Jugsharan Singh Virdi

MALDI-TOF mass spectrometry: an emerging technology for microbial identification and diagnosis

Currently microorganisms are best identified using 16S rRNA and 18S rRNA gene sequencing. However, in recent years matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has emerged as a potential tool for microbial identification and diagnosis. During the MALDI-TOF MS process, microbes are identified using either intact cells or cell extracts. The process is rapid, sensitive, and economical in terms of both labor and costs involved. The technology has been readily imbibed by microbiologists who have reported usage of MALDI-TOF MS for a number of purposes like, microbial identification and strain typing, epidemiological studies, detection of biological warfare agents, detection of water- and food-borne pathogens, detection of antibiotic resistance and detection of blood and urinary tract pathogens etc. The limitation of the technology is that identification of new isolates is possible only if the spectral database contains peptide mass fingerprints of the type strains of specific genera/species/subspecies/strains. This review provides an overview of the status and recent applications of mass spectrometry for microbial identification. It also explores the usefulness of this exciting new technology for diagnosis of diseases caused by bacteria, viruses, and fungi.

Emerging water-borne pathogens.

Abstract. Emerging water-borne pathogens constitute a major health hazard in both developed and developing nations. A new dimension to the global epidemiology of cholera—an ancient scourge—was provided by the emergence of Vibrio cholerae O139. Also, water-borne enterohaemorrhagic Escherichia coli (E. coli O157:H7), although regarded as a problem of the industrialized west, has recently caused outbreaks in Africa. Outbreaks of chlorine-resistant Cryptosporidium have motivated water authorities to reassess the adequacy of current water-quality regulations. Of late, a host of other organisms, such as hepatitis viruses (including hepatitis E virus), Campylobacter jejuni, microsporidia, cyclospora, Yersinia enterocolitica, calciviruses and environmental bacteria like Mycobacterium spp, aeromonads, Legionella pneumophila and multidrug-resistant Pseudomonas aeruginosa have been associated with water-borne illnesses. This review critically examines the potential of these as emerging water-borne pathogens. It also examines the possible reasons, such as an increase in the number of immunocompromised individuals, urbanization and horizontal gene transfer, that may underlie their emergence. Further, measures required to face the challenge posed by these pathogens are also discussed.

The Enigma of Yersinia enterocolitica biovar 1A

Yersinia enterocolitica, an important food- and water-borne enteropathogen causes acute diarrhea, terminal ileitis, and mesenteric lymphadenitis. It is represented by six biovars (1A, 1B, 2-5). The biovar 1A strains are generally regarded as avirulent as they lack pYV plasmid and major chromosomal virulence genes. Despite this, some biovar 1A strains produce disease symptoms indistinguishable from that produced by known pathogenic biovars (1B, 2-5). Suggested prospective studies to understand pathogenic potential of biovar 1A should focus on role of insecticidal toxins, urease, protease, superoxide dismutase, and host responses. These studies should also take into account the clonal groups of biovar 1A.

Detection of Yersinia enterocolitica in food: an overview

Yersinia enterocolitica is a gastrointestinal pathogen which causes yersiniosis, an illness characterized by diarrhea, ileitis, and mesenteric lymphadenitis. Y. enterocolitica is transmitted via the feco-oral route by the consumption of contaminated food or water. Several phenotypic and genotypic methods have been developed to reliably detect Y. enterocolitica in food. However, the source of infection of many recently reported foodborne outbreaks remains obscure. The detection of this pathogen in food is a challenging task, since it shares similarities with other enteric bacteria. The presence of other microorganisms in the food samples makes it even more difficult to identify this slow-growing pathogen. Therefore, the present-day emphasis is on the development of sensitive, easily automated methods suitable for in-situ detection, allowing quick and cost-effective characterization of food samples. This review summarizes and compares the currently available cultural, immunological, and molecular methods, particularly in relation to their specific merits or demerits when implemented for the detection of Y. enterocolitica in food.

Distribution and molecular characterization of genes encoding CTX-M and AmpC β-lactamases in Escherichia coli isolated from an Indian urban aquatic environment

Aquatic environments harboring antibiotic resistant Escherichia coli constitute an important public health concern. Thus, it is important to characterize the resistance genetic elements of waterborne E. coli. It is also important to identify the predominant clonal groups/phylogroups represented by resistant strains to understand the epidemiology of antibiotic resistant E. coli in natural environments, and to identify the role of well-established genotypes in the spread of resistance in a particular geographical area through natural environments. In the present investigation, E. coli strains (n=126) isolated from various points along the river Yamuna traversing through the National Capital Territory of Delhi (India) were grouped phylogenetically. A collection of 61 strains representing all phylogroups was investigated for extended-spectrum β-lactamase (ESBL) and AmpC production. blaTEM, blaSHV and blaCTX-M genes were detected and analyzed, promoter/attenuator mutations associated with chromosomally-mediated AmpC overexpression were identified, and plasmid-mediated ampC was determined. blaTEM was the most widespread (100%) gene followed by bla(CTX-M) (16%), and plasmid-mediated ampC (3%). bla(CTX-M-15) and bla(CMY-42) were identified as the genes encoding CTX-M type ESBL and CIT type AmpC β-lactamases, respectively. CTX-M-15 ESBL phenotype was most common in phylogroup D (50%), followed by phylogroups B1 (30%), and A (20%). E. coli that produce plasmid-mediated AmpC were rare and present only in phylogroup D. Presence of multi β-lactam resistance, bla(CTX-M-15) and bla(CMY-42) in waterborne E. coli belonging to virulence-associated phylogroup D highlights the need for routine surveillance of resistance determinants in aquatic environments. This is also the first report for the presence of bla(CMY-42) in waterborne E. coli.

Mycobacterium tuberculosis Cyclophilin A Uses Novel Signal Sequence for Secretion and Mimics Eukaryotic Cyclophilins for Interaction with Host Protein Repertoire

Cyclophilins are prolyl isomerases with multitude of functions in different cellular processes and pathological conditions. Cyclophilin A (PpiA) of Mycobacterium tuberculosis is secreted during infection in intraphagosomal niche. However, our understanding about the evolutionary origin, secretory mechanism or the interactome of M. tuberculosis PpiA is limited. This study demonstrates through phylogenetic and structural analyses that PpiA has more proximity to human cyclophilins than the prokaryotic counterparts. We report a unique N-terminal sequence (MADCDSVTNSP) present in pathogenic mycobacterial PpiA and absent in non-pathogenic strains. This sequence stretch was shown to be essential for PpiA secretion. The overexpression of full-length PpiA from M. tuberculosis in non-pathogenic Mycobacterium smegmatis resulted in PpiA secretion while truncation of the N-terminal stretch obstructed the secretion. In addition, presence of an ESX pathway substrate motif in M. tuberculosis PpiA suggested possible involvement of Type VII secretion system. Site-directed mutagenesis of key residues in this motif in full-length PpiA also hindered the secretion in M. smegmatis. Bacterial two-hybrid screens with human lung cDNA library as target were utilized to identify interaction partners of PpiA from host repertoire, and a number of substrates with functional representation in iron storage, signal transduction and immune responses were detected. The extensive host interactome coupled with the sequence and structural similarity to human cyclophilins is strongly suggestive of PpiA being deployed by M. tuberculosis as an effector mimic against the host cyclophilins.

Genetic relationships between clinical and non-clinical strains of Yersinia enterocolitica biovar 1A as revealed by multilocus enzyme electrophoresis and multilocus restriction typing

BackgroundGenetic relationships among 81 strains of Y. enterocolitica biovar 1A isolated from clinical and non-clinical sources were discerned by multilocus enzyme electrophoresis (MLEE) and multilocus restriction typing (MLRT) using six loci each. Such studies may reveal associations between the genotypes of the strains and their sources of isolation.ResultsAll loci were polymorphic and generated 62 electrophoretic types (ETs) and 12 restriction types (RTs). The mean genetic diversity (H) of the strains by MLEE and MLRT was 0.566 and 0.441 respectively. MLEE (DI = 0.98) was more discriminatory and clustered Y. enterocolitica biovar 1A strains into four groups, while MLRT (DI = 0.77) identified two distinct groups. BURST (Based Upon Related Sequence Types) analysis of the MLRT data suggested aquatic serotype O:6,30-6,31 isolates to be the ancestral strains from which, clinical O:6,30-6,31 strains might have originated by host adaptation and genetic change.ConclusionMLEE revealed greater genetic diversity among strains of Y. enterocolitica biovar 1A and clustered strains in four groups, while MLRT grouped the strains into two groups. BURST analysis of MLRT data nevertheless provided newer insights into the probable evolution of clinical strains from aquatic strains.

CBMAR: a comprehensive β-lactamase molecular annotation resource.

β-Lactam antibiotics are among the most widely used antibiotics against microbial pathogens. However, enzymatic hydrolysis of these antibiotics by bacterial β-lactamases is increasingly compromising their efficiency. Although new generation β-lactam antibiotics have been developed to combat antibiotic resistance, β-lactamases have also evolved along with the new variants of the substrate. A strong selection pressure from the newer generation of β-lactam antibiotics has resulted in evolution of different families within each class of β-lactamase. To facilitate detailed characterization of different families of β-lactamases, we have created a database, CBMAR, which facilitates comprehensive molecular annotation and discovery of novel β-lactamases. As against the limited scope of other existing similar databases, CBMAR provides information useful for molecular and biochemical characterization of each family of β-lactamase. The basic architecture of CBMAR is based on Ambler classification, which divides β-lactamases as serine (Classes A, C and D) and metallo-β-lactamases (Class B). Each class is further divided into several families on the basis of their hydrolytic character. In CBMAR, each family is annotated with (i) sequence variability, (ii) antibiotic resistance profile, (iii) inhibitor susceptibility, (iv) active site, (v) family fingerprints, (vi) mutational profile, (vii) variants, (viii) gene location, (ix) phylogenetic tree and several other features. Each entry also has external links to the relevant protein/nucleotide sequence and structure databases. The database also supports sequence similarity searches using BLAST and assigns a new β-lactamase protein to its respective family on the basis of family-specific fingerprint. Database URL: http://14.139.227.92/mkumar/lactamasedb

Amelioratory Effects of Zinc Supplementation on Salmonella-induced Hepatic Damage in the Murine Model

Zinc (Zn) has been reported to influence the susceptibility of the host to a diverse range of infectious pathogens, including viruses, bacteria, fungi and protozoa. We report here an evaluation of the effects of Zn supplementation on Salmonella enterica serovar Typhimurium (S. typhimurium)-induced hepatic injury in the murine model. Zinc levels in the plasma and liver tissues were measured by atomic absorption spectroscopy. The effect of Zn supplementation was evaluated by assessing the bacterial load and levels of lipid peroxidation (LPO), antioxidants and monokines present in the hepatic tissue as well as by histopathological studies. Zinc supplementation reduced the bacterial load in the liver and reversed hepatic microscopic abnormalities. It also decreased the levels of LPO but increased the levels of reduced glutathione (GSH) as well as the activities of superoxide-dismutase (SOD) and catalase in the livers of infected mice supplemented with Zn compared to the livers of infected mice not supplemented with Zn. Zinc supplementation was also able to modulate the levels of monokines such as tumour necrosis factor alpha (TNF-α), interleukin-1 (IL-1) and interleukin-6 (IL-6). Our results indicate a role for Zn in downregulating oxidative stress and upregulating antioxidant defense enzymes through the action of monokines, suggesting that supplementation with Zn has a protective function in Salmonella-induced liver injury.

Quinolone co-resistance in ESBL- or AmpC-producing Escherichia coli from an Indian urban aquatic environment and their public health implications

Quinolone and β-lactam antibiotics constitute major mainstay of treatment against infections caused by pathogenic Escherichia coli. Presence of E. coli strains expressing co-resistance to both these antibiotic classes in urban aquatic environments which are consistently being used for various anthropogenic activities represents a serious public health concern. From a heterogeneous collection of 61 E. coli strains isolated from the river Yamuna traversing through the National Capital Territory of Delhi (India), those harboring blaCTX-M-15 (n = 10) or blaCMY-42 (n = 2) were investigated for co-resistance to quinolones and the molecular mechanisms thereof. Resistance was primarily attributed to amino acid substitutions in the quinolone resistance-determining regions (QRDRs) of GyrA (S83L ± D87N) and ParC (S80I ± E84K). One of the E. coli strains, viz., IPE, also carried substitutions in GyrB and ParE at positions Ser492→Asn and Ser458→Ala, respectively. The phenotypically susceptible strains nevertheless carried plasmid-mediated quinolone resistance (PMQR) gene, viz., qnrS, which showed co-transfer to the recipient quinolone-sensitive E. coli J53 along with the genes encoding β-lactamases and led to increase in minimal inhibitory concentrations of quinolone antibiotics. To the best of our knowledge, this represents first report of molecular characterization of quinolone co-resistance in E. coli harboring genes for ESBLs or AmpC β-lactamases from a natural aquatic environment of India. The study warrants true appreciation of the potential of urban aquatic environments in the emergence and spread of multi-drug resistance and underscores the need to characterize resistance genetic elements vis-à-vis their public health implications, irrespective of apparent phenotypic resistance.