Vibrio cholerae Persisted in Microcosm for 700 Days Inhibits Motility but Promotes Biofilm Formation in Nutrient-Poor Lake Water Microcosms

Abstract

Toxigenic Vibrio cholerae, ubiquitous in aquatic environments, is responsible for cholera; humans can become infected after consuming food and/or water contaminated with the bacterium. The underlying basis of persistence of V. cholerae in the aquatic environment remains poorly understood despite decades of research. We recently described a “persister” phenotype of V. cholerae that survived in nutrient-poor “filter sterilized” lake water (FSLW) in excess of 700-days. Previous reports suggest that microorganisms can assume a growth advantage in stationary phase (GASP) phenotype in response to long-term survival during stationary phase of growth. Here we report a V. cholerae GASP phenotype (GASP-700D) that appeared to result from 700 day-old persister cells stored in glycerol broth at −80°C. The GASP-700D, compared to its wild-type N16961, was defective in motility, produced increased biofilm that was independent of vps (p<0.005) and resistant to oxidative stress when grown specifically in FSLW (p<0.005). We propose that V. cholerae GASP-700D represents cell populations that may better fit and adapt to stressful survival conditions while serving as a critical link in the cycle of cholera transmission.

Mutation in flrA and mshA Genes of Vibrio cholerae Inversely Involved in vps-Independent Biofilm Driving Bacterium Toward Nutrients in Lake Water

“…To determine if GASP-700D ( Jubair et al, 2014 ) sustained a mutation in the flrA gene, we PCR amplified the gene from the GASP-700D genome with two convergent PCR primers [aa778 and aa780 (Supplementary Table 1 )] using standard PCR conditions. The amplicon was purified using Qiagen PCR Purification Kit (Qiagen, Valencia, CA, United States) and sequenced with help from the Interdisciplinary Center for Biotechnology Research (ICBR) at University of Florida; sequence analysis revealed that GASP-700D sustained a 49-bp deletion mutation in flrA gene (we hereafter referred this strain as GSΔ flrA ).…”

“…Despite decades of investigations, the physiological and genetic basis of the persistence of V. cholerae in aquatic reservoirs, particularly during inter-epidemic periods, remains to be fully elucidated. Possible survival mechanisms in nutrient-poor and stressful aquatic reservoirs include: (a) adopting a viable but non-culturable (VBNC) state ( Xu et al, 1982 ; Colwell and Huq, 1994 ), (b) switching from planktonic to biofilm lifestyle ( Wai et al, 1998 ; Yildiz and Schoolnik, 1999 ; Ali et al, 2002 ), and/or (c) assuming “persister” and “growth advantage stationary phase” (“GASP”) phenotypes as described recently ( Jubair et al, 2012 , 2014 ).…”

“…Long-term persistence of bacteria in stressful and stationary growth phase can promote a “GASP” phenotype ( Finkel and Kolter, 1999 ; Zinser and Kolter, 2004 ; Finkel, 2006 ). We recently reported a V. cholerae GASP phenotype (GASP-700D) after its long-term persistence (700 days) in nutrient-poor lake water microcosms ( Jubair et al, 2014 ). Compared to its wild-type N16961S phenotype, the GASP-700D phenotype was defective in motility and produced significantly increased vps -independent biofilm when grown specifically in filter sterilized lake water (FSLW).…”

High-Frequency Rugose Exopolysaccharide Production by Vibrio cholerae Strains Isolated in Haiti

“…To determine if VpsA is essential for the expression of rugose colony phenotype in Haitian altered V. cholerae strain, we created a vpsA in-frame deletion mutation in the background of HC16R strain (Table 4), using genetic manipulations described recently [18]. An internal in-frame deletion in the vpsA [HC16RΔ vpsA (Table 4)] was created and the mutation was verified by PCR and DNA sequencing.…”

Altered Proteome of Burkholderia pseudomallei Colony Variants Induced by Exposure to Human Lung Epithelial Cells

“…The SCVs are known for their ability to resist antibiotics, remain persistent in mammalian cells and cause latent or recurrent infections in the infected host. Several pathogenic bacteria have been reported to produce SCV including, Burkholderia cepacia [ 2 ], Pseudomonas aeruginosa [ 5 ], Salmonella [ 4 ], Vibrio cholera [ 6 ], Brucella melitensis [ 7 ], Escherichia coli [ 8 ], Serratia marcescens [ 9 ], Staphylococcus aureus [ 10 ], and Staphylococcus epidermidis [ 11 ].…”