Molecular diversity of CTX prophage in Vibrio cholerae

Abstract

Aims: The objective of this study was to investigate the molecular diversity of CTX genetic element within toxigenic Vibrio cholerae genomes and to determine the genetic diversity of V. cholerae population collected in a 6‐year period (2004–2009) in Iran. Methods and Results: The results of mismatch amplification mutation assay (MAMA)‐PCR and sequencing showed cytosine nucleotide in positions 203 and 115 in all 50 El Tor V. cholerae strains, which is the same as classical ctxB sequence. One strain yielded amplicons with both El Tor and classical biotype primers in MAMA‐PCR indicative of presence of two copies of CTX phages with different genotypes (rstRET ctxBclass and rstRET ctxBET) integrated within the genome of this isolate, which suggested the integration of two different CTX phages at different occasions or point mutation in one copy of CTX. Sequencing and PCR analysis indicated the presence of hybrid CTX genotype (rstRET ctxclass) in 70·6% of the isolates; however, only El Tor RS1 phage has been integrated in flanking to the CTX phages with different genotypes. Conclusions: Enterobacterial repetitive intergenic consensus‐PCR (ERIC‐PCR) and ribosomal gene spacer‐PCR (RS‐PCR) showed a relatively homogenous population in different years. Our findings indicate that sequence analysis of RS and ctxB regions has more discriminative power than restriction‐based methods. Significance and Impact of the Study: Investigating the molecular diversity of CTX prophage among V. cholerae strains helps to establish a new valuable database of genetic information about isolates, which is of great importance for epidemiologic studies in Iran and other countries encountering cholera epidemics.

Correlation of the Native Inaba Strain With the Dominant Isolated Strains Obtained From Outbreaks in 2013 in Iran

“…7 Results of PFGE analysis in this study revealed the cholera isolates were in different clusters. Ogawa serotype was a main type of the outbreak of 2011.…”

“…This serotype was distributed through the country with 6 different patterns, while a few Inaba strains were only isolated with differentiated patterns from Ogawa serotype. 7 In the present study, the diversity of cholera strains in 2013 outbreak was studied and the results were compared with the homology of detected Inaba strains in 2011. We concluded the previous native Inaba strain could not have a role in 2013 outbreak.…”

“…The conditions used for separation were as follows: an isolated colony was streaked from test cultures to Trypticase soy agar with 5% defibrinated sheep blood (TSA-SB) plates incubated overnight for confluent growth. 7,13 Grown colonies within 14-18 hours were used to prepare cell suspension. Bacterial suspension was prepared in a cell suspension buffer (100 mM Tris:100 mM EDTA, pH 8.0) and adjusted to absorbance values of 0.8-1.0 at a wavelength of 610 nm after which plugs were prepared with SeaKem Gold Agarose (Lonza, Rockland, ME, USA) and proteinase K. Bacterial plugs were lysed (50 mM Tris: 50 mM EDTA, pH 8.0 + 1% sarcosyl, and 25 μL proteinase K 20 mg/mL) and washed with pre-heated sterile ultrapure water and sterile TE buffer 6 times in a 54-55°C water bath.…”

“…This method is able to support epidemiological data in describing how a V. cholerae O1 isolate can emerge from abroad or reemerge from native strains. Based on a previous study, 7 pulsotypes were reported from which 3 types were dominant throughout the country and 4 were sporadic, 7 while in previous studies just two types were detected. 5,6 Objectives The objective of this study was the analysis of Inaba strains isolated from 2013 outbreak to study the similarity of the isolated strains and compare their homology in order to find out the route of infection whether emerged from foreign strains or reemerged from domestic native strains.…”

“…Based on a previous study, a few Inaba had been isolated from sporadic cases. 7 Mo lecular typing techniques such as random amplified polymorphic DNA (RAPD), ribotyping, and multilocus enzyme electrophoresis (MEE) have been employed to study genetic relatedness. 8,9 However, PFGE has been able to play a considerable role in epidemiological investigations because it has a high discriminatory power.…”