Epidemic Clostridium difficile (027/BI/NAP1) rapidly emerged in the past decade as the leading cause of antibiotic-associated diarrhea worldwide. However, the key moments in the evolutionary history leading to its emergence and subsequent patterns of global spread remain unknown. Here we define the global population structure of C. difficile 027/BI/NAP1 based on whole-genome sequencing and phylogenetic analysis. We demonstrate that two distinct epidemic lineages, FQR1 and FQR2, not one as previously thought, emerged in North America within a relatively short period after acquiring the same fluoroquinolone resistance mutation and a highly-related conjugative transposon. The two epidemic lineages displayed distinct patterns of global spread, and the FQR2 lineage spread more widely leading to healthcare outbreaks in the UK, continental Europe and Australia. Our analysis identifies key genetic changes linked to the rapid trans-continental dissemination of epidemic C. difficile 027/BI/NAP1 and highlights the routes by which it spreads through the global healthcare system.

VBNC Vibrio cholerae O139 VC-280 obtained by incubation in 1% solution of artificial sea water IO at 4• C for 74 days converted to the culturable state when co-cultured with CHO cells. Other eukaryotic cell lines, including HT-29, Caco-2, T84, HeLa, and Intestine 407, also supported conversion of VBNC cells to the culturable state. Conversion of VBNC V. cholerae O1 N16961 and V. cholerae O139 VC-280/pG13 to the culturable state, under the same conditions, was also confirmed. When VBNC V. cholerae O139 VC-280 was incubated in 1% IO at 4• C for up to 91 days, the number of cells converted by co-culture with CHO cells declined with each additional day of incubation and after 91 days conversion was not observed.Key words co-culture, conversion to culturability, eukaryotic cell, viable but nonculturable (VBNC) Vibrio cholerae.The VBNC state in bacteria is defined as that in which the bacteria remain viable but the cells do not grow or divide on, or in, routinely used bacteriological media. VBNC state of Vibrio cholerae O1 was first reported by Xu et al.(1) and this finding was subsequently confirmed by many studies (2-7). Conversion of VBNC V. cholerae O1 to the culturable state was first demonstrated by Colwell et al. (8) who showed that inoculation of VBNC V. cholerae O1 into rabbit ileal loops resulted in fluid accumulation from which culturable V. cholerae O1 could be isolated. Furthermore, it was shown that VBNC V. cholerae O1 converted to the culturable state after ingestion during a human volunteer study (9). List of Abbreviations: APW, alkaline peptone water; FBS, fetal bovine serum; GFP, green fluorescent protein; IO, instant ocean; MEM, minimum essential medium; MEM-FBS, MEM supplemented with 10% fetal bovine serum, but without supplementation of streptomycin and penicillin; NA, nutrient agar; TCBS, thiosulfate citrate bile salts sucrose; VBNC, viable but nonculturable. Wai et al. (3) reported in vitro conversion of VBNC V. cholerae O1 to the culturable state by heat shock, but confirmation of this finding was not successful. In the present study, we report in vitro conversion of VBNC V. cholerae O139 and O1 to the culturable state by coculture with eukaryotic cells. MATERIALS AND METHODS Bacterial strains and culture mediaVibrio cholerae O139 VC-280 isolated in Kolkata from a cholera patient, V. cholerae O1 N16961 which is a stock 502

Vibrio cholerae O1 El Tor variant strains produced much more cholera toxin than did prototype El Tor strains. The amount of cholera toxin produced by El Tor variant strains both in vitro and in vivo was more or less equivalent to that produced by classical strains.

Viable but nonculturable (VBNC) Vibrio cholerae non-O1/non-O139, V. parahaemolyticus, enterohemorrhagic Escherichia coli, enterotoxigenic E. coli, enteropathogenic E. coli, Shigella flexneri, and Salmonella enterica were converted to the culturable state by co-culture with selected eukaryotic cells, e.g., HT-29, Caco-2, T84, HeLa, Intestine 407, and CHO cells.Key words conversion to culturability, enteric bacteria, viable but nonculturable.Viable but nonculturable (VBNC) bacteria are defined as bacteria that are living but unable to grow or divide when inoculated into routinely used bacteriological culture media. In 1982, Xu et al. first reported on VBNC bacteria, including Escherichia coli and Vibrio cholerae (1). Subsequently, many investigators have reported on VBNC bacteria, with more than 60 species of bacteria shown to demonstrate the phenomenon, including many human pathogens (2, 3).Conversion of VBNC cells to the culturable state was first demonstrated by Colwell et al., using rabbit ileal loop inoculation (4), followed by human volunteer studies (5). Several in vitro methods have been used to convert VBNC bacteria to the culturable state (6-10). Recently, we reported VBNC V. cholerae O1 and O139 cells could be converted to the culturable state by co-culture with eukaryotic cells, namely HT-29, Caco-2, T84, HeLa, Intestine 407, and CHO cells (11). We report here the conversion of several enteric bacteria in the VBNC state, including V. cholerae non-O1/non-O139, V. parahaemolyticus, enterohemorrhagic E. coli (EHEC), enteroCorrespondence

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