Sukhadeo B. Barbuddhe

Rapid Identification and Typing of Listeria Species by Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry†

ABSTRACT Listeria monocytogenes is a food-borne pathogen that is the causative agent of human listeriosis, an opportunistic infection that primarily infects pregnant women and immunologically compromised individuals. Rapid, accurate discrimination between Listeria strains is essential for appropriate therapeutic management and timely intervention for infection control. A rapid method involving matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) that shows promise for identification of Listeria species and typing and even allows for differentiation at the level of clonal lineages among pathogenic strains of L. monocytogenes is presented. A total of 146 strains of different Listeria species and serotypes as well as clinical isolates were analyzed. The method was compared with the pulsed-field gel electrophoresis analysis of 48 Listeria strains comprising L. monocytogenes strains isolated from food-borne epidemics and sporadic cases, isolates representing different serotypes, and a number of Listeria strains whose genomes have been completely sequenced. Following a short inactivation/extraction procedure, cell material from a bacterial colony was deposited on a sample target, dried, overlaid with a matrix necessary for the MALDI process, and analyzed by MALDI-TOF MS. This technique examines the chemistry of major proteins, yielding profile spectra consisting of a series of peaks, a characteristic “fingerprint” mainly derived from ribosomal proteins. Specimens can be prepared in a few minutes from plate or liquid cultures, and a spectrum can be obtained within 1 minute. Mass spectra derived from Listeria isolates showed characteristic peaks, conserved at both the species and lineage levels. MALDI-TOF MS fingerprinting may have potential for Listeria identification and subtyping and may improve infection control measures.

Reassessment of the Listeria monocytogenes pan-genome reveals dynamic integration hotspots and mobile genetic elements as major components of the accessory genome

BackgroundListeria monocytogenes is an important food-borne pathogen and model organism for host-pathogen interaction, thus representing an invaluable target considering research on the forces governing the evolution of such microbes. The diversity of this species has not been exhaustively explored yet, as previous efforts have focused on analyses of serotypes primarily implicated in human listeriosis. We conducted complete genome sequencing of 11 strains employing 454 GS FLX technology, thereby achieving full coverage of all serotypes including the first complete strains of serotypes 1/2b, 3c, 3b, 4c, 4d, and 4e. These were comparatively analyzed in conjunction with publicly available data and assessed for pathogenicity in the Galleria mellonella insect model.ResultsThe species pan-genome of L. monocytogenes is highly stable but open, suggesting an ability to adapt to new niches by generating or including new genetic information. The majority of gene-scale differences represented by the accessory genome resulted from nine hyper variable hotspots, a similar number of different prophages, three transposons (Tn916, Tn554, IS3-like), and two mobilizable islands. Only a subset of strains showed CRISPR/Cas bacteriophage resistance systems of different subtypes, suggesting a supplementary function in maintenance of chromosomal stability. Multiple phylogenetic branches of the genus Listeria imply long common histories of strains of each lineage as revealed by a SNP-based core genome tree highlighting the impact of small mutations for the evolution of species L. monocytogenes. Frequent loss or truncation of genes described to be vital for virulence or pathogenicity was confirmed as a recurring pattern, especially for strains belonging to lineages III and II. New candidate genes implicated in virulence function were predicted based on functional domains and phylogenetic distribution. A comparative analysis of small regulatory RNA candidates supports observations of a differential distribution of trans-encoded RNA, hinting at a diverse range of adaptations and regulatory impact.ConclusionsThis study determined commonly occurring hyper variable hotspots and mobile elements as primary effectors of quantitative gene-scale evolution of species L. monocytogenes, while gene decay and SNPs seem to represent major factors influencing long-term evolution. The discovery of common and disparately distributed genes considering lineages, serogroups, serotypes and strains of species L. monocytogenes will assist in diagnostic, phylogenetic and functional research, supported by the comparative genomic GECO-LisDB analysis server (http://bioinfo.mikrobio.med.uni-giessen.de/geco2lisdb).

Listeria as an enteroinvasive gastrointestinal pathogen.

The bacterium Listeria monocytogenes is the causative agent of listeriosis, a highly fatal opportunistic foodborne infection. Listeria spp. are isolated from a diversity of environmental sources, including soil, water, effluents, a large variety of foods, and the feces of humans and animals. Recent outbreaks demonstrated that L. monocytogenes can cause gastroenteritis in otherwise healthy individuals and more severe invasive disease in immunocompromised patients. Common symptoms include fever, watery diarrhea, nausea, headache, and pains in joints and muscles. The intestinal tract is the major portal of entry for L. monocytogenes, whereby strains penetrate the mucosal tissue either directly, via invasion of enterocytes, or indirectly, via active penetration of the Peyers patches. Studies have revealed the strategy taken by the bacteria to overcome changes in oxygen tension, osmolarity, acidity, and the sterilizing effects of bile or antimicrobial peptides to adapt to conditions in the gut. In addition, L. monocytogenes has evolved species-specific strategies for intestinal entry by exploiting the interaction between the internalin protein and its receptor E-cadherin, or inducing diarrhea and an inflammatory response via the activity of its hemolytic toxin, listeriolysin. The ability of these bacteria to survive in bile-rich environments, and to induce depletion of sentinel cells such as Paneth cells that monitor the luminal burden of commensal bacteria, suggest strategies that have evolved to promote intestinal survival. Preexisting gastrointestinal disease may be a risk factor for infection of the gastrointestinal tract with L. monocytogenes. Currently, there is enough evidence to warrant consideration of L. monocytogenes as a possible etiology in outbreaks of febrile gastroenteritis, and for further studies to examine the genetic structure of Listeria strains that have a propensity to cause gastrointestinal versus systemic infections.

Effect of nisin and its combination with sodium chloride on the survival of Listeria monocytogenes added to raw buffalo meat mince

Antilisterial activity of nisin (Nisaplin), alone at concentrations of 400 and 800 IU/g and in combination with 2% sodium chloride was incorporated in raw buffalo meat mince. Samples of the raw meat mince were inoculated with 10(3) colony forming units (cfu)/g of L. monocytogenes and stored at 4°C for 16 days and at 37°C for 36 h. Initial estimates of pH, extract release volume, mesophilic and psychrophilic counts were found to be 5.74, 48 ml, 3.5×10(5) and 1.0×10(5) cfu/g of meat, respectively. The growth of L. monocytogenes in the treated groups was significantly (P<0.05) inhibited compared to the control group. The degree of inhibition increased with increasing concentration of nisin and decreasing storage temperature. Addition of 2% sodium chloride in combination with nisin increased the efficacy of nisin at both storage temperatures. The pH in the treated groups remained significantly lower (P<0.01) than in the control groups at both 4 and 37°C.

Listeria monocytogenes in spontaneous abortions in humans and its detection by multiplex PCR

Aim: To assess the extent of Listeria monocytogenes in causation of human spontaneous abortions by isolation methods and PCR analysis for the presence of virulence‐associated genes.

PCR-based detection of genes encoding virulence determinants in Staphylococcus aureus from bovine subclinical mastitis cases

The present study was carried out to genotypically characterize Staphylococcus aureus (S. aureus) isolated from bovine mastitis cases. A total of 37 strains of S. aureus were isolated during processing of 552 milk samples from 140 cows. The S. aureus strains were characterized phenotypically, and were further characterized genotypically by polymerase chain reaction using oligonucleotide primers that amplified genes encoding coagulase (coa), clumping factor (clfA), thermonuclease (nuc), enterotoxin A (entA), and the gene segments encoding the immunoglobulin G binding region and the X region of protein A gene spa. All of the isolates yielded an amplicon with a size of approximately 1,042 bp of the clfA gene. The amplification of the polymorphic spa gene segment encoding the immunoglobulin G binding region was observed in 34 isolates and X-region binding was detected in 26 isolates. Amplification of the coa gene yielded three different products in 20, 10, and 7 isolates. The amplification of the thermonuclease gene, nuc, was observed in 36 out of 37 isolates. All of the samples were negative for the entA gene. The phenotypic and genotypic findings of the present strategies might provide an understanding of the distribution of the prevalent S. aureus clones among bovine mastitis isolates, and might aid in the development of steps to control S. aureus infections in dairy herds.

Multiplex PCR assay for species identification of bovine mastitis pathogens

Aim:  To develop and evaluate a multiplex PCR (mPCR) assay for simultaneous detection of 10 bacterial species causing bovine mastitis namely, Staphylococcus aureus, Staphylococcus chromogenes, Staphylococcus epidermidis, Staphylococcus sciuri, Staphylococcus haemolyticus, Staphylococcus simulans, Streptococcus agalactiae, Streptococcus dysgalactiae, Streptococcus uberis and Escherichia coli in milk.

Detection of intercellular adhesion genes and biofilm production in Staphylococcus aureus isolated from bovine subclinical mastitis

Biofilm production by Staphylococcus aureus, an important virulence factor was investigated employing phenotypic and genotypic methods. A total of 102 S. aureus isolates from bovine subclinical mastitis cases were included in the study. Maximum number of biofilm producing strains were detected by Congo red agar (CRA) method (48.03%) followed by tube method (36.27%). Tissue culture plate method (TCP) without and with destaining identified 19.60 and 29.41% of S. aureus as biofilm producers, respectively. A polymerase chain reaction for detection of intercellular adhesion genes, icaA and icaD, responsible for biofilm formation was standardized. Of the 102 S. aureus isolates investigated, 36 (35.29%) strains revealed presence of both the genes. Considering polymerase chain reaction as a standard test, CRA and TCP without destaining were the most sensitive and specific, respectively. PCR technique standardized for detection of the icaA and icaD genes is reliable for identifying biofilm producing potential of S. aureus which may help in rapid detection of biofilm-producer Staphylococci. This would allow the early application of control measures.

Comparison of PCR, Immunofluorescence Assay, and Pathogen Isolation for Diagnosis of Q Fever in Humans with Spontaneous Abortions

ABSTRACT Coxiella burnetii, an obligate intracellular parasite with a worldwide distribution, is the causative agent of Q fever in humans. We tested a total of 368 samples (placental bits, genital swabs, fecal swabs, and urine and serum samples) collected from women (n = 74) with spontaneous abortions for C. burnetii by a PCR assay targeting IS1111, the repetitive transposon-like region of C. burnetii (trans-PCR); real-time PCR; an indirect immunofluorescence assay (IFA); and the isolation of the pathogen. The IFA showed seropositivity for 25.68% of the women with spontaneous abortions, whereas trans-PCR and real-time PCR each detected the pathogen in 21.62% of cases. Overall, 25.68% of the subjects were positive by one or more assays. Real-time PCR showed a slightly higher level of sensitivity than trans-PCR. With the IFA as the reference, the two PCR assays showed a higher level of sensitivity (84.21%) than pathogen isolation (26.31%), while both the PCR assays and pathogen isolation were specific (100%). The detection of high numbers of C. burnetii cells in clinical samples and the frequent association of the pathogen with cases of spontaneous abortions observed in this study revealed that Q fever remains underdiagnosed and that the prevalence in India is underestimated.

Biofilm-Forming Abilities of Listeria monocytogenes Serotypes Isolated from Different Sources.

A total of 98 previously characterized and serotyped L. monocytogenes strains, comprising 32 of 1/2a; 20 of 1/2b and 46 of 4b serotype, from clinical and food sources were studied for their capability to form a biofilm. The microtiter plate assay revealed 62 (63.26%) strains as weak, 27 (27.55%) strains as moderate, and 9 (9.18%) strains as strong biofilm formers. Among the strong biofilm formers, 6 strains were of serotype 1/2a and 3 strains were of serotype 1/2b. None of the strain from 4b serotype exhibited strong biofilm formation. No firm correlation (p = 0.015) was noticed between any serotype and respective biofilm formation ability. Electron microscopic studies showed that strong biofilm forming isolates could synthesize a biofilm within 24 h on surfaces important in food industries such as stainless steel, ceramic tiles, high-density polyethylene plastics, polyvinyl chloride pipes, and glass. Cell enumeration of strong, moderate, and weak biofilm was performed to determine if the number of cells correlated with the biofilm-forming capabilities of the isolates. Strong, moderate, and weak biofilm showed 570±127× 103 cells/cm2, 33±26× 103 cells/cm2, 5±3× 103 cells/cm2, respectively, indicating that the number of cells was directly proportional to the strength of the biofilm. The hydrophobicity index (HI) analysis revealed higher hydrophobicity with an increased biofilm formation. Fatty acid methyl esterase analysis revealed the amount of certain fatty acids such as iso-C15:0, anteiso-C15:0, and anteiso-C17:0 fatty acids correlated with the biofilm-forming capability of L. monocytogenes. This study showed that different strains of L. monocytogenes form biofilm of different intensities which did not completely correlate with their serotype; however, it correlated with the number of cells, hydrophobicity, and amount of certain fatty acids.