Cryo-EM structure of GABA transporter 1 reveals substrate recognition and transport mechanism.

The inhibitory neurotransmitter γ-aminobutyric acid (GABA) is cleared from the synaptic cleft by the sodium- and chloride-coupled GABA transporter GAT1. Inhibition of GAT1 prolongs the GABAergic signaling at the synapse and is a strategy to treat certain forms of epilepsy. In this study, we present the cryo-electron microscopy structure of Rattus norvegicus GABA transporter 1 (rGAT1) at a resolution of 3.1 Å. The structure elucidation was facilitated by epitope transfer of a fragment-antigen binding (Fab) interaction site from the Drosophila dopamine transporter (dDAT) to rGAT1. The structure reveals rGAT1 in a cytosol-facing conformation, with a linear density in the primary binding site that accommodates a molecule of GABA, a displaced ion density proximal to Na site 1 and a bound chloride ion. A unique insertion in TM10 aids the formation of a compact, closed extracellular gate. Besides yielding mechanistic insights into ion and substrate recognition, our study will enable the rational design of specific antiepileptics.

Spectrum of ctxB genotypes, antibiogram profiles and virulence genes of Vibrio cholerae serogroups isolated from environmental water sources from Odisha, India.

BACKGROUND: The present study reports on the comprehensive analysis of Vibrio cholerae O1 and non-O1/non-O139 serogroups isolated from environmental water sources during cholera outbreaks, epidemics and surveillance studies between years 2007 to 2019 from different districts of Odisha, India. METHODS: A total of 85 stocked cultures of V. cholerae O1 and non-O1/non-O139 strains were analyzed for different ctxB genotypes, toxic genes, antibiogram profiles through PCR assays and pulsotyped by pulsed-field gel electrophoresis (PFGE). RESULTS: From all V. cholerae strains tested, 51 isolates were O1 Ogawa and the rest 34 strains were non-O1/non-O139. All the V. cholerae O1 strains were altered El Tor variants carrying ctxB1, ctxB3 and ctxB7 genotypes. However, only ctxB1 genotypes were present in V. cholerae non-O1/non-O139. Though non-O1/non-O139 strains were negative by O1 antisera, 20% strains were positive for rfbO1 gene by PCR assay. All the V. cholerae isolates possessed a variety of virulence genes including ace, ctxAB, toxR, zot, hlyA which were in higher percentage in the case of V. cholerae O1. The Vibrio cholerae O1 and non-O1-/non-O139 strains showed multiple antibiotic resistances in 2007 and 2012. The PCR detection of four resistance associated genes (strB, dfrA1, sulll, SXT) confirmed higher prevalence in V. cholerae non-O1/non-O139 strains. The PFGE analysis revealed 3 pulsotypes having 93% similarity among V. cholerae O1 strains. CONCLUSION: This study indicates the changing epidemiology, antibiogram patterns and continuous genetic variation in environmental V. cholerae strains of Odisha over the years. So continuous surveillance is necessary to understand the changing patterns of V. cholerae different serogroups isolated from stool and water samples from Odisha.

Sequential outbreaks due to mixed ctxB alleles of Vibrio cholerae O1 in Mayurbhanj district of Odisha, India.

INTRODUCTION: Cholera is a significant threat causing outbreaks/epidemics with high morbidity and mortality in coastal and tribal districts of Odisha. A sequential cholera outbreak reported from four places in Mayurbhanj district of Odisha during June to July 2009 was investigated. METHODOLOGY: Rectal swabs from diarrhea patients were analyzed for the identification, antibiogram profiles and detection of ctxB genotypes by double mismatch amplification mutation (DMAMA) polymerase chain reaction (PCR) assays and sequenced. The different virulent and drug resistant genes were detected by multiplex PCR assays. The clonality analysis on selected strains was done by pulse field gel electrophoresis (PFGE). RESULTS: Bacteriological analysis of rectal swabs revealed the presence of V. cholerae O1 Ogawa biotype El Tor which were resistant to co-trimoxazole, chloramphenicol, streptomycin, ampicillin, nalidixic acid, erythromycin, furazolidone and polymyxin B. DMAMA-PCR assay revealed that the cholera outbreak in Mayurbhanj district was due to both ctxB1 and ctxB7 alleles of V. cholerae O1 El Tor strains. All the V. cholerae O1 strains were positive for all virulence genes. The multiplex PCR assay on V. cholerae O1 strains revealed the presence of antibiotic resistance genes like dfrA1 (100%), intSXT (100%), sulII (62.5%) and StrB (62.5%). PFGE results on V. cholerae O1 strains exhibited two different pulsotypes with 92% similarity. CONCLUSIONS: This outbreak was a transition phase where both ctxB genotypes were prevalent after which the ctxB7 genotype gradually became dominant in Odisha. Therefore, close monitoring and continuous surveillance on diarrheal disorders is essential to prevent the future diarrheal outbreaks in this region.

Structural insights into GABA transport inhibition using an engineered neurotransmitter transporter.

γ-aminobutyric acid (GABA) is the major inhibitory neurotransmitter, and its levels in the synaptic space are controlled by the GABA transporter isoforms (GATs). GATs are structurally related to biogenic amine transporters but display interactions with distinct inhibitors used as anti-epileptics. In this study, we engineer the binding pocket of Drosophila melanogaster dopamine transporter to resemble GAT1 and determine high-resolution X-ray structures of the modified transporter in the substrate-free state and in complex with GAT1 inhibitors NO711 and SKF89976a that are analogs of tiagabine, a medication prescribed for the treatment of partial seizures. We observe that the primary binding site undergoes substantial shifts in subsite architecture in the modified transporter to accommodate the two GAT1 inhibitors. We also observe that SKF89976a additionally interacts at an allosteric site in the extracellular vestibule, yielding an occluded conformation. Interchanging SKF89976a interacting residue in the extracellular loop 4 between GAT1 and dDAT suggests a role for this motif in the selective control of neurotransmitter uptake. Our findings, therefore, provide vital insights into the organizational principles dictating GAT1 activity and inhibition.

Genomic diversities of ctxB, tcpA and rstR alleles of Vibrio cholerae O139 strains isolated from Odisha, India.

The genome of Vibrio cholerae O139 strains has undergone cryptic changes since its first emergence in 1992 in South India. This study aimed to determine the presence of genotypic changes marked in ctxB, tcpA and rstR genes located within the CTX prophages among the strains of V. cholerae O139 isolated from 1999 to 2017 in Odisha. Antibiotic susceptibility test was conducted on 59 V. cholerae O139 strains. A conventional PCR assay was done for ctxB gene typing followed by sequencing along with identification of rstR and tcpA gene. Pulsed-field gel electrophoresis (PFGE) was carried out to reveal clonal variations among the V. cholerae O139 strains. Among V. cholerae O139 isolates more than 60% showed resistance to ampicillin, co-trimoxazole, furazolidone, streptomycin, neomycin and nalidixic acid. The ctxB sequencing and rstR allele-specific PCR assay revealed the presence of three genotypes 1, 3 and 4 with at least one copy of CTX Calc φ in addition to CTX ET and CTX Cl prophages in V. cholerae O139 isolates. PFGE analysis revealed 13 pulsotypes with two clades having 60% similarity among V. cholerae O139 strains. The circulating V. cholerae O139 strains in Odisha showed variation in genotypes with multiple clonal expansions over the years.

Variants of ctxB alleles of Vibrio cholerae O1 caused sequential cholera outbreaks in the tribal areas of Odisha, India.

Cholera localized outbreaks/epidemics accounting for high morbidity and mortality have been reported in different years both from the coastal and tribal districts of Odisha. In the present study, the emergence and spread of two sequential cholera outbreaks reported in July to October 2012 from Rayagada and Kalahandi districts of Odisha was investigated. Environmental water samples from different sources and rectal swabs from diarrhoea patients were analysed for identification, antibiogram profiles and molecular studies using DMAMA-PCR assays. The pulsed field gel electrophoresis (PFGE) was done on some selected Vibrio cholerae O1 strains isolated from these cholera outbreak areas. Results showed 42% of rectal swabs and 2.3% of water samples collected from both the districts were positive for Vibrio cholerae O1 Ogawa biotype El Tor carrying both ctxB1 and ctxB7 genotypes. The common resistance profile of V. cholerae O1 strains was ampicillin, nalidixic acid, furazolidone and co-trimoxazole. The PFGE analysis on selected V. cholerae O1 strains of ctxB1 and ctxB7 genotypes showed three pulsotypes with 96% similarity matrix exhibiting the relationship with their respective water sources. Hence, continuous surveillance is highly essential to monitor the antibiogram profile and changing pattern of ctxB genotypes of V. cholerae O1 in this region.

Origin and Dissemination of Altered El Tor Vibrio cholerae O1 Causing Cholera in Odisha, India: Two and Half Decade's View.

The origin, spread and molecular epidemiology of altered El Tor Vibrio cholerae O1 strains isolated from cholera outbreaks/surveillance studies between 1995 and 2019 from different district of Odisha were analyzed. The stock cultures of V. cholerae O1 strains from 1995 to 2019 were analyzed through molecular analysis using different PCR assays and pulse field gel electrophoresis (PFGE) analysis. The spread map (month, year and place) was constructed to locate the dissemination of altered El Tor variants of V. cholerae O1 in this region. A total of 13 cholera outbreaks were caused by V. cholerae O1 Ogawa biotype El Tor carrying ctxB1 and ctxB7 genotypes. The ctxB1 alleles of V. cholerae O1 mostly confined to the coastal areas, whereas the ctxB7 genotypes, though originating in the coastal region of Odisha, concentrated more in the tribal areas. The positive correlation between virulence-associated genes (VAGs) was found through Pearson's correlation model, indicative of a stronger association between the VAGs. The clonal relationship through PFGE between ctxB1 and ctxB7 genotypes of V. cholerae O1 strains exhibited 80% similarity indicating single- or multi-clonal evolution. It is evident from this study that the spread of multidrug-resistant V. cholerae O1-altered El Tor was dominant over the prototype El Tor strains in this region. The origin of altered El Tor variants of V. cholerae O1 occurred in the East Coast of Odisha established that the origin of cholera happened in the Gangetic belts of Bay of Bengal where all new variants of V. cholerae O1 might have originated from the Asian countries.

Haitian variant Vibrio cholerae O1 Ogawa caused cholera outbreaks in Odisha.

PURPOSE: Diarrheal disorders particularly cholera cause a significant threat resulting in high morbidity and mortality in the coastal and tribal areas of Odisha. Two sequential diarrheal outbreaks reported in 2016 from Balasore and Rayagada districts of Odisha were investigated to find out the causative organisms, antibiogram profile and molecular analysis of the isolated pathogens. METHOD: Bacteriological analysis and antibiogram profiles of the pathogens were carried out as per the standard procedure followed. The double mismatch amplification mutation (DMAMA) PCR for ctxB gene, sequencing and pulse-field gel electrophoresis (PFGE) were carried out on Vibrio cholerae O1 strains. RESULTS: The rectal swabs and water samples from these districts were positive for V. cholerae O1 Ogawa biotype El Tor. The V. cholerae O1 strains isolated from Balasore district were multidrug resistant to many antibiotics which differed from the isolates of Rayagada district. The DMAMA PCR assay on all clinical and water isolates from these areas and some strains from other districts exhibited ctxB7 allele of V. cholerae O1 which correlates with the sequencing results having different pulsotypes. The Haitian variant of V. cholerae O1 strains which were compared with the V. cholerae O1 strains of 1999 and 2000 exhibited different pulsotypes. CONCLUSION: The present study reports cholera outbreaks due to multidrug resistant ctxB7 allele of V. cholerae O1 from both coastal (Balasore) and tribal (Rayagada) areas of Odisha.

Emergence and spread of different ctxB alleles of Vibrio cholerae O1 in Odisha, India.

This study reports variants of the ctxB allele of Vibrio cholerae O1 isolated between 1995 and 2019 in Odisha, India. ctxB1 genotypes dominated from 1995 to 2016. The Haitian variant and El Tor ctxB3 genotypes of V. cholerae O1 emerged in 1999, and were most common in 2018-2019 and 2005-2011, respectively. The ctxB7 genotype of the Haitian variant of V. cholerae O1 was quiescent from 2000 to 2006, but further spread was noted from 2007 to 2019.

Dissemination of Vibrio cholerae O1 isolated from Odisha, India.

The present study reported the antimicrobial susceptibility trends, virulence genes, and drug resistance genes of Vibrio cholerae O1 strains isolated from outbreaks and epidemics over two and half decades (1995-2019) from Odisha, India. Antimicrobial susceptibility testing was performed by disc diffusion method. Virulence and drug resistance genes were detected by multiplex PCR assays. All V. cholerae O1 strains were sensitive to gentamicin, chloramphenicol, norfloxacin and ciprofloxacin while resistant to one or more antibiotics used. About 90% of the isolates of V. cholerae O1 carried antibiotic drug resistant genes (SulII, dfrA1 and strB) and SXT elements and the results correlated with the phenotypic antibiotic data obtained through disc diffusion assay. The tcpA Haitian variant V. cholerae O1 first appeared in 1999, gradually showing its increasing number upto 2019. TcpA El Tor strains only prevailed from 1995 to 2006; whereas the tcpA classical strains of V.choleraeO1 were found in less number from 1995 to 2016. Two multiplex PCR assays confirmed the presence of various toxigenic and virulence genes (toxR, ompU, ace, rtxC, ctxA, tcpA, rfbO1 and ompW) in all isolate of V. cholerae O1 strains. The present findings demonstrated the origin and spread of Haitian variants tcpA in V. cholerae O1 strains over two and half decades.

Environmental reservoirs of Vibrio cholerae serogroups in the flowing freshwater environs from the tribal areas of Odisha, Eastern India.

The environmental reservoirs of different serogroups of Vibrio cholerae causing cholera in the flowing freshwater bodies of the tribal areas of Odisha are not known. So the present study was conducted from June 2017 to March 2020 to find out the environmental reservoirs of V. cholerae serogroups in the water and plankton samples collected from the river, nala, stream and chua from Rayagada district. Similarly, rectal swabs were collected from diarrhoea patients and correlation was established among the V. cholerae strains isolated from diarrhoea patients and environmental V. cholerae isolates through routine culture, different multiplex PCR assays and pulse field gel electrophoresis (PFGE) analysis using standard techniques. The multiplex PCR assays on biotypes and different toxic genes exhibited similar correlation between the clinical and water isolates, which was further strengthened by PFGE analysis. The planktonic DNA was positive for ctxA gene which established that the environmental water bodies were the reservoirs for virulence genes of V. cholerae serogroups. The detection of environmental reservoirs of V. cholerae serogroups in temporarily stagnant condition of water; partially encircled by stones, and near the bank of the river, nala and stream were the reservoirs which is a rare report from Odisha, India and Globe.

Dissemination of Polymyxin B Sensitivity in El Tor Vibrio cholerae O1 Strains in Odisha, India.

The Vibrio species undergo cryptic changes in their genetic material for better adaptability, which accounts for antibiotic resistance. In the present study, we investigated the emergence and spread of sensitivity to polymyxin B (PB) by El Tor V. cholerae O1 strains from 1995 to 2019 in Odisha, India. The results showed that out of 1200 V. cholerae O1 strains, 89.4% were resistant and the remaining 10.6% strains were sensitive to PB. The sensitivity to PB of V. cholerae O1 strains emerged from 2005 to 2019, except in 2015, clearly signifying the presence of classical biotype characteristics in the El Tor variant of V. cholerae O1 strains. The Etest assay revealed some interesting traits of PB susceptibility in the ctxB1 and ctxB7 genotypes of V. cholerae O1 strains. The minimum inhibitory concentration (MIC) of ctxB7 genotypes showed reduced MIC values of ≤ 4 µg/mL, whereas ctxB1 genotypes exhibited higher MIC values of 24 and 32 µg/mL.

Spread of Haitian Variant Vibrio cholerae O1 Causing Cholera Outbreaks in Odisha, India.

Cholera posed a significant threat causing outbreaks/epidemics with high morbidity and mortality in Odisha. This study envisages the characterisation of isolated pathogen from two cholera outbreaks reported in 2018 and 2019 from Bargarh and Rayagada districts of Odisha respectively. Vibrio cholerae O1 were isolated following standard techniques. The different virulent and drug resistant genes were detected by multiplex PCR assays; whereas the ctxB genotypes were characterised through double mismatch amplification mutation (DMAMA) PCR assay. The ctxB genes were further sequenced and pulse-field gel electrophoresis (PFGE) was done on some selected strains. The clinical and water isolates of Haitian variant (HCT) V. cholerae O1 Ogawa biotype El Tor with multi drug resistant strains were isolated from both the places. All the V. cholerae O1 strains were positive for virulence genes. The antibiotic resistant genes like dfrA1 (100%), strB (76.9%), intSXT (61.5%) were detected. The PFGE results on V. cholerae O1 strains exhibited two different pulsotypes. These cholera outbreaks were due to multidrug resistant HCT variant V. cholerae O1 strains which were circulating and caused the cholera outbreaks in Odisha. So continuous surveillance on diarrheal disorders is highly essential to prevent the future diarrheal outbreaks in this region.

Incidence of Bacterial Enteropathogens among Diarrhea Patients from Tribal Areas of Odisha.

Infectious diarrheal diseases remain a leading cause of morbidity and mortality in developing and underdeveloped countries. The present study documented the etiology of bacterial enteropathogens in three tribal districts of Odisha from July 2010 to September 2013. A total of 1427 rectal swabs were collected and bacteriologically analyzed by following standard procedure. Among the 930 (65.2%) culture positive samples, Escherichia coli (E. coli) constituted 636 (44.6%); Vibrio cholerae (V. cholerae) O1, 146 (10.2%); Salmonella species (spp.), 10 (0.7%); Shigella spp., 79 (5.5%); and Aeromonas spp., 59 (4.1%). Of the 729 environmental water samples taken from river, open well, Nala (a small stream), and Chua (a shallow pit on a river bed), 14 (1.9%) contained non-O1/non-O139 V. cholerae and 13 (1.8%) had V. cholerae O1 strains. An analysis of the demographics showed that people in the 14 to 40-year age group were highly susceptible to diarrhea caused by V. cholerae which occurred mainly during the rainy and post-rainy seasons. All enteropathogens were multidrug-resistant and found throughout the study period. The V. cholerae strains isolated were El Tor variants carrying the classical, El Tor, and Haitian cholera toxin subunit B (ctxB) genes. The classical ctxB was the dominant allele, and the prevalence of the Haitian ctxB allele increased during the test period. These findings indicate that active surveillance is needed to monitor the changing antibiotic resistance patterns of V. cholerae serogroups and biotypes present in this region.

Epidemiology and Antibiogram Profile of Vibrio cholerae Isolates between 2004-2013 from Odisha, India.

Cholera is an acute diarrheal disease caused by Vibrio cholerae serogroups O1 and O139, which are known to cause epidemics of cholera in Odisha. The present study was intended to document the antibiotic resistance pattern among clinical isolates of both serogroups of V. cholerae (O1 and O139) isolated during 2004-2013. Nine-hundred nine isolates of V. cholerae were included in this study and were identified by standard procedures. An antibiotic sensitivity test was performed by the disc diffusion method. The seasonality of cholera in this region indicated that there was one peak in the rainy season only. The number of cholera cases started increasing from July and declined starting from the month of October onward. The adult age group of patients was the worst affected among all age groups of patients. The 2 different serogroups of V. cholerae (O1 and O139) showed different prevalence rates (%) of resistance to all the antibiotics in each year. Serogroup O1 showed uniformly high resistance to co-trimoxazole, furazolidone, and nalidixic acid throughout the study. Chloramphenicol encountered resistance only during 2009, but the strains were sensitive in the other years. The emergence of multiple drug-resistant V. cholerae strains may significantly influence the control of future outbreaks and epidemics of cholera in this region.

Vibrio cholerae O1 Ogawa Strains Carrying the ctxB7 Allele Caused a Large Cholera Outbreak during 2014 in the Tribal Areas of Odisha, India.

The large outbreak of cholera reported during July to September 2014 in the Narla block of Kalahandi district, India, was investigated to determine the causative organism. Rectal swabs collected from patients with diarrhea and environmental water samples were cultured following standard techniques. The causative organism was identified as Vibrio cholerae O1 Ogawa biotype El Tor, and analysis by double mismatch mutation assay PCR confirmed that all strains were the ctxB7 variant of Haitian V. cholerae O1. The environmental water samples were negative for V. cholerae. The V. cholerae O1 strains were sensitive to tetracycline, ciprofloxacin, norfloxacin, ofloxacin, doxycycline, and azithromycin, but were resistant to erythromycin, gentamicin, chloramphenicol, furazolidone, neomycin, cotrimoxazole, nalidixic acid, and ampicillin. In the 2014 cholera outbreak, the early reporting of the pathogen enabled the government authorities to implement adequate control measures in time to curtail the spread of the disease. That was the second large cholera outbreak due to Haitian variants of V. cholerae O1 after the 2010 Haiti cholera outbreak reported from Odisha, India, and other locations globally. Active surveillance is required to track the spread of this strain in the Odisha region.