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.

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.

Proposal of a novel selective enrichment broth, NCT-mTSB, for isolation of Escherichia albertii from poultry samples.

AIMS: Escherichia albertii is an emerging diarrheagenic pathogen causing food- and water-borne infection in humans. However, no selective enrichment broths for E. albertii have ever been reported. In this study, we tested several basal media, selective supplements and culture conditions which enabled selective enrichment of E. albertii. METHODS AND RESULTS: We developed a selective enrichment broth, novobiocin-cefixime-tellurite supplemented modified tryptic soy broth (NCT-mTSB). NCT-mTSB supported the growth of 22 E. albertii strains, while inhibited growth of other Enterobacteriaceae at 37°C, except for Escherichia coli and Shigella spp. Enrichment of E. albertii was improved further by growth at 44°C, a temperature that suppresses growth of several strains of E. coli/Shigella. Combined use of NCT-mTSB with XR-DH-agar, xylose-rhamnose supplemented deoxycholate hydrogen sulphide agar, enabled isolation of E. albertii when at least 1 CFU of the bacterium was present per gram of chicken meat. This level of enrichment was superior to those obtained using buffered peptone water, modified-EC broth, or mTSB (with novobiocin). CONCLUSIONS: Novobiocin-cefixime-tellurite supplemented modified tryptic soy broth enabled effective enrichment of E. albertii from poultry samples and was helpful for isolation of this bacterium. SIGNIFICANCE AND IMPACT OF STUDY: To our knowledge, this is the first report of selective enrichment of E. albertii from poultry samples.

Additional Og-Typing PCR Techniques Targeting Escherichia coli-Novel and Shigella-Unique O-Antigen Biosynthesis Gene Clusters.

The O-serogrouping of pathogenic Escherichia coli is a standard method for subtyping strains for epidemiological studies and controls. O-serogroup diversification shows a strong association with the genetic diversity in some O-antigen biosynthesis gene clusters. Through genomic studies, in addition to the types of O-antigen biosynthesis gene clusters (Og-types) from conventional O-serogroup strains, a number of novel Og-types have been found in E. coli isolates. To assist outbreak investigations and surveillance of pathogenic E. coli at inspection institutes, in previous studies, we developed PCR methods that could determine almost all conventional O-serogroups and some novel Og-types. However, there are still many Og-types that may not be determined by simple genetic methods such as PCR. Thus, in the present study, we aimed to develop an additional Og-typing PCR system. Based on the novel Og-types, including OgN32, OgN33, and OgN34, presented in this study, we designed an additional 24 PCR primer pairs targeting 14 novel and 2 diversified E. coli Og-types and 8 Shigella-unique Og-types. Subsequently, we developed 5 new multiplex PCR sets consisting of 33 primers, including the aforementioned 24 primers and 9 primers reported in previous studies. The accuracy and specificity of the PCR system was validated using approximately 260 E. coli and Shigella O-serogroup and Og-type reference strains. The Og-typing PCR system reported here can determine a wide range of Og-types of E. coli and may help epidemiological studies, in addition to the surveillance of pathogenic E. coli.

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.

Escherichia coli H-Genotyping PCR: a Complete and Practical Platform for Molecular H Typing.

In Escherichia coli, more than 180 O groups and 53 H types have been recognized. The O:H serotyping of E. coli strains is an effective method for identifying strains with pathogenic potential and classifying them into clonal groups. In particular, the serotyping of Shiga toxin-producing E. coli (STEC) strains provides valuable information to evaluate the routes, sources, and prevalence of agents in outbreak investigations and surveillance. Here, we present a complete and practical PCR-based H-typing system, E. coli H-genotyping PCR, consisting of 10 multiplex PCR kits with 51 single PCR primer pairs. Primers were designed based on a detailed comparative analysis of sequences from all H-antigen (flagellin)-encoding genes, fliC and its homologs. The specificity of this system was confirmed by using all H type reference strains. Additionally, 362 serotyped wild strains were also used to evaluate its practicality. All 277 H-type-identified isolates gave PCR products that corresponded to the results of serological H typing. Moreover, 76 nonmotile and nine untypeable strains could be successfully subtyped into any H type by the PCR system. The E. coli H-genotyping PCR developed here allows broader, rapid, and low-cost subtyping of H types and will assist epidemiological studies as well as surveillance of pathogenic E. coli.

Subtilase cytotoxin produced by locus of enterocyte effacement-negative Shiga-toxigenic Escherichia coli induces stress granule formation.

Subtilase cytotoxin (SubAB) is mainly produced by locus of enterocyte effacement (LEE)-negative strains of Shiga-toxigenic Escherichia coli (STEC). SubAB cleaves an endoplasmic reticulum (ER) chaperone, BiP/Grp78, leading to induction of ER stress. This stress causes activation of ER stress sensor proteins and induction of caspase-dependent apoptosis. We found that SubAB induces stress granules (SG) in various cells. Aim of this study was to explore the mechanism by which SubAB induced SG formation. Here, we show that SubAB-induced SG formation is regulated by activation of double-stranded RNA-activated protein kinase (PKR)-like endoplasmic reticulum kinase (PERK). The culture supernatant of STEC O113:H21 dramatically induced SG in Caco2 cells, although subAB knockout STEC O113:H21 culture supernatant did not. Treatment with phorbol 12-myristate 13-acetate (PMA), a protein kinase C (PKC) activator, and lysosomal inhibitors, NH4 Cl and chloroquine, suppressed SubAB-induced SG formation, which was enhanced by PKC and PKD inhibitors. SubAB attenuated the level of PKD1 phosphorylation. Depletion of PKCδ and PKD1 by siRNA promoted SG formation in response to SubAB. Furthermore, death-associated protein 1 (DAP1) knockdown increased basal phospho-PKD1(S916) and suppressed SG formation by SubAB. However, SG formation by an ER stress inducer, Thapsigargin, was not inhibited in PMA-treated cells. Our findings show that SubAB-induced SG formation is regulated by the PERK/DAP1 signalling pathway, which may be modulated by PKCδ/PKD1, and different from the signal transduction pathway that results in Thapsigargin-induced SG formation.

Phylogenetic Analysis of Enteroaggregative Escherichia coli (EAEC) Isolates from Japan Reveals Emergence of CTX-M-14-Producing EAEC O25:H4 Clones Related to Sequence Type 131.

Enteroaggregative Escherichia coli (EAEC) causes acute or persistent diarrhea. The aggR gene is widely used as a marker for typical EAEC. The heterogeneity of EAEC is well known; however, there are few reports on the phylogenetic relationships of EAEC. Recently, CTX-M extended-spectrum ß-lactamase (ESBL)-producing EAEC strains have been reported worldwide. To characterize EAEC strains in Japan, we investigated the population structure of EAEC. A total of 167 aggR-positive strains isolated from stool specimens from diarrheal patients in Kagoshima (139 strains) and Osaka (28 strains), Japan, between 1992 and 2010 were examined for the prevalence of EAEC virulence markers, the blaCTX-M gene, and the capacity to form biofilms. Multilocus sequence typing was also conducted. EAEC strains were widely distributed across four major E. coli phylogroups. Strains of O111:H21/clonal group 40 (CG40) (30 strains), O126:H27/CG200 (13 strains), and O86a:H27/CG3570 (11 strains) in phylogroup B1 are the historical EAEC clones in Japan, and they exhibited strong biofilm formation. Twenty-nine strains of EAEC O25:H4/CG131 were identified in phylogroup B2, 79% of which produced CTX-M-14. This clone has emerged since 2003. The clone harbored plasmid-encoded EAEC virulence genes but not chromosomal virulence genes and had lower biofilm-forming capacity than historical EAEC strains. This clone most likely emerged from a pandemic uropathogenic O25:H4/sequence type 131 clone by acquiring an EAEC virulence plasmid from canonical EAEC. Surveillance of the horizontal transfer of both virulence and ESBL genes among E. coli strains is important for preventing a worldwide increase in antimicrobial drug resistance.

Escherichia coli O-Genotyping PCR: a Comprehensive and Practical Platform for Molecular O Serogrouping.

The O serogrouping of pathogenic Escherichia coli is a standard method for subtyping strains for epidemiological studies and enhancing phylogenetic studies. In particular, the identification of strains of the same O serogroup is essential in outbreak investigations and surveillance. In a previous study, we analyzed the O-antigen biosynthesis gene cluster in all known E. coli O serogroups (A. Iguchi et al., DNA Res, 22:101-107, 2015, http://dx.doi.org/10.1093/dnares/dsu043). Based on those results, we have arranged 162 PCR primer pairs for the identification or classification of O serogroups. Of these, 147 pairs were used to identify 147 individual O serogroups with unique O-antigen biosynthesis genes, and the other 15 pairs were used to identify 15 groups of strains (Gp1 to Gp15). Each of these groups consisted of strains with identical or very similar O-antigen biosynthesis genes, and the groups represented a total of 35 individual O serogroups. We then used the 162 primer pairs to create 20 multiplex PCR sets. Each set contained six to nine primer pairs that amplify products of markedly different sizes. This genetic methodology (E. coli O-genotyping PCR) allowed for comprehensive, rapid, and low-cost typing. Validation of the PCR system using O-serogroup references and wild strains showed that the correct O serogroups were specifically and accurately identified for 100% (182/182) and 90.8% (522/575) of references and wild strains, respectively. The PCR-based system reported here might be a promising tool for the subtyping of E. coli strains for epidemiological studies as well as for the surveillance of pathogenic E. coli during outbreaks.

Characterization of Third-Generation-Cephalosporin-Resistant Shiga Toxin-Producing Strains of Escherichia coli O157:H7 in Japan.

We isolated Shiga toxin-producing Escherichia coli O157:H7 strains resistant to third-generation cephalosporins. The resistant strains harbored blaCMY-2, a plasmid-mediated AmpC ß-lactamase. Genotyping of isolates revealed the possible spread of this problematic bacterium. Results suggested the importance of the investigation and surveillance of enterobacteria with plasmids harboring blaCMY-2.

First report in Thailand of a stx-negative Escherichia Coli 0157 strain from a patient with diarrhea.

E. coli serotype 0157 is well known to cause serious illnesses in humans. However, there has been no case report to date of this serotype in Thailand. In this study, we report for the first time E. coli 0157 (designated as PSU120) isolated from a stool sample among 228 diarrheal swab samples at Hat Yai Hospital, Songkhla Province, Thailand. This PSU120 was identified as being stx-negative and lacked eae but carried escV, a marker for the locus of enterocyte effacement. Of the five reported integration sites frequently occupied by stx phages, the sbcB and yehV loci were occupied, suggesting that PSU120 is active in horizontal genetic transfer. Antimicrobial susceptibility assay revealed that E. coli 0157 strain PSU120 was resistant to cephalothin, erythromycin, methicillin and vancomycin. Using pulsed- field gel-electrophoresis to compare the genetic relatedness of E. coli 0157 strain PSU120 to two other E. coli 0157 strains, namely, the well-established EHEC strain EDL933 and PSU2, a surrogate of E. coli 0157:H7 whose genotype stx1-, stx2+, eae+ is frequently obtained from the environment in this area during the last decade, revealed 88.6% in similarity. We suggest that PSU120 was originally stx+ but lostthe gene after establishing infection.

Combined usage of serodiagnosis and O antigen typing to isolate Shiga toxin-producing Escherichia coli O76:H7 from a hemolytic uremic syndrome case and genomic insights from the isolate.

IMPORTANCE: Hemolytic uremic syndrome (HUS) is a life-threatening disease caused by Shiga toxin-producing Escherichia coli (STEC) infection. The treatment approaches for STEC-mediated typical HUS and atypical HUS differ, underscoring the importance of rapid and accurate diagnosis. However, specific detection methods for STECs other than major serogroups, such as O157, O26, and O111, are limited. This study focuses on the utility of PCR-based O-serotyping, serum agglutination tests utilizing antibodies against the identified Og type, and isolation techniques employing antibody-conjugated immunomagnetic beads for STEC isolation. By employing these methods, we successfully isolated a STEC strain of a minor serotype, O76:H7, from a HUS patient.

Modulation of O-antigen chain length by the wzz gene in Escherichia coli O157 influences its sensitivities to serum complement.

Escherichia coli O157 strains belonging to a distinct lineage and expressing different O-antigen (Oag) lengths were isolated. Although the function of wzz in E. coli has not been adequately investigated, this gene is known to be associated with regulation of Oag length. Using E. coli O157:H7 ATCC43888 (wild-type), several wzz mutants of E. coli O157, including a wzz deletion mutant, were generated and the relationship between the length of Oag modulated by the wzz gene and sensitivities to serum complement investigated. SDS-PAGE, immunoblot analyses and sensitivity tests to human serum complement were performed on these strains. The lengths of the O157-antigen could be modulated by the wzz gene mutations and were classified into long, intermediate and short groups. The short chain mutant was more serum sensitive than the wild-type strain and the other wzz mutants (P < 0.001). In conclusion, Oag chain length modulated by the wzz gene in E. coli O157 influences its sensitivities to serum complement. The present findings suggest that E. coli O157 strains with intermediate or long length Oag chains might show greater resistance to serum complement than those with short chains.

Real-time PCR assays to detect 10 Shiga toxin subtype (Stx1a, Stx1c, Stx1d, Stx2a, Stx2b, Stx2c, Stx2d, Stx2e, Stx2f, and Stx2g) genes.

To develop subtyping methods for Shiga toxin (Stx)1a, Stx1c, Stx1d, Stx2a, Stx2b, Stx2c, Stx2d, Stx2e, Stx2f, and Stx2g genes for epidemiological analyses of Shiga toxin-producing Escherichia coli (STEC), we developed 10 simplex real-time polymerase chain reaction (PCR) assays with reference to 284 valid stx sequences and evaluated their specificity and quantitative accuracy using STEC and non-STEC isolates and recombinant plasmids, respectively. Three stx1 and 5 stx2 subtype genes, except for stx2c and stx2d, were detected with high specificity using STEC isolates. However, some stx2a sequences potentially being close to both Stx2a and Stx2d cluster in neighbor-joining cluster analysis were positive for stx2a and stx2d by real-time PCR. For the stx2c assay, the number of real-time PCR cycles was reduced to avoid unnecessary false-positive results. Based on these considerations, the real-time PCR assays developed here might aid epidemiological investigations of infections or outbreaks caused by STEC harboring any of the stx subtype genes.

A Salmonella enterica Serovar Oranienburg Clone Caused a Cluster of Bacteremia Cases in Persons With No Recognizable Underlying Diseases in Japan.

Background: Salmonella enterica subspecies enterica serovar Oranienburg (SO) is a foodborne pathogen but rarely causes systemic infections such as bacteremia. Between July and September 2018, bacteremia cases caused by SO were identified in 12 persons without any underlying medical conditions in the southern Kyushu area of Japan. Methods: Randomly amplified polymorphic DNA (RAPD) analysis was performed to investigate the genetic similarity of the 12 bacteremia-related strains and other Japanese isolates. Furthermore, a series of whole-genome sequence (WGS)-based phylogenetic analyses was performed with a global SO strain set (n = 1648). Results: The resolution power of RAPD was insufficient to investigate the genetic similarity between the bacteremia-related strains and other strains. WGS-based phylogenetic analyses revealed that the bacteremia-related strains formed a tight cluster along with 2 strains isolated from asymptomatic carriers in 2018 in the same area, with a maximum within-cluster single-nucleotide polymorphism (SNP) distance of 11. While several strains isolated in the United States and the United Kingdom were found to be closely related to the bacteremia-related strains, 2 strains isolated in 2016 in the southern Kyushu area were most closely related, with SNP distances of 4-11 and 5-10, and had the same plasmids as the bacteremia-related strains. Conclusions: The 12 bacteremia cases identified were caused by a single SO clone. As none of the bacteremia patients had any underlying diseases, this clone may be prone to cause bacteremia. Although further analyses are required to understand its virulence, particular attention should be given to this clone and its close relatives in the surveillance of nontyphoidal salmonellae.

Global population structure, genomic diversity and carbohydrate fermentation characteristics of clonal complex 119 (CC119), an understudied Shiga toxin-producing E. coli (STEC) lineage including O165:H25 and O172:H25.

Among Shiga toxin (Stx)-producing Escherichia coli (STEC) strains of various serotypes, O157:H7 and five major non-O157 STEC (O26:H11, O111:H8, O103:H2, O121:H19 and O145:H28) can be selectively isolated by using tellurite-containing media. While human infections by O165:H25 STEC strains have been reported worldwide, their detection and isolation are not easy, as they are not resistant to tellurite. Systematic whole-genome sequencing (WGS) analyses have not yet been conducted. Here, we defined O165:H25 strains and their close relatives, including O172:H25 strains, as clonal complex 119 (CC119) and performed a global WGS analysis of the major lineage of CC119, called CC119 sensu stricto (CC119ss), by using 202 CC119ss strains, including 90 strains sequenced in this study. Detailed comparisons of 13 closed genomes, including 7 obtained in this study, and systematic analyses of Stx phage genomes in 50 strains covering the entire CC119ss lineage, were also conducted. These analyses revealed that the Stx2a phage, the locus of enterocyte effacement (LEE) encoding a type III secretion system (T3SS), many prophages encoding T3SS effectors, and the virulence plasmid were acquired by the common ancestor of CC119ss and have been stably maintained in this lineage, while unusual exchanges of Stx1a and Stx2c phages were found at a single integration site. Although the genome sequences of Stx2a phages were highly conserved, CC119ss strains exhibited notable variation in Stx2 production levels. Further analyses revealed the lack of SpLE1-like elements carrying the tellurite resistance genes in CC119ss and defects in rhamnose, sucrose, salicin and dulcitol fermentation. The genetic backgrounds underlying these defects were also clarified.

Clinical significance of Escherichia albertii.

Discriminating Escherichia albertii from other Enterobacteriaceae is difficult. Systematic analyses showed that E. albertii represents a substantial portion of strains currently identified as eae-positive Escherichia coli and includes Shiga toxin 2f-producing strains. Because E. albertii possesses the eae gene, many strains might have been misidentified as enterohemorrhagic or enteropathogenic E. coli.

Subtilase cytotoxin from Shiga-toxigenic Escherichia coli impairs the inflammasome and exacerbates enteropathogenic bacterial infection.

Subtilase cytotoxin (SubAB) is an AB5 toxin mainly produced by the locus of enterocyte effacement-negative Shiga-toxigenic Escherichia coli (STEC) strain such as O113:H21, yet the contribution of SubAB to STEC infectious disease is unclear. We found that SubAB reduced activation of the STEC O113:H21 infection-induced non-canonical NLRP3 inflammasome and interleukin (IL)-1ß and IL-18 production in murine macrophages. Downstream of lipopolysaccharide signaling, SubAB suppressed caspase-11 expression by inhibiting interferon-ß/STAT1 signaling, followed by disrupting formation of the NLRP3/caspase-1 assembly. These inhibitions were regulated by PERK/IRE1α-dependent endoplasmic reticulum (ER) stress signaling initiated by cleavage of the host ER chaperone BiP by SubAB. Our murine model of SubAB-producing Citrobacter rodentium demonstrated that SubAB promoted C. rodentium proliferation and worsened symptoms such as intestinal hyperplasia and diarrhea. These findings highlight the inhibitory effect of SubAB on the NLRP3 inflammasome via ER stress, which may be associated with STEC survival and infectious disease pathogenicity in hosts.

Emergence of a novel Shiga toxin-producing Escherichia coli O serogroup cross-reacting with Shigella boydii type 10.

This is the first report of the isolation of Shiga toxin-producing Escherichia coli (STEC) strains whose O antigens were genetically and serologically identical to those of Shigella boydii type 10, from human feces. The novel STEC O serogroup may be widespread in Japan and associated with diarrhea and hemorrhagic colitis.

Molecular epidemiological investigation of a diffuse outbreak caused by Salmonella enterica serotype Montevideo isolates in Osaka Prefecture, Japan.

In Osaka Prefecture, Japan, three foodborne outbreaks were caused by Salmonella enterica serotype Montevideo in rapid succession between September 2007 and May 2008. Further, Salmonella Montevideo was also isolated from several sporadic diarrhea patients and asymptomatic carriers examined during approximately the identical period. To investigate the relatedness of the isolates, we performed antimicrobial susceptibility testing, pulsed-field gel electrophoresis (PFGE) analysis, and multiple-locus variable-number tandem repeat (VNTR) analysis (MLVA) for 29 Salmonella Montevideo isolates obtained in this region between 1991 and 2008. Although antimicrobial susceptibility tests had low discriminatory power, PFGE patterns revealed 17 unique types with <90% similarity in combined analyses involving XbaI and BlnI. Moreover, we detected three VNTR loci that were useful to genotype Salmonella Montevideo isolates, with our method ultimately classifying the isolates into 11 MLVA types based on differences in repeat unit number in each examined locus. Six isolates obtained from patients of two separate foodborne disease outbreaks, one sporadic patient, and three different carriers between 2007 and 2008 had nearly identical PFGE patterns and were classified into the identical MLVA type; further, the isolates with this PFGE and MLVA pattern appeared only at that time between 1991 and 2008. These data strongly suggest that genetically identical Salmonella Montevideo strains may have caused the 2007 and 2008 outbreaks in Osaka Prefecture. Our results demonstrate that PFGE using XbaI and BlnI is useful for discriminating between Salmonella Montevideo isolates, even within a limited area, and reconfirm that continuous epidemiological surveillance for bacterial intestinal infections such as salmonellosis may be useful to not only monitor changes in the genetic diversity of isolates, but to also detect diffuse outbreaks.