequences of bovine commensal E. coli are limited, and the emerging process of STEC and EPEC is largely unknown. Here, we performed a large-scale genomic comparison of bovine commensal E. coli with human commensal and clinical strains, including EPEC and STEC, at a global level. The analyses identified two distinct lineages, in which bovine and human commensal strains are enriched, respectively, and revealed that STEC and EPEC strains have emerged in multiple sublineages of the bovine-associated lineage. In addition to the bovine-associated lineage-specific genes, including fimbriae, capsule, and nutrition utilization genes, specific virulence gene communities have been accumulated in stx- and LEE-positive strains, respectively, with notable overlaps of community members. Functional associations of these genes probably confer benefits to these E. coli strains in inhabiting and/or adapting to the bovine intestinal environment and drive their evolution to highly virulent human pathogens under the bovine-adapted genetic background. Our data highlight the importance of large-scale genome sequencing of animal strains in the studies of zoonotic pathogens.

Insertion Sequence (IS)-Excision Enhancer (IEE)-Mediated IS Excision from the lacZ Gene Restores the Lactose Utilization Defect of Shiga Toxin-Producing Escherichia coli O121:H19 Strains and Is Responsible for Their Delayed Lactose Utilization Phenotype.

Lactose utilization is one of the general biochemical characteristics of Escherichia coli, and the lac operon is responsible for this phenotype, which can be detected on lactose-containing media, such as MacConkey agar, after 24 h of incubation. However, some Shiga toxin-producing E. coli (STEC) O121:H19 strains exhibit an unusual phenotype called delayed lactose utilization (DLU), in which lactose utilization can be detected after 48 h of cultivation but not after only 24 h of cultivation. Insertion of an insertion sequence (IS), IS600, into the lacZ gene appears to be responsible for the DLU phenotype, and exposure to lactose has been reported to be necessary to observe this phenotype, but the mechanism underlying these phenomena remains to be elucidated. Here, we performed detailed analyses of the lactose utilization abilities of a set of O121:H19 strains and their mutants and found that IS-excision enhancer (IEE)-mediated excision of IS600 reactivates the lacZ gene and that the selective proliferation of IS-cured subclones in lactose-supplemented culture medium is responsible for the expression of the DLU phenotype. In addition, we analyzed the patterns of IS insertion into the lacZ and iee genes in the global O121:H19 population and revealed that while there are O121:H19 strains or lineage/sublineages that contain the IS insertion into iee or intact lacZ and thus do not show the DLU phenotype, most currently circulating O121:H19 strains contain IS600-inserted lacZ and intact iee and thus exhibit this phenotype. IMPORTANCE Insertion sequences (ISs) can modulate gene expression by gene inactivation or activation. While phenotypic changes due to IS insertion/transposition are frequently observed, gene reactivation by precise or simple IS excision rarely occurs. In this study, we show that IS600 is excised from the lacZ gene by IS-excision enhancer (IEE) during the cultivation of Shiga toxin-producing Escherichia coli (STEC) O121:H19 strains that show an unusual phenotype called delayed lactose utilization (DLU). This excision rescued their lactose utilization defect, and the subsequent selective proliferation of IS-cured subclones in lactose-containing medium resulted in the expression of the DLU phenotype. As we also show that most currently circulating O121:H19 strains exhibit this phenotype, this study not only provides information helpful for the isolation and identification of O121:H19 STEC but also offers novel insights into the roles of IS and IEE in the generation of phenotypic variation in bacterial populations.

Within-host evolution of a Klebsiella pneumoniae clone: selected mutations associated with the alteration of outer membrane protein expression conferred multidrug resistance.

BACKGROUND: A patient repeatedly developed bacteraemia despite the continuous use of antibiotics. We obtained two Klebsiella pneumoniae isolates from the patient's blood on Days 72 and 105 after hospitalization. Each of the two isolates belonged to ST45, but while the first isolate was susceptible to most antibiotics, the second one was resistant to multiple drugs including carbapenems. OBJECTIVES: To identify the genetic differences between the two isolates and uncover alterations formed by the within-host bacterial evolution leading to the antimicrobial resistance. METHODS: Whole-genome comparison of the two isolates was carried out to identify their genetic differences. We then profiled their outer membrane proteins related to membrane permeability to drugs. To characterize a ramR gene mutation found in the MDR isolate, its WT and mutant genes were cloned and expressed in the MDR isolate. RESULTS: The two isolates showed only three genomic differences, located in mdoH, ramR and upstream of ompK36. In the MDR isolate, a single nucleotide substitution in the ompK36 upstream region attenuated OmpK36 expression. A single amino acid residue insertion in RamR in the MDR isolate impaired its function, leading to the down-regulation of OmpK35 and the subsequent up-regulation of the AcrAB-TolC transporter, which may contribute to the MDR. CONCLUSIONS: We identified very limited genomic changes in the second K. pneumoniae clone during within-host evolution, but two of the three identified mutations conferred the MDR phenotype on the clone by modulating drug permeability.

"Candidatus Mesenet longicola": Novel Endosymbionts of Brontispa longissima that Induce Cytoplasmic Incompatibility.

Intracellular bacteria that are mainly transmitted maternally affect their arthropod hosts' biology in various ways. One such effect is known as cytoplasmic incompatibility (CI), and three bacterial species are known to induce CI: Wolbachia, Cardinium hertigii, and a recently found alphaproteobacterial symbiont. To clarify the taxonomic status and provide the foundation for future studies to reveal CI mechanisms and other phenotypes, we investigated genetic and morphological properties of the third CI inducer that we have previously reported inducing CI in the coconut beetle Brontispa longissima. The draft genome of the bacteria was obtained from the oocytes of two isofemale lines of B. longissima infected with the bacteria: one from Japan (GL2) and the other from Vietnam (L5). Genome features of the symbionts (sGL2 and sL5) were highly similar, showing 1.3 Mb in size, 32.1% GC content, and 99.83% average nucleotide sequence. A phylogenetic study based on 43 universal and single-copy phylogenetic marker genes indicates that they formed a distinct clade in the family Anaplasmataceae. 16S rRNA gene sequences indicate that they are different from the closest known relatives, at least at the genus level. Therefore, we propose a new genus and species, "Candidatus Mesenet longicola", for the symbionts of B. longissima. Morphological analyses showed that Ca. M. longicola is an intracellular bacterium that is ellipsoidal to rod-shaped and 0.94 ± 0.26 µm (mean ± SD) in length, and accumulated in the anterior part of the oocyte. Candidates for the Ca. M. longicola genes responsible for CI induction are also described.

16S rRNA-based amplicon analysis of changes in the bacterial population in the lesions of papillomatous digital dermatitis in dairy cattle after topical treatment with allyl isothiocyanate.

Papillomatous digital dermatitis (PDD) is a foot disease causing lameness in dairy cattle. It is regarded as a polymicrobial infection, although its etiology is not fully understood. PDD is treated by the topical or systemic administration of antibiotics such as lincomycin (LCM); however, the milk of the cows cannot be marketed during the treatment and withdrawal period due to the residual antibiotics in milk. Allyl isothiocyanate (AITC), an extract of Wasabia japonica (known as wasabi or Japanese horseradish) widely employed as a food additive, can be used as an alternative antimicrobial agent that overcomes this problem. We previously showed that AITC is as effective as LCM in PDD treatment. Here, using the samples obtained in the previous clinical study, we analyzed changes in the bacterial population in the PDD-associated microbiota after AITC treatment and compared those with that following LCM treatment by 16S ribosomal RNA (rRNA)-based amplicon analysis. Both treatments induced major changes in the bacterial population, and Treponema species, which have been regarded as the major causative agents of PDD, were efficiently eliminated by both agents. However, the AITC-treated samples exhibited higher diversity compared with pretreatment samples, but this trend was not observed for LCM treatment, probably reflecting different antibacterial activities of the two agents. Importantly, this analysis detected population changes before morphological changes in PDD lesions (clinical signs of healing) became evident, indicating that 16S rRNA-based amplicon analysis represents an efficient strategy for analyzing and monitoring the treatment efficiency of PDD as well as other polymicrobial diseases.

Genomic Characterization of ß-Glucuronidase-Positive Escherichia coli O157:H7 Producing Stx2a.

Among Shiga toxin (Stx)-producing Escherichia coli (STEC) O157:H7 strains, those producing Stx2a cause more severe diseases. Atypical STEC O157:H7 strains showing a ß-glucuronidase-positive phenotype (GP STEC O157:H7) have rarely been isolated from humans, mostly from persons with asymptomatic or mild infections; Stx2a-producing strains have not been reported. We isolated, from a patient with bloody diarrhea, a GP STEC O157:H7 strain (PV15-279) that produces Stx2a in addition to Stx1a and Stx2c. Genomic comparison with other STEC O157 strains revealed that PV15-279 recently emerged from the stx1a/stx2c-positive GP STEC O157:H7 clone circulating in Japan. Major virulence genes are shared between typical (ß-glucuronidase-negative) and GP STEC O157:H7 strains, and the Stx2-producing ability of PV15-279 is comparable to that of typical STEC O157:H7 strains; therefore, PV15-279 presents a virulence potential similar to that of typical STEC O157:H7. This study reveals the importance of GP O157:H7 as a source of highly pathogenic STEC clones.

Emergence of a Multidrug-Resistant Shiga Toxin-Producing Enterotoxigenic Escherichia coli Lineage in Diseased Swine in Japan.

EnterotoxigenicEscherichia coli(ETEC) and Shiga toxin-producingE. coli(STEC) are important causes of diarrhea and edema disease in swine. The majority of swine-pathogenicE. colistrains belong to a limited range of O serogroups, including O8, O138, O139, O141, O147, O149, and O157, which are the most frequently reported strains worldwide. However, the circumstances of ETEC and STEC infections in Japan remain unknown; there have been few reports on the prevalence or characterization of swine-pathogenicE. coli In the present study, we determined the O serogroups of 967E. coliisolates collected between 1991 and 2014 from diseased swine in Japan, and we found that O139, O149, O116, and OSB9 (O serogroup ofShigella boydiitype 9) were the predominant serogroups. We further analyzed these four O serogroups using pulsed-field gel electrophoresis (PFGE), multilocus sequence typing, and virulence factor profiling. Most of the O139 and O149 strains formed serogroup-specific PFGE clusters (clusters I and II, respectively), whereas the O116 and OSB9 strains were grouped together in the same cluster (cluster III). All of the cluster III strains belonged to a single sequence type (ST88) and carried genes encoding both enterotoxin and Shiga toxin. This PFGE cluster III/ST88 lineage exhibited a high level of multidrug resistance (to a median of 10 antimicrobials). Notably, these bacteria were resistant to fluoroquinolones. Thus, this lineage should be considered a significant risk to animal production due to the toxigenicity and antimicrobial resistance of these bacteria.

Genetic relatedness and virulence properties of enteropathogenic Escherichia coli strains of serotype O119:H6 expressing localized adherence or localized and aggregative adherence-like patterns on HeLa cells.

Enteropathogenic Escherichia coli (EPEC) induce attaching and effacing (A/E) lesions in enterocytes and produce the bundle-forming pilus (BFP) contributing to the localized adherence (LA) pattern formation on HeLa cells. Enteroaggregative E. coli (EAEC) produce aggregative adherence (AA) on HeLa cells and form prominent biofilms. The ability to produce LA or AA is an important hallmark to classify fecal E. coli isolates as EPEC or EAEC, respectively. E. coli strains of serotype O119:H6 exhibit an LA+ phenotype and have been considered as comprising a clonal group of EPEC strains. However, we have recently identified O119:H6 EPEC strains that produce LA and an AA-like pattern concurrently (LA/AA-like+). In this study, we evaluated the relatedness of three LA/AA-like+ and three LA+ O119:H6 strains by comparing their virulence and genotypic properties. We first found that the LA/AA-like+ strains induced actin accumulation in HeLa cells (indicative of A/E lesions formation) and formed biofilms on abiotic surfaces more efficiently than the LA+ strains. MLST analysis showed that the six strains all belong to the ST28 complex. All strains carried multiple plasmids, but as plasmid profiles were highly variable, this cannot be used to differentiate LA/AA-like+ and LA+ strains. We further obtained their draft genome sequences and the complete sequences of four plasmids harbored by one LA/AA-like+ strain. Analysis of these sequences and comparison with 37 fully sequenced E. coli genomes revealed that both O119:H6 groups belong to the E. coli phylogroup B2 and are very closely related with only 58-67 SNPs found between LA/AA-like+ and LA+ strains. Search of the draft sequences of the six strains for adhesion-related genes known in EAEC and other E. coli pathotypes detected no genes specifically present in LA/AA-like+ strains. Unexpectedly however, we found that a large plasmid distinct from pEAF is responsible for the AA-like phenotype of the LA/AA-like+ strains. Although we have not identified any plasmid genes specifically present in all LA/AA-like+ strains and absent in the LA+ strains, these results suggest the presence of an unknown mechanism to promote the AA-like pattern production and biofilm formation by the LA/AA-like+ strains. Because their ability to produce A/E lesions and biofilm concomitantly could exacerbate the clinical condition of the patient and lead to persistent diarrhea, the mechanism underlying the enhanced biofilm formation by the LA/AA-like+ O119:H6 strains and their spread and involvement in severe diarrheal diseases should be more intensively investigated.

A comprehensive list of genes required for the efficient conjugation of plasmid Rts1 was determined by systematic deletion analysis.

While conjugation-related genes have been identified in many plasmids by genome sequencing, functional analyses have not yet been performed in most cases, and a full set of conjugation genes has been identified for only a few plasmids. Rts1, a prototype IncT plasmid, is a conjugative plasmid that was originally isolated from Proteus vulgaris. Here, we conducted a systematic deletion analysis of Rts1 to fully understand its conjugation system. Through this analysis along with complementation assays, we identified 32 genes that are required for the efficient conjugation of Rts1 from Escherichia coli to E. coli. In addition, the functions of the 28 genes were determined or predicted; 21 were involved in mating-pair formation, three were involved in DNA transfer and replication, including a relaxase gene belonging to the MOBH12 family, one was involved in coupling, and three were involved in transcriptional regulation. Among the functionally well-analysed conjugation systems, most of the 28 genes showed the highest similarity to those of the SXT element, which is an integrative conjugative element of Vibrio cholerae. The Rts1 conjugation gene set included all 23 genes required for the SXT system. Two groups of plasmids with conjugation systems nearly identical or very similar to that of Rts1 were also identified.

Mobile genetic element-driven genomic changes in a community-associated methicillin-resistant Staphylococcus aureus clone during its transmission in a regional community outbreak in Japan.

Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) infections are now a public health concern in both community and healthcare settings worldwide. We previously identified a suspected case of a maternity clinic-centred outbreak of CA-MRSA skin infection in a regional community in Japan by PFGE-based analysis. In this study, we performed genome sequence-based analyses of 151 CA-MRSA isolates, which included not only outbreak-related isolates that we previously defined based on identical or similar PFGE patterns but also other isolates obtained during the same period in the same region. Our analysis accurately defined 133 isolates as outbreak-related isolates, collectively called the TDC clone. They belonged to a CA-MRSA lineage in clonal complex (CC) 30, known as the South West Pacific (SWP) clone. A high-resolution phylogenetic analysis of these isolates combined with their epidemiological data revealed that the TDC clone was already present and circulating in the region before the outbreak was recognized, and only the isolates belonging to two sublineages (named SL4 and SL5) were directly involved in the outbreak. Long persistence in patients/carriers and frequent intrahousehold transmission of the TDC clone were also revealed by this analysis. Moreover, by systematic analyses of the genome changes that occurred in this CA-MRSA clone during transmission in the community, we revealed that most variations were associated with mobile genetic elements (MGEs). Variant PFGE types were generated by alterations of prophages and genomic islands or insertion sequence (IS)-mediated insertion of a plasmid or a sequence of unknown origin. Dynamic changes in plasmid content, which were linked to changes in antimicrobial resistance profiles in specific isolates, were generated by frequent gain and loss of plasmids, most of which were self-transmissible or mobilizable. The introduction of IS256 by a plasmid (named pTDC02) into sublineage SL5 led to SL5-specific amplification of IS256, and amplified IS256 copies were involved in some of the structural changes of chromosomes and plasmids and generated variations in the repertoire of virulence-related genes in limited isolates. These data revealed how CA-MRSA genomes change during transmission in the community and how MGEs are involved in this process.

Complete genome sequences of 11 Streptococcus pneumoniae strains isolated from acute infectious purpura fulminans, sepsis, and pneumonia.

The complete genome sequences of 11 Japanese Streptococcus pneumoniae isolates were determined by hybrid assembly of long and short reads, including two strains isolated from patients with acute infectious purpura fulminans, six strains from patients with sepsis, and three strains from patients with pneumonia.

Generation and maintenance of the circularized multimeric IS26-associated translocatable unit encoding multidrug resistance.

In gram-negative bacteria, IS26 often exists in multidrug resistance (MDR) regions, forming a pseudocompound transposon (PCTn) that can be tandemly amplified. It also generates a circular intermediate called the "translocatable unit (TU)", but the TU has been detected only by PCR. Here, we demonstrate that in a Klebsiella pneumoniae MDR clone, mono- and multimeric forms of the TU were generated from the PCTn in a preexisting MDR plasmid where the inserted form of the TU was also tandemly amplified. The two modes of amplification were reproduced by culturing the original clone under antimicrobial selection pressure, and the amplified state was maintained in the absence of antibiotics. Mono- and multimeric forms of the circularized TU were generated in a RecA-dependent manner from the tandemly amplified TU, which can be generated in RecA-dependent and independent manners. These findings provide novel insights into the dynamic processes of genome amplification in bacteria.

Diversity of Shiga toxin transducing phages in Escherichia coli O145:H28 and the different Shiga toxin 2 production levels associated with short- or long-tailed phages.

Shiga toxin (Stx)-producing Escherichia coli (STEC) causes serious gastrointestinal illness, including hemorrhagic colitis and hemolytic uremic syndrome. Two types of Stxs (Stx1 and Stx2) are known and both are encoded by bacteriophages (Stx phages), but the production of Stx2 is known to be a major risk factor for severe STEC infections. The production of Stx2, but not Stx1, is tightly coupled with the induction of Stx phages, and Stx2 production levels vary between STEC strains even within the same serotype. Here, we analyzed the genomic diversity of all Stx phages in 71 strains representing the entire O145:H28 lineage, one of the often highly pathogenic STECs, and the relationship between the variations in Stx phage genomes and the levels of Stx2 production by host strains. Our analysis reveals highly dynamic natures of Stx phages in O145:H28, including the independent acquisition of similar Stx phages by different sublineages, the recent transfer of Stx phage between different sublineages, and the frequent gain and loss of Stx phages in some sublineages. We also show the association of the Stx2 phage types with the Stx2 production levels of host strains: strains carrying short-tailed Stx2 phages exhibited significantly higher Stx2 production levels than those carrying long-tailed Stx2 phages. Detailed analyses of the Stx2 phage genomes revealed that both of short- and long-tailed phages exhibited sequence diversification and they were divided into two groups, respectively, based on the sequence similarity of the phage early region encoding genes responsible for phage induction, short-tailed phages contained early regions clearly different in genetic organization from those in long-tailed phages. Therefore, the variations in the early regions between short-and long-tailed Stx2 phages appeared to be linked to a striking difference in Stx2 production levels in their host strains. These results broaden our understanding of the diversification and dynamism of Stx phages in O145:H28 and the association of Stx2 phage types with the Stx2 production level in this STEC lineage.

Dynamic changes in Shiga toxin (Stx) 1 transducing phage throughout the evolution of O26:H11 Stx-producing Escherichia coli.

Shiga toxin (Stx) is the key virulence factor of Stx-producing Escherichia coli (STEC). All known Stxs (Stx1 and Stx2) are encoded by bacteriophages (Stx phages). Although the genetic diversity of Stx phages has frequently been described, systematic analyses of Stx phages in a single STEC lineage are limited. In this study, focusing on the O26:H11 STEC sequence type 21 (ST21) lineage, where the stx1a gene is highly conserved, we analysed the Stx1a phages in 39 strains representative of the entire ST21 lineage and found a high level of variation in Stx1a phage genomes caused by various mechanisms, including replacement by a different Stx1a phage at the same or different locus. The evolutionary timescale of events changing Stx1a phages in ST21 was also determined. Furthermore, by using an Stx1 quantification system developed in this study, we found notable variations in the efficiency of Stx1 production upon prophage induction, which sharply contrasted with the conserved iron regulated Stx1 production. These variations were associated with the Stx1a phage alteration in some cases but not in other cases; thus, Stx1 production in this STEC lineage was determined by differences not only in Stx1 phages but also in host-encoded factors.

Isolation and Genomic Characterization of a Heat-Labile Enterotoxin 1-Producing Escherichia fergusonii Strain from a Human.

Escherichia fergusonii strains have been isolated from patients with diarrhea, but their virulence determinant has not been well elucidated. Here, we report the first isolation of a heat-labile enterotoxin 1 (LT1)-producing E. fergusonii strain (strain 30038) from a patient in Japan. The complete genome sequence of strain 30038 was determined and subjected to comparative genomics and phylogenetic analyses with 195 publicly available genomes of E. fergusonii. In addition to strain 30038, the elt1 gene was also identified in an E. fergusonii strain that is phylogenetically distinct and which was isolated from poultry in the United Kingdom. Fine genomic comparison revealed that these two strains share comparable elt1-bearing plasmids. However, an intriguing distinction arises in strain 30038, wherein the plasmid has integrated into the chromosome via a recombination process mediated by an insertion sequence. The production of active LT1 toxin by strain 30038 was verified through an in vitro assay using cultured cells. A large plasmid carrying 11 antimicrobial resistance genes was also identified in strain 30038. Our results indicate that extensive surveillance of elt1-positive E. fergusonii strains as diarrheagenic pathogens is needed. IMPORTANCE Escherichia fergusonii, a species closely related to Escherichia coli, is known to cause sporadic conditions in humans, including diarrhea. However, the critical virulence factors in E. fergusonii clinical isolates remain to be identified. This study shows the first isolation of an E. fergusonii strain carrying the elt1 gene, which encodes heat-labile enterotoxin 1, from a patient with diarrhea. Our analysis of public databases also revealed the presence of elt1-positive E. fergusonii strains isolated from poultry in the United Kingdom. Interestingly, while the elt1 gene in the poultry isolate was present on a large plasmid, in the human isolate it was integrated into the chromosome, which may confer stability on the elt1-carrying genetic element. Our findings highlight the need for extensive surveillance of elt1-positive E. fergusonii strains in livestock animals.