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Comparative genomic analysis of Vibrio cholerae El Tor associated with endemic cholera in Asia revealed two distinct lineages, one dominant in Bangladesh and the other in India. An in-depth whole-genome study of V. cholerae El Tor strains isolated during endemic cholera in Bangladesh (1991 to 2017) included reference genome sequence data obtained online. Core genome phylogeny established using single nucleotide polymorphisms (SNPs) showed V. cholerae El Tor strains comprised two lineages, BD-1 and BD-2, which, according to Bayesian phylodynamic analysis, originated from paraphyletic group BD-0 around 1981. BD-1 and BD-2 lineages overlapped temporally but were negatively associated as causative agents of cholera during 2004 to 2017. Genome-wide association study (GWAS) revealed 140 SNPs and 31 indels, resulting in gene alleles unique to BD-1 and BD-2. Regression analysis of root to tip distance and year of isolation indicated early BD-0 strains at the base, whereas BD-1 and BD-2 subsequently emerged and progressed by accumulating SNPs. Pangenome analysis provided evidence of gene acquisition by both BD-1 and BD-2, of which six crucial proteins of known function were predominant in BD-2. BD-1 and BD-2 diverged and have distinctively different genomic traits, namely, heterogeneity in VSP-2, VPI-1, mobile elements, toxin encoding elements, and total gene abundance. In addition, the observed phage-inducible chromosomal island-like element (PLE1), and SXT ICE elements (ICETET) in BD-2 presumably provided a fitness advantage for the lineage to outcompete BD-1 as the etiological agent of endemic cholera in Bangladesh, with implications for global cholera epidemiology. IMPORTANCE Cholera is a global disease with specific reference to the Bay of Bengal Ganges Delta where Vibrio cholerae O1 El Tor, the causative agent of the disease showed two circulating lineages, one dominant in Bangladesh and the other in India. Results of an in-depth genomic study of V. cholerae associated with endemic cholera during the past 27 years (1991 to 2017) indicate emergence and succession of the two lineages, BD-1 and BD-2, arising from a common ancestral paraphyletic group, BD-0, comprising the early strains and short-term evolution of the bacterium in Bangladesh. Among the two V. cholerae lineages, BD-2 supersedes BD-1 and is predominant in the most recent endemic cholera in Bangladesh. The BD-2 lineage contained significantly more SNPs and indels, and showed richness in gene abundance, including antimicrobial resistance genes, gene cassettes, and PLE to fight against bacteriophage infection, acquired over time. These findings have important epidemic implications on a global scale.

Vibrio cholerae is a significant threat to global public health in part due to its propensity for large-scale evolutionary sweeps where lineages emerge and are replaced. These sweeps may originate from the Bay of Bengal, where bacteriophage predation and the evolution of antiphage counterdefenses is a recurring theme. The bacteriophage ICP1 is a key predator of epidemic V. cholerae and is notable for acquiring a CRISPR-Cas system to combat PLE, a defensive subviral parasite encoded by its V. cholerae host. Here, we describe the discovery of four previously unknown PLE variants through a retrospective analysis of >3,000 publicly available sequences as well as one additional variant (PLE10) from recent surveillance of cholera patients in Bangladesh. In recent sampling we also observed a lineage sweep of PLE-negative V. cholerae occurring within the patient population in under a year. This shift coincided with a loss of ICP1's CRISPR-Cas system in favor of a previously prevalent PLE-targeting endonuclease called Odn. Interestingly, PLE10 was resistant to ICP1-encoded Odn, yet it was not found in any recent V. cholerae strains. We also identified isolates from within individual patient samples that revealed both mixed PLE(+)/PLE(-) V. cholerae populations and ICP1 strains possessing CRISPR-Cas or Odn with evidence of in situ recombination. These findings reinforce our understanding of the successive nature of V. cholerae evolution and suggest that ongoing surveillance of V. cholerae, ICP1, and PLE in Bangladesh is important for tracking genetic developments relevant to pandemic cholera that can occur over relatively short timescales. IMPORTANCE With 1 to 4 million estimated cases annually, cholera is a disease of serious global concern in regions where access to safe drinking water is limited by inadequate infrastructure, inequity, or natural disaster. The Global Task Force on Cholera Control (GTFCC.org) considers outbreak surveillance to be a primary pillar in the strategy to reduce mortality from cholera worldwide. Therefore, developing a better understanding of temporal evolutionary changes in the causative agent of cholera, Vibrio cholerae, could help in those efforts. The significance of our research is in tracking the genomic shifts that distinguish V. cholerae outbreaks, with specific attention paid to current and historical trends in the arms race between V. cholerae and a cooccurring viral (bacteriophage) predator. Here, we discover additional diversity of a specific phage defense system in epidemic V. cholerae and document the loss of a phage-encoded CRISPR-Cas system, underscoring the dynamic nature of microbial populations across cholera outbreaks.

Bacteriophage predation selects for diverse antiphage systems that frequently cluster on mobilizable defense islands in bacterial genomes. However, molecular insight into the reciprocal dynamics of phage-bacterial adaptations in nature is lacking, particularly in clinical contexts where there is need to inform phage therapy efforts and to understand how phages drive pathogen evolution. Using time-shift experiments, we uncovered fluctuations in Vibrio cholerae's resistance to phages in clinical samples. We mapped phage resistance determinants to SXT integrative and conjugative elements (ICEs), which notoriously also confer antibiotic resistance. We found that SXT ICEs, which are widespread in γ-proteobacteria, invariably encode phage defense systems localized to a single hotspot of genetic exchange. We identified mechanisms that allow phage to counter SXT-mediated defense in clinical samples, and document the selection of a novel phage-encoded defense inhibitor. Phage infection stimulates high-frequency SXT ICE conjugation, leading to the concurrent dissemination of phage and antibiotic resistances.

BackgroundOne of the most significant public health concerns in today's world is the persistent upsurge of infections caused by multidrug resistant bacteria. As a result, clinicians are being forced to intervene with either less effective backup drugs or ones with substantial side-effects. Colistin is a last resort antimicrobial agent for the treatment of infections caused by multi-drug resistant gram-negative bacteria.MethodsEscherichia coli (n = 65) isolated from street food (n = 20), hand rinse (n = 15), surface water (n = 10), and healthy human stool (n = 20) were tested for colistin resistance gene mcr-1 and response to antimicrobial agents. Antimicrobial resistance genes and virulence genes were detected by employing polymerase chain reaction. DNA fingerprinting of the strains were determined by pulsed-field gel electrophoresis.ResultsScreening of E. coli allowed us to confirm colistin resistance marker gene mcr-1 in 13 strains (street food, n = 4; hand rinse, n = 2; surface water, n = 4; and stool, n = 3); and two of these E. coli strains carrying mcr-1 harbored bla TEM gene encoding extended spectrum beta lactamase. Antibiotic assay results revealed all 13 E. coli strains carrying mcr-1 to be multi-drug resistant (MDR), including to colistin. The minimum inhibitory concentration (MIC) for colistin ranged from 2 to 6 μg/ml. DNA sequencing confirmed homogeneity of the nucleotide sequence for mcr-1, but the E. coli strains were heterogenous, as confirmed by pulsed-field gel electrophoresis suggesting horizontal transmission of colistin resistance in Bangladesh.ConclusionWidespread dissemination of E. coli strains carrying mcr-1 encoding resistance to colistin in the present study is alarming as this is the last resort drug for the treatment of infections caused by MDR gram-negative bacteria resistant to almost all drugs used commonly.

Phage defense systems are often found on mobile genetic elements (MGEs), where they constitutively defend against invaders or are induced to respond to new assaults. Phage satellites, one type of MGE, are induced during phage infection to promote their own transmission, reducing phage production and protecting their hosts in the process. One such satellite in Vibrio cholerae, phage-inducible chromosomal island-like element (PLE), sabotages the lytic phage ICP1, which triggers PLE excision from the bacterial chromosome, replication, and transduction to neighboring cells. Analysis of patient stool samples from different geographic regions revealed that ICP1 has evolved to possess one of two syntenic loci encoding an SF1B-type helicase, either of which PLE exploits to drive replication. Further, loss of PLE mobilization limits anti-phage activity because of phage-mediated degradation of the bacterial genome. Our work provides insight into the unique challenges facing parasites of lytic phages and underscores the adaptions of satellites to their ever-evolving target phage.

(1) Shigella spp. (Shigella) is known for causing dysentery with blood in stool, but most children infected with Shigella have non-dysentery Shigella-associated diarrhea (NDSD). The World Health Organization recommends the use of antibiotics when diarrhea is bloody, leaving most NDSD cases untreated. The absence of dysentery may not indicate a low risk of death and severe morbidity. Rapid diagnosis and treatment of shigellosis in vulnerable, young children may be lifesaving. INSIGHT aims to determine the potential benefit of identifying NDSD cases (n = 296) and their outcomes compared to cases of Shigella dysentery [DS (n = 148)] and non-Shigella watery diarrhea [WD (n = 148)]. (2) Children seeking care at hospitals in Bangladesh will be enrolled and followed for 1 year (NDSD and DS) or 90 days (WD). We will determine the impact of NDSD on morbidity, mortality, gut health, nutritional status, and cognitive development compared to DS and WD in children and whether the simple "Rapid LAMP-based Diagnostic Test (RLDT)" can accelerate the detection and treatment of shigellosis in the clinical settings of rural Bangladesh. (3) INSIGHT will determine the impact of NDSD in children and determine if the treatment guidelines of shigellosis need to be revisited to improve clinical outcomes and the development of these children. [ABSTRACT FROM AUTHOR]

Enterotoxigenic Escherichia coli (ETEC) infections undeniably continue to have substantial morbidity and mortality in younger children; however, limited data are available on the disease burden of older children and adults and on ETEC epidemiology by geographical location at the subnational level. Facility-based surveillance over the years was established to identify patients with ETEC diarrhea in two geographically distinct areas in rural Bangladesh, Chhatak in the north and Mathbaria in the southern coastal area. ETEC was highly prevalent in both areas, while the proportions, toxin types and colonization factors varied by location, season and age groups. Children < 5 years old and adults between 20 and 60 years old were at the highest risk of ETEC diarrhea which required urgent care. This study underscores the importance of capturing subnational and seasonal variations in ETEC epidemiology. ETEC vaccine developers and public health stakeholders may need to target adults between 20 and 60 years of age in addition to young children as new vaccines currently under development become licensed and introduction begins. [ABSTRACT FROM AUTHOR]

Author(s): Zillur Rahman[sup.1] , Aminul Islam[sup.1] , Mahamud-ur Rashid[sup.1] , Fatema-Tuz Johura[sup.1] , Shirajum Monira[sup.1] , Haruo Watanabe[sup.2] , Niyaz Ahmed[sup.1] , Andrew Camilli[sup.3] and Munirul Alam[sup.1] Background Acquisition of [...]

To the Editor: Cholera, caused by Vibrio cholerae, continues to affect millions of persons in disease-endemic areas where safe drinking water is scarce and sanitation is poor. Of 7 cholera [...]