Roy D. Sleator

'Big data', Hadoop and cloud computing in genomics

Since the completion of the Human Genome project at the turn of the Century, there has been an unprecedented proliferation of genomic sequence data. A consequence of this is that the medical discoveries of the future will largely depend on our ability to process and analyse large genomic data sets, which continue to expand as the cost of sequencing decreases. Herein, we provide an overview of cloud computing and big data technologies, and discuss how such expertise can be used to deal with biologys big data sets. In particular, big data technologies such as the Apache Hadoop project, which provides distributed and parallelised data processing and analysis of petabyte (PB) scale data sets will be discussed, together with an overview of the current usage of Hadoop within the bioinformatics community.

High-pressure processing--effects on microbial food safety and food quality.

High-pressure processing (HPP) is a nonthermal process capable of inactivating and eliminating pathogenic and food spoilage microorganisms. This novel technology has enormous potential in the food industry, controlling food spoilage, improving food safety and extending product shelf life while retaining the characteristics of fresh, preservative-free, minimally processed foods. As with other food processing methods, such as thermal processing, HPP has somewhat limited applications as it cannot be universally applied to all food types, such as some dairy and animal products and shelf-stable low-acid foods. Herein, we discuss the effects of high-pressure processing on microbial food safety and, to a lesser degree, food quality.

Acinetobacter baumannii: an emerging opportunistic pathogen.

Acinetobacter baumannii is an opportunistic bacterial pathogen primarily associated with hospital-acquired infections. The recent increase in incidence, largely associated with infected combat troops returning from conflict zones, coupled with a dramatic increase in the incidence of multidrug-resistant (MDR) strains, has significantly raised the profile of this emerging opportunistic pathogen. Herein, we provide an overview of the pathogen, discuss some of the major factors that have led to its clinical prominence and outline some of the novel therapeutic strategies currently in development.

Contribution of Three Bile-Associated Loci, bsh, pva, and btlB, to Gastrointestinal Persistence and Bile Tolerance of Listeria monocytogenes

ABSTRACT Listeria monocytogenes must resist the deleterious actions of bile in order to infect and subsequently colonize the human gastrointestinal tract. The molecular mechanisms used by the bacterium to resist bile and the influence of bile on pathogenesis are as yet largely unexplored. This study describes the analysis of three genes—bsh, pva, and btlB—previously annotated as bile-associated loci in the sequenced L. monocytogenes EGDe genome (lmo2067, lmo0446, and lmo0754, respectively). Analysis of deletion mutants revealed a role for all three genes in resisting the acute toxicity of bile and bile salts, particularly glycoconjugated bile salts at low pH. Mutants were unaffected in the other stress responses examined (acid, salt, and detergents). Bile hydrolysis assays demonstrate that L. monocytogenes possesses only one bile salt hydrolase gene, namely, bsh. Transcriptional analyses and activity assays revealed that, although it is regulated by both PrfA and σB, the latter appears to play the greater role in modulating bsh expression. In addition to being incapable of bile hydrolysis, a sigB mutant was shown to be exquisitely sensitive to bile salts. Furthermore, increased expression of sigB was detected under anaerobic conditions and during murine infection. A gene previously annotated as a possible penicillin V amidase (pva) or bile salt hydrolase was shown to be required for resistance to penicillin V but not penicillin G but did not demonstrate a role in bile hydrolysis. Finally, animal (murine) studies revealed an important role for both bsh and btlB in the intestinal persistence of L. monocytogenes.

Bacterial stress response in Listeria monocytogenes: jumping the hurdles imposed by minimal processing

Minimal processing relies on the use of multiple sub-lethal stresses (or processes) to achieve a similar level of microbial control as that traditionally achieved using a single lethal stress. The benefit to the consumer is products which are less obviously processed than a frozen or canned, acidified or heavily salted food item. However, our increasing understanding of how bacteria can adapt to sub-lethal stresses in a manner which can render them less susceptible to additional insults, should be borne in mind when designing safety or extended shelf-life into a minimally processed product. Listeria monocytogenes is a target organism for many minimally processed food manufacturers because of its ability to tolerate adverse conditions such as low Aw and low temperature. In this communication we use L. monocytogenes as a model system to describe some of the consequences of stress adaptation in terms of improved survival in minimally processed foods and, importantly, the consequences in terms of the virulence of the target organism.

Analysis of the Role of OpuC, an Osmolyte Transport System, in Salt Tolerance and Virulence Potential of Listeria monocytogenes

ABSTRACT The success of Listeria monocytogenes as a food-borne pathogen owes much to its ability to survive a variety of stresses, both in the external environment prior to ingestion and subsequently within the animal host. Growth at high salt concentrations and low temperatures is attributed mainly to the accumulation of organic solutes such as glycine betaine and carnitine. We utilized a novel system for generating chromosomal mutations (based on a lactococcal pWVO1-derived Ori+ RepA− vector, pORI19) to identify a listerial OpuC homologue. Mutating the operon in two strains of L. monocytogenes revealed significant strain variation in the observed activity of OpuC. Radiolabeled osmolyte uptake studies, together with growth experiments in defined media, linked OpuC to carnitine and glycine betaine uptake inListeria. We also investigated the role of OpuC in contributing to the growth and survival of Listeria in an animal (murine) model of infection. Altering OpuC resulted in a significant reduction in the ability of Listeria to colonize the upper small intestine and cause subsequent systemic infection following peroral inoculation.

A Postgenomic Appraisal of Osmotolerance in Listeria monocytogenes

A characteristic feature of the intracellular food-borne pathogen Listeria monocytogenes is its ability to survive and even proliferate under a variety of hostile environmental conditions, particularly elevated osmolarity (10% NaCl) ([47][1]) and reduced temperature (−0.1°C) ([75][2]). Early

A PrfA-regulated bile exclusion system (BilE) is a novel virulence factor in Listeria monocytogenes.

The ability to colonize the gall bladder has recently been shown to be an important feature of virulent Listeria monocytogenes (J. Hardy, K. P. Francis, M. DeBoer, P. Chu, K. Gibbs, C. H. Contag. Science 303: 851–853, 2004). We suggest that the cytotoxic effects of bile may be increased upon release from the gall bladder into the upper small intestine, and report the identification of a novel bile exclusion system which plays an essential role in intestinal colonization and virulence of L. monocytogenes. In silico analysis of the L. monocytogenes EGDe genome revealed a two‐gene operon (formerly opuB) exhibiting significant sequence similarity to members of the betaine carnitine choline transporter (BCCT) family. The operon, herein designated bilE (bile Exclusion) is preceded by consensus σA‐ and σB‐dependent promoter‐binding sites and is transcriptionally upregulated at elevated osmolarities and reduced temperatures (stresses known to induce sigB). Furthermore, a significant reduction in the level of bilE transcription was observed in the absence of σB. In addition, we demonstrate an important role for PrfA, the master regulator of virulence potential in L. monocytogenes, in coordinating bilE expression. Computational structural analysis suggests that, rather than functioning as a compatible solute uptake system as was previously believed, BilE is more likely to be an exclusion system, a conclusion substantiated by radiolabelled bile accumulation studies. In addition, functionally inactivating BilE resulted in a five‐log reduction in the ability of the bacterium to tolerate lethal concentrations of bovine bile (oxgall) and also significantly increased sensitivity to physiological concentrations of human bile, a phenotype which translates to a significant reduction in virulence potential when administered to a murine model by the oral route. Thus, this novel bile exclusion locus bilE, coordinately regulated by σB and PrfA, represents a new and important virulence factor in L. monocytogenes.

Heterologous Expression of BetL, a Betaine Uptake System, Enhances the Stress Tolerance of Lactobacillus salivarius UCC118

ABSTRACT Given the increasing commercial and clinical relevance of probiotic cultures, improving the technological robustness of what are often process-sensitive cultures is an important biological goal. The nisin-controlled expression system was used to direct the heterologous expression of the listerial betaine uptake system BetL in the probiotic strain Lactobacillus salivarius UCC118. Following nisin induction, strains expressing betL exhibited a significant increase in resistance to several stresses, including elevated osmo-, cryo-, baro-, and chill tolerance, as well as increased resistance to spray- and freeze-drying. The ability to confer additional stress tolerance on a probiotic culture may be an important step in delivering viable cultures for maximal efficacy.

Multiple Deletions of the Osmolyte Transporters BetL, Gbu, and OpuC of Listeria monocytogenes Affect Virulence and Growth at High Osmolarity

ABSTRACT The success of Listeria monocytogenes as a food-borne pathogen owes much to its ability to survive a variety of stresses, both in the food environment and, after ingestion, within the animal host. Growth at high salt concentrations is attributed mainly to the accumulation of organic solutes such as glycine betaine and carnitine. We characterized L. monocytogenes LO28 strains with single, double, and triple deletions in the osmolyte transport systems BetL, Gbu, and OpuC. When single deletion mutants were tested, Gbu was found to have the most drastic effect on the rate of growth in brain heart infusion (BHI) broth with 6% added NaCl. The highest reduction in growth rate was found for the triple mutant LO28BCG (ΔbetL ΔopuC Δgbu), although the mutant was still capable of growth under these adverse conditions. In addition, we analyzed the growth and survival of this triple mutant in an animal (murine) model. LO28BCG showed a significant reduction in its ability to cause systemic infection following peroral coinoculation with the wild-type parent. Altering OpuC alone resulted in similar effects (R. D. Sleator, J. Wouters, C. G. M. Gahan, T. Abee, and C. Hill, Appl. Environ. Microbiol. 67:2692-2698, 2001), leading to the assumption that OpuC may play an important role in listerial pathogenesis. Analysis of the accumulation of osmolytes revealed that betaine is accumulated up to 300 μmol/g (dry weight) when grown in BHI broth plus 6% NaCl whereas no carnitine accumulation could be detected. Radiolabeled-betaine uptake studies revealed an inability of BGSOE (ΔbetL Δgbu) and LO28BCG to transport betaine. Indeed, for LO28BCG, no accumulated betaine was found, but carnitine was accumulated in this strain up to 600 μmol/g (dry weight) of cells, indicating the presence of a possible fourth osmolyte transporter.