ProBac-seq, a bacterial single-cell RNA sequencing methodology using droplet microfluidics and large oligonucleotide probe sets.

Methods that measure the transcriptomic state of thousands of individual cells have transformed our understanding of cellular heterogeneity in eukaryotic cells since their introduction in the past decade. While simple and accessible protocols and commercial products are now available for the processing of mammalian cells, these existing technologies are incompatible with use in bacterial samples for several fundamental reasons including the absence of polyadenylation on bacterial messenger RNA, the instability of bacterial transcripts and the incompatibility of bacterial cell morphology with existing methodologies. Recently, we developed ProBac sequencing (ProBac-seq), a method that overcomes these technical difficulties and provides high-quality single-cell gene expression data from thousands of bacterial cells by using messenger RNA-specific probes. Here we provide details for designing large oligonucleotide probe sets for an organism of choice, amplifying probe sets to produce sufficient quantities for repeated experiments, adding unique molecular indexes and poly-A tails to produce finalized probes, in situ probe hybridization and single-cell encapsulation and library preparation. This protocol, from the probe amplification to the library preparation, requires ~7 d to complete. ProBac-seq offers several advantages over other methods by capturing only the desired target sequences and avoiding nondesired transcripts, such as highly abundant ribosomal RNA, thus enriching for signal that better informs on cellular state. The use of multiple probes per gene can detect meaningful single-cell signals from cells expressing transcripts to a lesser degree or those grown in minimal media and other environmentally relevant conditions in which cells are less active. ProBac-seq is also compatible with other organisms that can be profiled by in situ hybridization techniques.

Altered molecular attributes and antimicrobial resistance patterns of Vibrio cholerae O1 El Tor strains isolated from the cholera endemic regions of India.

AIMS: The present study aimed to document the comparative analysis of differential hypervirulent features of Vibrio cholerae O1 strains isolated during 2018 from cholera endemic regions in Gujarat and Maharashtra (Western India) and West Bengal (Eastern India). METHODS AND RESULTS: A total of 87 V. cholerae O1 clinical strains from Western India and 48 from Eastern India were analysed for a number of biotypic and genotypic features followed by antimicrobial resistance (AMR) profile. A novel polymerase chain reaction was designed to detect a large fragment deletion in the Vibrio seventh pandemic island II (VSP-II) genomic region, which is a significant genetic feature of the V. cholerae strains that have caused Yemen cholera outbreak. All the strains from Western India belong to the Ogawa serotype, polymyxin B-sensitive, hemolytic, had a deletion in VSP-II (VSP-IIC) region and carried Haitian genetic alleles of ctxB, tcpA and rtxA. Conversely, 14.6% (7/48) of the strains from Eastern India belonged to the Inaba serotype, polymyxin B-resistant, nonhemolytic, harboured VSP-II other than VSP-IIC type, classical ctxB, Haitian tcpA and El Tor rtxA alleles. Resistance to tetracycline and chloramphenicol has been observed in strains from both regions. CONCLUSIONS: This study showed hypervirulent, polymyxin B-sensitive epidemic causing strains in India along with the strains with polymyxin B-resistant and nonhemolytic traits that may spread and cause serious disease outcomes in future. SIGNIFICANCE AND IMPACT OF THE STUDY: The outcomes of this study can help to improve the understanding of the hyperpathogenic property of recently circulating pandemic Vibrio cholerae strains in India. Special attention is also needed for the monitoring of AMR surveillance because V. cholerae strains are losing susceptibility to many antibiotics used as a second line of defence in the treatment of cholera.

Elucidating the correlation between the number of TTTTGAT heptamer repeats and cholera toxin promoter activity in Vibrio cholerae O1 pandemic strains.

A complex virulence-regulatory cascade controls expression of the cholera toxin genes (ctxAB) in Vibrio cholerae, which eventually leads to the production and secretion of choleragen (CT), responsible for rice watery diarrhoea in infected individuals. The cholera toxin promoter (PctxAB) contains a series of heptad repeats (5'-TTTTGAT-3'), which has previously been shown to play a crucial role in transcriptional regulation of ctxAB by recruiting the transcriptional activators ToxT, ToxR and the nucleoid-associated protein H-NS along the ctx promoter. The number of these repeats differs not only between the two biotypes of V. cholerae O1 strains, but also among the strains belonging to the same biotype. In this study, we examined if regulation of PctxAB is influenced in any way by the number of these repeats. Based on our observations, we posit that ctx activation indeed depends on the number of TTTTGAT heptad repeats within PctxAB, and occupation of the distal repeats by H-NS could prevent transcriptional activation of the ctx genes in V. cholerae O1 pandemic isolates. Our results suggest that ToxT-dependent transcriptional activation may not require entire displacement of H-NS and supports a recently described revised model of ToxT and H-NS mediated PctxAB transcriptional regulation.

Characterization of diarrhoeagenic Escherichia coli with special reference to antimicrobial resistance isolated from hospitalized diarrhoeal patients in Kolkata (2012-2019), India.

AIMS: This study analyses the prevalence and antimicrobial resistance (AMR) of major diarrhoeagenic Escherichia coli (DEC) pathotypes detected in hospitalized diarrhoeal patients in Kolkata, India, during 2012-2019. METHODS AND RESULTS: A total of 8891 stool samples were collected from the Infectious Diseases Hospital, Kolkata and screened for the presence of enteric pathogens. Multiplex PCR identified the presence of DEC in 7.8% of the samples, in which ETEC was most common (47.7%) followed by EAEC (38.4%) and EPEC (13.9%). About 54% cases were due to sole DEC infections. Majority of the mixed DEC infections were caused by the Vibrio spp. (19.1%) followed by Rotavirus (14.1%) and Campylobacter spp. (8.4%). ETEC and EAEC were associated significantly with diarrhoea in children <5 years of age, whereas EPEC and also ETEC were prevalent in patients aged between 5 and 14 years. AMR profile showed high prevalence of multidrug resistance (MDR) among DEC (56.9%) in which 9% were resistant to antibiotics of six different antimicrobial classes. Screening of the AMR conferring genes of DEC showed the presence of blaCTX-M3 (30.2%) in highest number followed by blaTEM (27.5%), tetB (18%), sul2 (12.6%), strA (11.8%), aadA1 (9.8%), blaOXA-1 (9%), dfrA1 (1.6%) and blaSHV (1.2%). CONCLUSIONS: These findings highlighted the high prevalence of MDR in major DEC pathotypes that could be considered as the leading aetiological bacterial agent responsible for diarrhoea and suggests a significant public health threat. SIGNIFICANCE AND IMPACT OF THE STUDY: The results of this study can help to improve the understanding of the epidemiology of DEC infections in patients with diarrhoea. Monitoring of AMR surveillance needs special attention because the DEC isolates were highly resistant to commonly used antimicrobials in the treatment of diarrhoea.

Recent Vibrio cholerae O1 Epidemic Strains Are Unable To Replicate CTXΦ Prophage Genome.

Cholera, an acute diarrheal disease, is caused by pathogenic strains of Vibrio cholerae generated by the lysogenization of the filamentous cholera toxin phage CTXΦ. Although CTXΦ phage in the classical biotype are usually integrated solitarily or with a truncated copy, those in El Tor biotypes are generally found in tandem and/or with related genetic elements. Due to this structural difference in the CTXΦ prophage array, the prophage in the classical biotype strains does not yield extrachromosomal CTXΦ DNA and does not produce virions, whereas the El Tor biotype strains can replicate the CTXΦ genome and secrete infectious CTXΦ phage particles. However, information on the CTXΦ prophage array structure of pathogenic V. cholerae is limited. Therefore, we investigated the complete genomic sequences of five clinical V. cholerae isolates obtained in Kolkata (India) during 2007 to 2011. The analysis revealed that recent isolates possessed an altered CTXΦ prophage array of the prototype El Tor strain. These strains were defective in replicating the CTXΦ genome. All recent isolates possessed identical rstA and intergenic sequence 1 (Ig-1) sequences and comparable rstA expression in the prototype El Tor strain, suggesting that the altered CTXΦ array was responsible for the defective replication of the prophage. Therefore, CTXΦ structures available in the database and literatures can be classified as replicative and nonreplicative. Furthermore, V. cholerae epidemic strains became capable of producing CTXΦ phage particles since the 1970s. However, V. cholerae epidemic strains again lost the capacity for CTXΦ production around the year 2010, suggesting that a significant change in the dissemination pattern of the current cholera pandemic occurred. IMPORTANCE Cholera is an acute diarrheal disease caused by pathogenic strains of V. cholerae generated by lysogenization of the filamentous cholera toxin phage CTXΦ. The analysis revealed that recent isolates possessed altered CTXΦ prophage array of prototype El Tor strain and were defective in replicating the CTXΦ genome. Classification of CTXΦ structures in isolated years suggested that V. cholerae epidemic strains became capable of producing CTXΦ phage particles since the 1970s. However, V. cholerae epidemic strains again lost the capacity for CTXΦ production around the year 2010, suggesting that a critical change had occurred in the dissemination pattern of the current cholera pandemic.

Whole-Genome Analysis of Clinical Vibrio cholerae O1 in Kolkata, India, and Dhaka, Bangladesh, Reveals Two Lineages of Circulating Strains, Indicating Variation in Genomic Attributes.

Vibrio cholerae serogroup O1 is responsible for epidemic and pandemic cholera and remains a global public health threat. This organism has been well established as a resident flora of the aquatic environment that alters its phenotypic and genotypic attributes for better adaptation to the environment. To reveal the diversity of clinical isolates of V. cholerae O1 in the Bay of Bengal, we performed whole-genome sequencing of isolates from Kolkata, India, and Dhaka, Bangladesh, collected between 2009 and 2016. Comparison with global isolates by phylogenetic analysis placed the current isolates in two Asian lineages, with lineages 1 and 2 predominant in Dhaka and Kolkata, respectively. Each lineage possessed different genetic traits in the cholera toxin B subunit gene, Vibrio seventh pandemic island II, integrative and conjugative element, and antibiotic-resistant genes. Thus, although recent global transmission of V. cholerae O1 from South Asia has been attributed only to isolates of lineage 2, another distinct lineage exists in Bengal.IMPORTANCE Cholera continues to be a global concern, as large epidemics have occurred recently in Haiti, Yemen, and countries of sub-Saharan Africa. A single lineage of Vibrio cholerae O1 has been considered to be introduced into these regions from South Asia and to cause the spread of cholera. Using genomic epidemiology, we showed that two distinct lineages exist in Bengal, one of which is linked to the global lineage. The other lineage was found only in Iran, Iraq, and countries in Asia and differed from the global lineage regarding cholera toxin variant and drug resistance profile. Therefore, the potential transmission of this lineage to other regions would likely cause worldwide cholera spread and may result in this lineage replacing the current global lineage.

Deciphering the possible role of ctxB7 allele on higher production of cholera toxin by Haitian variant Vibrio cholerae O1.

Cholera continues to be an important public health concern in developing countries where proper hygiene and sanitation are compromised. This severe diarrheal disease is caused by the Gram-negative pathogen Vibrio cholerae belonging to serogroups O1 and O139. Cholera toxin (CT) is the prime virulence factor and is directly responsible for the disease manifestation. The ctxB gene encodes cholera toxin B subunit (CTB) whereas the A subunit (CTA) is the product of ctxA gene. Enzymatic action of CT depends on binding of B pentamers to the lipid-based receptor ganglioside GM1. In recent years, emergence of V. cholerae Haitian variant strains with ctxB7 allele and their rapid spread throughout the globe has been linked to various cholera outbreaks in Africa and Asia. These strains produce classical type (WT) CTB except for an additional mutation in the signal sequence region where an asparagine (N) residue replaces a histidine (H) at the 20th amino acid position (H20N) of CTB precursor (pre-CTB). Here we report that Haitian variant V. cholerae O1 strains isolated in Kolkata produced higher amount of CT compared to contemporary O1 El Tor variant strains under in vitro virulence inducing conditions. We observed that the ctxB7 allele, itself plays a pivotal role in higher CT production. Based on our in silico analysis, we hypothesized that higher accumulation of toxin subunits from ctxB7 allele might be attributed to the structural alteration at the CTB signal peptide region of pre-H20N CTB. Overall, this study provides plausible explanation regarding the hypertoxigenic phenotype of the Haitian variant strains which have spread globally, possibly through positive selection for increased pathogenic traits.

A Point Mutation in carR Is Involved in the Emergence of Polymyxin B-Sensitive Vibrio cholerae O1 El Tor Biotype by Influencing Gene Transcription.

Antimicrobial peptides play an important role in host defense against Vibrio cholerae Generally, the V. cholerae O1 classical biotype is polymyxin B (PB) sensitive and El Tor is relatively resistant. Detection of classical biotype traits like the production of classical cholera toxin and PB sensitivity in El Tor strains has been reported in recent years, including in the devastating Yemen cholera outbreak during 2016-2018. To investigate the factor(s) responsible for the shift in the trend of sensitivity to PB, we studied the two-component system encoded by carRS, regulating the lipid A modification of El Tor vibrios, and found that only carR contains a single nucleotide polymorphism (SNP) in recently emerged PB-sensitive strains. We designated the two alleles present in PB-resistant and -sensitive strains carRr and carRs alleles, respectively, and replaced the carRs allele of a sensitive strain with the carRr allele, using an allelic-exchange approach. The sensitive strain then became resistant. The PB-resistant strain N16961 was made susceptible to PB in a similar fashion. Our in silico CarR protein models suggested that the D89N substitution in the more stable CarRs protein brings the two structural domains of CarR closer, constricting the DNA binding cleft. This probably reduces the expression of the carR-regulated almEFG operon, inducing PB susceptibility. Expression of almEFG in PB-sensitive strains was found to be downregulated under natural culturing conditions. In addition, the expression of carR and almEG decreased in all strains with increased concentrations of extracellular Ca2+ but increased with a rise in pH. The downregulation of almEFG in CarRs strains confirmed that the G265A mutation is responsible for the emergence of PB-sensitive El Tor strains.

Haitian-like genetic traits with creeping MIC of Azithromycin in Vibrio cholerae O1 isolates from Puducherry, India.

Introduction. The emergence of novel strains of Vibrio cholerae O1 El Tor biotype has gained attention due to causing several epidemics around the world. Variant strains have evolved as a result of the acquisition of genes that confer extended virulence and pathogenicity.Aim. This study aimed to determine the presence of the most recently emerging Haitian-like genetic traits among the isolates from Jawaharlal Institute of Postgraduate Medical Education and Research, Pondicherry, Southern India. We also wanted to detect the prevalence of the sulfamethoxazole and trimethoprim (SXT) element, which is an integrating conjugative element (ICE) and the antimicrobial resistance genes present in our isolates.Methodology. Identification of Haitian-specific alleles was done by mismatched amplification mutation assay PCR (MAMA-PCR). The presence of SXT elements was carried out by PCR by detecting int, eex, att-prfC and setR genes. Detection of antibiotic resistance determinant, sul(1,2,3); dfr(A1,18,5) for trimethoprim resistance, tet(A,B,C,D,E,Y,G,M), tet34 for tetracycline resistance and erm(A,B,C), mph(A,B), ere(A,B), msr(A,D) for azithromycin resistance were targeted by PCR. The MIC of tetracycline, ciprofloxacin and azithromycin was determined by the E-test method.Results. Of the 95 isolates, 60 % of the isolates were found to carry Haitian-specific alleles of ctxB, tcpA and rtxA gene, 100 % of the isolates were found to carry SXT elements. All the isolates harboured the four conserved genes of the SXT element, except one which had only eex, att-prfC, setR genes. About 99 % harboured sul2 and dfrA1 genes. No tet and macrolide genes were detected. We observed a progressive increase in the MIC of azithromycin ranging from 0.75 µg ml-1 to 2 µg ml-1.Conclusion. None of the isolates were the prototype El Tor biotype. All the isolates were a Haitian variant. The presence of SXT elements across all our isolates and their creeping MIC of azithromycin is a matter of concern. Further testing for other genetic determinants of resistance will be carried out in our future studies.

Post-monsoon waterlogging-associated upsurge of cholera cases in and around Kolkata metropolis, 2015.

The Infectious Diseases and Beliaghata General Hospital, Kolkata, India witnessed a sudden increase in admissions of diarrhoea cases during the first 2 weeks of August 2015 following heavy rainfall. This prompted us to investigate the event. Cases were recruited through hospital-based surveillance along with the collection of socio-demographic characteristics and clinical profile using a structured questionnaire. Stool specimens were tested at bacteriological laboratory of the National Institute of Cholera and Enteric Diseases (NICED), Kolkata. Admission of 3003 diarrhoea cases, clearly indicated occurrence of outbreak in Kolkata municipal area as it was more than two standard deviation of the mean number (911; s.d. = 111) of diarrhoea admissions during the same period in previous 7 years. Out of 164 recruited cases, 25% were under-5 children. Organisms were isolated from 80 (49%) stool specimens. Vibrio cholerae O1 was isolated from 50 patients. Twenty-eight patients had this organism as the sole pathogen. Among 14 infants, five had cholera. All V. cholerae O1 isolates were resistant to nalidixic acid, followed by co-trimoxazole (96%), streptomycin (92%), but sensitive to fluroquinolones. We confirmed the occurrence of a cholera outbreak in Kolkata during August 2015 due to V. cholerae O1 infection, where infants were affected.

Vibrio cholerae O6 gastroenteritis in a patient with lupus nephritis - a report from coastal Karnataka, South India.

INTRODUCTION: Vibrio cholerae O1 strains are responsible for pandemics of cholera and major epidemics in the world. All the remaining V. cholerae non-O1/non-O139 strains are less virulent and are responsible for sporadic cases of gastroenteritis. These non-O1/non-O139 serogroups have more than 200 somatic antigens, and mostly lack cholera toxin and toxin co-regulated pilus encoding genes. Toxigenic and non-toxigenic non-O1/non-O139 V. cholerae have caused several diarrhoeal outbreaks in India and other countries. Acute gastroenteritis is the typical clinical sign and symptom of non-O1/non-O139 V. cholerae infection for both periodical and outbreak cases; in contrast, these V. cholerae are rarely associated with extraintestinal infections. CASE PRESENTATION: Here, we present a case of a 27-year-old female with underlying kidney disease (lupus nephritis) presenting with loose stools, vomiting and fever. V. cholerae O6 was isolated from a faecal sample, which was positive for hlyA and the type III secretion system. The present case is, to the best of our knowledge, the first such case to be reported from South India. CONCLUSION: The V. cholerae O6 associated with autoimmune disease in the present study demonstrates the role of this pathogen in acute gastroenteritis, and if it is left undiagnosed it can lead to septicaemia and other complications. The pathogenic mechanisms of non-O1/non-O139 V. cholerae are multivariate, virulence factors being naturally present in these strains. Therefore, further epidemiological studies are necessary to determine the virulence factors and their pathogenic mechanisms. Non-O1/non-O139 V. cholerae can undoubtedly be the cause of diarrhoea and it would be important to extend bacteriological identification in this line as well as in all cases of gastroenteritis of unknown aetiology.

Haitian Variant Vibrio cholerae O1 Strains Manifest Higher Virulence in Animal Models.

Vibrio cholerae causes fatal diarrheal disease cholera in humans due to consumption of contaminated water and food. To instigate the disease, the bacterium must evade the host intestinal innate immune system; penetrate the mucus layer of the small intestine, adhere and multiply on the surface of microvilli and produce toxin(s) through the action of virulence associated genes. V. cholerae O1 that has caused a major cholera outbreak in Haiti contained several unique genetic signatures. These novel traits are used to differentiate them from the canonical El Tor strains. Several studies reported the spread of these Haitian variant strains in different parts of the world including Asia and Africa, but there is a paucity of information on the clinical consequence of these genetic changes. To understand the impact of these changes, we undertook a study involving mice and rabbit models to evaluate the pathogenesis. The colonization ability of Haitian variant strain in comparison to canonical El Tor strain was found to be significantly more in both suckling mice and rabbit model. Adult mice also displayed the same results. Besides that, infection patterns of Haitian variant strains showed a completely different picture. Increased mucosal damaging, colonization, and inflammatory changes were observed through hematoxylin-eosin staining and transmission electron microscopy. Fluid accumulation ability was also significantly higher in rabbit model. Our study indicated that these virulence features of the Haitian variant strain may have some association with the severe clinical outcome of the cholera patients in different parts of the world.

Dissemination of newly emerged polymyxin B sensitive Vibrio cholerae O1 containing Haitian-like genetic traits in different parts of India.

PURPOSE: Two natural epidemic biotypes of Vibrio cholerae O1, classical and El Tor, exhibit different patterns of sensitivity against the antimicrobial peptide polymyxin B. This difference in sensitivity has been one of the major markers in biotype classification system for several decades. A recent report regarding the emergence of polymyxin B-sensitive El Tor V. cholerae O1 in Kolkata has motivated us to track the spread of the strains containing this important trait, along with Haitian-like genetic content, in different parts of India. METHODOLOGY: We have collected 260 clinical V. cholerae O1 strains from 12 states in India and screened them for polymyxin B susceptibility. Genetic characterization was also performed to study the tcpA, ctxB and rtxA genotypes by allele-specific polymerase chain reaction (PCR) and nucleotide sequencing. RESULTS: Interestingly, 88.85 % of the isolates were found to be sensitive to polymyxin B. All of the states, with the exception of Assam, had polymyxin B-sensitive V. cholerae strains and complete replacement with this strain was found in eight of the states. However, from 2016 onwards, all the strains tested showed sensitivity to polymyxin B. Allele-specific PCR and sequencing confirmed that all strains possessed Haitian-like genetic traits. CONCLUSION: Polymyxin B-sensitive strains have begun to spread throughout India and may lead to the revision of the biotype classification. The dissemination of these new variant strains needs to be carefully monitored in different endemic populations through active holistic surveillance to understand their clinical and epidemiological consequences.

Characterization of Vibrio cholerae O1 strains that trace the origin of Haitian-like genetic traits.

Vibrio cholerae O1 is the etiological agent of the severe diarrheal disease cholera. The bacterium has recently been causing outbreaks in Haiti with catastrophic effects. Numerous mutations have been reported in V. cholerae O1 strains associated with the Haitian outbreak. These mutations encompass among other the genes encoding virulence factors such as the pilin subunit of the toxin-co-regulated pilus (tcpA), cholera toxin B subunit (ctxB), repeat in toxins (rtxA), and other genes such as the quinolone resistance-determining region (QRDR) of gyrase A (gyrA), rstB of RS element along with the alteration in the number of repeat sequences at the promoter region of ctxAB. Given the numerous genetic changes in those Haitian isolates, we decided to investigate the possible origins of those variations in the Indian subcontinent. Thus, we determined the genetic traits among V. cholerae O1 strains in Delhi, India. A total of 175 strains isolated from cholera patients during 2004 to 2012 were analysed in the present study. Our results showed that all the tested strains carried Haitian type tcpA (tcpACIRS) and variant gyrA indicating their first appearance before 2004 in Delhi. The Haitian variant rtxA and ctxB7 were first detected in Delhi during 2004 and 2006, respectively. Interestingly, not a single strain with the combination of El Tor rtxA and ctxB7 was detected in this study. The Delhi strains carried four heptad repeats (TTTTGAT) in the CT promoter region whereas Haitian strains carried 5 such repeats. Delhi strains did not have any deletion mutations in the rstB like Haitian strains. Overall, our study demonstrates the sequential accumulation of Haitian-like genetic traits among V. cholerae O1 strains in Delhi at different time points prior to the Haitian cholera outbreak.