Leptospira interrogans is a pathogenic, spirochetal bacterium that is responsible for leptospirosis, an emerging worldwide zoonosis. Leptospires colonize the renal proximal tubules and chronically infect the kidney. Live bacteria are excreted into urine, contaminating the environment. While it is well known that leptospires can persist in the kidneys without signs of disease for several months, the interactions of leptospires with the proximal renal epithelial tubule cells that allow the chronic renal colonization have not been elucidated yet. In the present study, we compared the interactions between a virulent, low passage (LP) strain and a cultured-attenuated, high passage (HP) strain with renal proximal tubule epithelial cells (RPTECs) to elucidate the strategies used by Leptospira to colonize the kidney. Kinetics analysis of kidney colonization in a mouse model of chronic infection performed by quantitative real-time PCR and immunofluorescence, showed that the LP strain reached the kidney by 3 days post infection (pi) and attached to the basal membrane side of the renal epithelial cells. At 10 days pi, some leptospires were attached to the luminal side of the tubular epithelia and the number of colonizing leptospires gradually increased. On the other hand, the HP strain was cleared during hematogenous dissemination and did not colonize the kidney. Transmission electron microscopy analysis of LP-infected kidneys at 25 days pi showed aggregated leptospires and membrane vesicles attached to the epithelial brush border. Leptospiral kidney colonization altered the organization of the RPTEC brush border. An in vitro model of infection using TCMK-1 cells, showed that leptospiral infection induced a host stress response, which is delayed in LP-infected cells. After hematogenous dissemination, leptospires create protective and replicative niches in the base membrane and luminal sides of the RPTECs. During the long-term colonization, leptospires attached to the RPTEC brush borders and membrane vesicles might be involved in the formation of a biofilm-like structure in vivo. Our results also suggested that the virulent strain is able to manipulate host cell stress responses to promote renal colonization.

Leptospirosis is one of the most significant zoonoses across the world not only because of its impact on human and animal health but also because of the economic and social impact on agrarian communities. Leptospirosis is endemic in Sri Lanka where paddy farming activities, the use of draught animals in agriculture, and peridomestic animals in urban and rural areas play important roles in maintaining the infection cycle of pathogenic Leptospira, especially concerning animals as a potential reservoir. In this study, an environmental DNA (eDNA) metabarcoding methodology was applied in two different agro-ecological regions of Sri Lanka to understand the eco-epidemiology of leptospirosis. Irrigation water samples were collected in Kandy District (wet zone mid-country region 2) and Girandurukotte, Badulla District (intermediate zone low-country region 2); and analysed for the presence of pathogenic Leptospira, associated microbiome and the potential reservoir animals. Briefly, we generated PCR products for high-throughput sequencing of multiple amplicons through next-generation sequencing. The analysis of eDNA showed different environmental microbiomes in both regions and a higher diversity of Leptospira species circulating in Kandy than in Girandurukotte. Moreover, the number of sequence reads of pathogenic Leptospira species associated with clinical cases such as L. interrogans was higher in Kandy than in Girandurukotte. Kandy also showed more animal species associated with pathogenic bacterial species than Girandurukotte. Finally, several pathogenic bacterial species including Arcobacter cryaerophilus, responsible for abortion in animals, was shown to be associated with pathogenic Leptospira. Leptospirosis has been considered to be endemic in wet regions, consistently, leptospiral sequences were detected strongly in Kandy. The great Leptospira species diversity in Kandy observed in this study shows that the etiological agents of leptospirosis in Sri Lanka might be underestimated. Furthermore, our eDNA metabarcoding can be used to discriminate bacterial and animal species diversity in different regions and to explore environmental microbiomes to identify other associated bacterial pathogens in the environment. Leptospirosis is a widespread bacterial zoonosis with increasing importance due to its vast range of reservoir hosts. Early symptoms are shared by other infectious diseases common in tropical and sub-tropical regions, where the real burden and risk factors need to be known. In Sri Lanka, leptospirosis is mostly an occupational disease associated with freshwater or animal exposure in agriculture communities. Thus, there is a need for understanding the epidemiology of leptospirosis in agrarian regions of the country for developing better prevention and intervention strategies. In this study, we applied an environmental DNA metabarcoding methodology to understand the environmental microbiome, potential reservoir animals and the Leptospira species circulating in two different agro-ecological regions of Sri Lanka: Kandy (wet region mid-country region 2) and Girandurukotte (intermediate region low-country region 2). It is known that pathogenic Leptospira are excreted through the urine of reservoir animals in the environment, where they can persist in humid conditions. Congruently, this study showed a higher detection of pathogenic Leptospira in the environment of Kandy where the environmental microbiome showed a higher diversity than Girandurukotte. Potential animal reservoirs were also detected in samples positive for pathogenic Leptospira, suggesting that environmental DNA metabarcoding can provide important information for management and intervention strategies to control leptospirosis.

Background: Diarrheagenic Escherichia coli (DEC) is a group of bacterial pathogens that causes life-threatening diarrhea in children in developing countries. However, there is limited information on the characteristics of DEC isolated from patients in these countries. A detailed genomic analysis of 61 DEC-like isolates from infants with diarrhea was performed to clarify and share the characteristics of DEC prevalent in Vietnam. Principal findings: DEC was classified into 57 strains, including 33 enteroaggregative E. coli (EAEC) (54.1%), 20 enteropathogenic E. coli (EPEC) (32.8%), two enteroinvasive E. coli (EIEC) (3.3%), one enterotoxigenic E. coli (ETEC), and one ETEC/EIEC hybrid (1.6% each), and surprisingly into four Escherichia albertii strains (6.6%). Furthermore, several epidemic DEC clones showed an uncommon combination of pathotypes and serotypes, such as EAEC Og130:Hg27, EAEC OgGp9:Hg18, EAEC OgX13:H27, EPEC OgGp7:Hg16, and E. albertii EAOg1:HgUT. Genomic analysis also revealed the presence of various genes and mutations associated with antibiotic resistance in many isolates. Strains that demonstrate potential resistance to ciprofloxacin and ceftriaxone, drugs recommended for treating childhood diarrhea, accounted for 65.6% and 41%, respectively. Significance: Our finding indicate that the routine use of these antibiotics has selected resistant DECs, resulting in a situation where these drugs do not provide in therapeutic effects for some patients. Bridging this gap requires continuous investigations and information sharing regarding the type and distribution of endemic DEC and E. albertii and their antibiotic resistance in different countries.

Cholera epidemics have been recorded periodically in Vietnam during the seventh cholera pandemic. Since cholera is a water-borne disease, systematic monitoring of environmental waters for Vibrio cholerae presence is important for predicting and preventing cholera epidemics. We conducted monitoring, isolation, and genetic characterization of V. cholerae strains in Nam Dinh province of Northern Vietnam from Jul 2013 to Feb 2015. In this study, four V. cholerae O1 strains were detected and isolated from 110 analyzed water samples (3.6%); however, none of them carried the cholera toxin gene, ctxA, in their genomes. Whole genome sequencing and phylogenetic analysis revealed that the four O1 isolates were separated into two independent clusters, and one of them diverged from a common ancestor with pandemic strains. The analysis of pathogenicity islands (CTX prophage, VPI-I, VPI-II, VSP-I, and VSP-II) indicated that one strain (VNND_2014Jun_6SS) harbored an unknown prophage-like sequence with high homology to vibriophage KSF-1 phi and VCY phi, identified from Bangladesh and the USA, respectively, while the other three strains carried tcpA gene with a distinct sequence demonstrating a separate clonal lineage. These results suggest that the aquatic environment can harbor highly divergent V. cholera strains and serve as a reservoir for multiple V. cholerae virulence-associated genes which may be exchanged via mobile genetic elements. Therefore, continuous monitoring and genetic characterization of V. cholerae strains in the environment should contribute to the early detection of the sources of infection and prevention of cholera outbreaks as well as to understanding the natural ecology and evolution of V. cholerae.

To clarify the mechanism of Seoul orthohantavirus (SEOV) persistence, we compared the humoral and cell-mediated immune responses to SEOV in experimentally and naturally infected brown rats. Rats that were experimentally infected by the intraperitoneal route showed transient immunoglobulin M (IgM) production, followed by an increased anti-SEOV immunoglobulin G (IgG) antibody response and maturation of IgG avidity. The level of SEOV-specific cytotoxic T lymphocytes (CTLs) peaked at 6 days after inoculation and the viral genome disappeared from serum. In contrast, naturally infected brown rats simultaneously had a high rate of SEOV-specific IgM and IgG antibodies (28/43). Most of the IgM-positive rats (24/27) had the SEOV genome in their lungs, suggesting that chronic SEOV infection was established in those rats. In female rats with IgG avidity maturation, the viral load in the lungs was decreased. On the other hand, there was no relationship between IgG avidity and viral load in the lungs in male rats. A CTL response was not detected in naturally infected rats. The difference between immune responses in the experimentally and naturally infected rats is associated with the establishment of chronic infection in natural hosts.

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