The lion's share of bacteria in various environments cannot be cloned in the laboratory and thus cannot be sequenced using existing technologies. A major goal of single-cell genomics is to complement gene-centric metagenomic data with whole-genome assemblies of uncultivated organisms. Assembly of single-cell data is challenging because of highly non-uniform read coverage as well as elevated levels of sequencing errors and chimeric reads. We describe SPAdes, a new assembler for both single-cell and standard (multicell) assembly, and demonstrate that it improves on the recently released E+V-SC assembler (specialized for single-cell data) and on popular assemblers Velvet and SoapDeNovo (for multicell data). SPAdes generates single-cell assemblies, providing information about genomes of uncultivatable bacteria that vastly exceeds what may be obtained via traditional metagenomics studies. SPAdes is available online ( http://bioinf.spbau.ru/spades ). It is distributed as open source software.

SPAdes, a new genome assembly algorithm and its applications to single-cell sequencing ( 7th Annual SFAF Meeting, 2012)

The causes of antibiotic resistance are complex and include human behaviour at many levels of society; the consequences affect everybody in the world. Similarities with climate change are evident. Many efforts have been made to describe the many different facets of antibiotic resistance and the interventions needed to meet the challenge. However, coordinated action is largely absent, especially at the political level, both nationally and internationally. Antibiotics paved the way for unprecedented medical and societal developments, and are today indispensible in all health systems. Achievements in modern medicine, such as major surgery, organ transplantation, treatment of preterm babies, and cancer chemotherapy, which we today take for granted, would not be possible without access to effective treatment for bacterial infections. Within just a few years, we might be faced with dire setbacks, medically, socially, and economically, unless real and unprecedented global coordinated actions are immediately taken. Here, we describe the global situation of antibiotic resistance, its major causes and consequences, and identify key areas in which action is urgently needed.

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Antibiotic resistance—the need for global solutions

Summary Background Not all patients infected with NDM-1-positive bacteria have a history of hospital admission in India, and extended-spectrum β-lactamases are known to be circulating in the Indian community. We therefore measured the prevalence of the NDM-1 gene in drinking water and seepage samples in New Delhi. Methods Swabs absorbing about 100 μL of seepage water (ie, water pools in streets or rivulets) and 15 mL samples of public tap water were collected from sites within a 12 km radius of central New Delhi, with each site photographed and documented. Samples were transported to the UK and tested for the presence of the NDM-1 gene, bla NDM-1 , by PCR and DNA probing. As a control group, 100 μL sewage effluent samples were taken from the Cardiff Wastewater Treatment Works, Tremorfa, Wales. Bacteria from all samples were recovered and examined for bla NDM-1 by PCR and sequencing. We identified NDM-1-positive isolates, undertook susceptibility testing, and, where appropriate, typed the isolates. We undertook Inc typing on bla NDM-1 -positive plasmids. Transconjugants were created to assess plasmid transfer frequency and its relation to temperature. Findings From Sept 26 to Oct 10, 2010, 171 seepage samples and 50 tap water samples from New Delhi and 70 sewage effluent samples from Cardiff Wastewater Treatment Works were collected. We detected bla NDM-1 in two of 50 drinking-water samples and 51 of 171 seepage samples from New Delhi; the gene was not found in any sample from Cardiff. Bacteria with bla NDM-1 were grown from 12 of 171 seepage samples and two of 50 water samples, and included 11 species in which NDM-1 has not previously been reported, including Shigella boydii and Vibrio cholerae . Carriage by enterobacteria, aeromonads, and V cholera was stable, generally transmissible, and associated with resistance patterns typical for NDM-1; carriage by non-fermenters was unstable in many cases and not associated with typical resistance. 20 strains of bacteria were found in the samples, 12 of which carried bla NDM-1 on plasmids, which ranged in size from 140 to 400 kb. Isolates of Aeromonas caviae and V cholerae carried bla NDM-1 on chromosomes. Conjugative transfer was more common at 30°C than at 25°C or 37°C. Interpretation The presence of NDM-1 β-lactamase-producing bacteria in environmental samples in New Delhi has important implications for people living in the city who are reliant on public water and sanitation facilities. International surveillance of resistance, incorporating environmental sampling as well as examination of clinical isolates, needs to be established as a priority. Funding European Union.

https://www.thelancet.com/pdfs/journals/laninf/PIIS1473-3099(11)70059-7.pdf

Dissemination of NDM-1 positive bacteria in the New Delhi environment and its implications for human health: an environmental point prevalence study

Although Escherichia coli can be an innocuous resident of the gastrointestinal tract, it also has the pathogenic capacity to cause significant diarrheal and extraintestinal diseases. Pathogenic variants of E. coli (pathovars or pathotypes) cause much morbidity and mortality worldwide. Consequently, pathogenic E. coli is widely studied in humans, animals, food, and the environment. While there are many common features that these pathotypes employ to colonize the intestinal mucosa and cause disease, the course, onset, and complications vary significantly. Outbreaks are common in developed and developing countries, and they sometimes have fatal consequences. Many of these pathotypes are a major public health concern as they have low infectious doses and are transmitted through ubiquitous mediums, including food and water. The seriousness of pathogenic E. coli is exemplified by dedicated national and international surveillance programs that monitor and track outbreaks; unfortunately, this surveillance is often lacking in developing countries. While not all pathotypes carry the same public health profile, they all carry an enormous potential to cause disease and continue to present challenges to human health. This comprehensive review highlights recent advances in our understanding of the intestinal pathotypes of E. coli.

Recent Advances in Understanding Enteric Pathogenic Escherichia coli

The global problem of antimicrobial resistance is particularly pressing in developing countries, where the infectious disease burden is high and cost constraints prevent the widespread application of newer, more expensive agents. Gastrointestinal, respiratory, sexually transmitted, and nosocomial infections are leading causes of disease and death in the developing world, and management of all these conditions has been critically compromised by the appearance and rapid spread of resistance. In this first part of the review, we have summarised the present state of resistance in these infections from the available data. Even though surveillance of resistance in many developing countries is suboptimal, the general picture is one of accelerating rates of resistance spurred by antimicrobial misuse and shortfalls in infection control and public health. Reservoirs for resistance may be present in healthy human and animal populations. Considerable economic and health burdens emanate from bacterial resistance, and research is needed to accurately quantify the problem and propose and evaluate practicable solutions. In part II, to be published next month, we will review potential containment strategies that could address this burgeoning problem.

https://www.thelancet.com/pdfs/journals/laninf/PIIS1473-3099(05)70189-4.pdf

Antimicrobial resistance in developing countries. Part I: recent trends and current status

Under stress conditions, many species of bacteria enter into starvation mode of metabolism or a physiologically viable but non-culturable (VBNC) state. Several human pathogenic bacteria have been reported to enter into the VBNC state under these conditions. The pathogenic VBNC bacteria cannot be grown using conventional culture media, although they continue to retain their viability and express their virulence. Though there have been debates on the VBNC concept in the past, several molecular studies have shown that not only can the VBNC state be induced under in vitro conditions but also that resuscitation from this state is possible under appropriate conditions. The most notable advance in resuscitating VBNC bacteria is the discovery of resuscitation-promoting factor (Rpf), which is a bacterial cytokines found in both Gram-positive and Gram-negative organisms. VBNC state is a survival strategy adopted by the bacteria, which has important implication in several fields, including environmental monitoring, food technology, and infectious disease management; and hence it is important to investigate the association of bacterial pathogens under VBNC state and the water/foodborne outbreaks. In this review, we describe various aspects of VBNC bacteria, which include their proteomic and genetic profiles under the VBNC state, conditions of resuscitation, methods of detection, antibiotic resistance, and observations on Rpf.

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Current Perspectives on Viable but Non-Culturable (VBNC) Pathogenic Bacteria.

Shigella species represent one of the growing numbers of antimicrobial-resistant bacteria in developing countries. Fluoroquinolone-resistant strains of Shigella dysenteriae type1 and Shigella flexneri type 2a emerged in India during 2002 and 2003, respectively. Sixty strains of Shigella from different parts of India were analysed for antimicrobial susceptibility, the presence of the qnr plasmid, mutations in the quinolone resistance determining regions (QRDRs), fluoroquinolone accumulation, and the presence of other genes encoding resistance to various antimicrobials. Fluoroquinolone-resistant strains had mutations in gyrA and parC genes and had an active efflux system. They were also resistant to several other antimicrobials but were susceptible to azithromycin and ceftriaxone. The majority of the strains harboured genes encoding resistance to ampicillin (97 %), tetracycline (95 %), streptomycin (95 %) and chloramphenicol (94 %). PFGE analysis revealed clonality among strains of S. dysenteriae types 1 and 5, S. flexneri type 2a and Shigella boydii type 12.

Molecular characterization of multidrug-resistant Shigella species isolated from epidemic and endemic cases of shigellosis in India.

We identified 281 Vibrio cholerae non-O1, non-O139 strains from patients with diarrhea in Kolkata, India. Cholera-like diarrhea was the major symptom (66.0%); some patients (20.3%) had severe dehydration. These strains lacked the ctxA gene but many had hlyA, rtxA, and rtxC genes. Pulsed-field gel electrophoresis showed no genetic link among strains.

https://ousar.lib.okayama-u.ac.jp/files/public/5/56677/20190515172531265927/Emerg_Infect_Dis_19_3_464.pdf

Vibrio cholerae non-O1, non-O139 serogroups and cholera-like diarrhea, Kolkata, India

Vibrio fluvialis is a pathogen commonly found in coastal environs. Considering recent increase in numbers of diarrheal outbreaks and sporadic extraintestinal cases, V. fluvialis has been considered as an emerging pathogen. Though this pathogen can be easily isolated by existing culture methods, its identification is still a challenging problem due to close phenotypic resemblance either with Vibrio cholerae or Aeromonas spp. However, using molecular tools, it is easy to identify V. fluvialis from clinical and different environmental samples. Many putative virulence factors have been reported, but its mechanisms of pathogenesis and survival fitness in the environment are yet to be explored. This chapter covers some of the major discoveries that have been made to understand the importance of V. fluvialis.

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Vibrio fluvialis: an emerging human pathogen.

A PCR-based assay was developed to discriminate the classical, El Tor, and Haitian types of ctxB alleles. Our retrospective study using this newly developed PCR showed that Haitian ctxB first appeared in Kolkata during April 2006, and 93.3% of strains isolated during 2011 carried the new allele. Dendrogram analysis showed a pulsed-field gel electrophoresis (PFGE) pattern of the new variant strains isolated recently that was distinct from the PFGE pattern of the strains carrying classical ctxB that closely matched the 2006 to 2007 variant strains.

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Development and Evaluation of a PCR Assay for Tracking the Emergence and Dissemination of Haitian Variant ctxB in Vibrio cholerae O1 Strains Isolated from Kolkata, India

Gururaja P. Pazhani

Papers

Current Perspectives on Viable but Non-Culturable (VBNC) Pathogenic Bacteria.

Molecular characterization of multidrug-resistant Shigella species isolated from epidemic and endemic cases of shigellosis in India.

Vibrio cholerae non-O1, non-O139 serogroups and cholera-like diarrhea, Kolkata, India

Vibrio fluvialis: an emerging human pathogen.

Development and Evaluation of a PCR Assay for Tracking the Emergence and Dissemination of Haitian Variant ctxB in Vibrio cholerae O1 Strains Isolated from Kolkata, India