Biomedical subjects

S Kathariou

Publications and source records attributed to S Kathariou.

At least 19 recordsLinked to original sources

PubMed2006

Resistance of Listeria monocytogenes biofilms to sanitizing agents in a simulated food processing environment.

The objective of this study was to evaluate the resistance of biofilms of Listeria monocytogenes to sanitizing agents under laboratory conditions simulating a food processing environment. Biofilms were initially formed on stainless steel and Teflon coupons using a five-strain mixture of L. monocytogenes. The coupons were then subjected to repeated 24-h daily cycles. Each cycle consisted of three sequential steps: (i) a brief (60 s) exposure of the coupons to a sanitizing agent (a mixture of peroxides) or saline as a control treatment, (ii) storage of the coupons in sterile plastic tubes without any nutrients or water for 15 h, (iii) and incubation of the coupons in diluted growth medium for 8 h. This regimen was repeated daily for up to 3 weeks and was designed to represent stresses encountered by bacteria in a food processing environment. The bacteria on the coupons were reduced in number during the first week of the simulated food processing (SFP) regimen, but then adapted to the stressful conditions and increased in number. Biofilms repeatedly exposed the peroxide sanitizer in the SFP regimen developed resistance to the peroxide sanitizer as well as other sanitizers (quaternary ammonium compounds and chlorine). Interestingly, cells that were removed from the biofilms on peroxide-treated and control coupons were not significantly different in their resistance to sanitizing agents. These data suggest that the resistance of the treated biofilms to sanitizing agents may be due to attributes of extracellular polymeric substances and is not an intrinsic attribute of the cells in the biofilm.

Bacterial Adhesion↗

PubMed2006

Involvement of closely related strains of a new clonal group of Listeria monocytogenes in the 1998-99 and 2002 multistate outbreaks of foodborne listeriosis in the United States.

In 1998-99, a multistate outbreak of listeriosis in the United States was associated with contaminated hot dogs and was caused by a strain of Listeria monocytogenes serotype 4b that had been only rarely encountered before in the national PulseNet database. Upon further characterization, the strains from this outbreak were designated as Epidemic Clone II (ECII). ECII isolates exhibited diversification in a genomic region ("region 18") that was otherwise conserved among L. monocytogenes of serotype 4b. Additional unique genetic markers were identified through genome sequencing of one of the isolates from the 1998-99 outbreak. In 2002, another multistate outbreak of listeriosis also involved bacteria of serotype 4b and was attributed to contaminated turkey deli meats. Molecular subtyping data revealed that the macrorestriction patterns of the isolates from the 1998-99 and 2002 outbreaks were closely related. In addition, the 2002 outbreak isolates harbored chromosomal genetic markers found to be unique to, and typical of, the 1998-99 outbreak isolates, including diversification in genomic region 18. Macroarray- based subtyping using chromosomal sequences confirmed the close genetic relatedness between the isolates from the two outbreaks. Genomic content was highly conserved among isolates from each outbreak, with differences detected only in prophage and internalin-like gene sequences. However, since these differences were observed among isolates from each of the outbreaks, they did not differentiate the 1998-99 isolates as a group from those of the 2002 outbreak. Two of 15 randomly chosen serotype 4b clinical isolates from a non-outbreak period (calendar year 2003) appeared to be closely related to the 1998-99 and 2002 outbreak isolates. These findings suggest that both multistate outbreaks of listeriosis in the United States involved closely related members of a single clonal group (ECII) that had not been identified in outbreaks prior to 1998. Since the outbreaks involved different food vehicles and processing plants, the findings suggest establishment of ECII in a still unidentified reservoir in the United States, from which the organisms were introduced to different processing plants.

Animals↗

PubMed2006

Effect of direct culture versus selective enrichment on the isolation of thermophilic campylobacter from feces of mature cattle at harvest.

Campylobacterjejuni and Campylobacter coli are leading bacterial causes of human gastroenteritis in the United States and other industrialized nations. These organisms frequently colonize avian hosts, including commercial poultry, but are also found in the gastrointestinal tract of other warm-blooded animals, including swine, sheep, and cattle. This study investigated the effect of direct culture versus selective enrichment on the isolation of thermophilic Campylobacter from the colon of 610 cattle. Fecal samples were taken from the colon of mature cattle (older than 30 months of age) immediately after slaughter in a commercial abattoir over a period of 17 months. Campylobacter was isolated from 23.4% of the animals. Most (93%) of the culture-confirmed Campylobacter isolates were C. jejuni, with the remaining 7% being C. coli. Additionally, of the 143 samples from which pure cultures of Campylobacter could be isolated, 72 (50.3%) were positive only with selective enrichment, 18 (12.6%) were positive only with direct plating, and 53 (37.1%) were positive by both methods. The data suggest that, even though selective enrichment was more effective than direct plating, both direct plating and selective enrichment protocols might need to be employed for optimal surveillance of C. jejuni in fecal material from cattle.

Abattoirs↗

PubMed2005

Molecular characterization of Listeria monocytogenes of the serotype 4b complex (4b, 4d, 4e) from two turkey processing plants.

Most foodborne outbreaks of listeriosis have been found to involve a small number of closely related strains of Listeria monocytogenes serotype 4b. The ecology of these organisms and their reservoirs in nature or in the processing plant environment, however, remain poorly understood. Surveys of environmental samples from two turkey processing plants in the United States indicated presence of L. monocytogenes of the serotype 4b complex (serotype 4b and the closely related serotypes 4d and 4e). In addition, environmental and raw product samples from one plant repeatedly yielded isolates with genetic markers typical of two major serotype 4b epidemic clonal groups, ECI and ECII. The pulsed field gel electrophoresis (PFGE) profiles of these isolates, however, were clearly distinct from those of confirmed epidemic-associated strains. Furthermore, we observed minor but consistent differences in PFGE profiles of isolates that harbored ECI- or ECII-specific genetic markers, and that were obtained at different sampling times from the same plant. The findings suggest processing plant persistence (or repeated introductions) and genomic diversification of L. monocytogenes serotype 4b isolates that harbor ECI- or ECII-specific genetic markers. Such diversification would need to be taken into consideration in further efforts to elucidate the evolution and epidemiology of these organisms.

Animals↗

PubMed2004

Effects of carcass washers on Campylobacter contamination in large broiler processing plants.

Campylobacter, a major foodborne pathogen found in poultry products, remains a serious problem facing poultry processors. Campylobacter research has primarily focused on detection methods, prevalence, and detection on carcasses; limited research has been conducted on intervention. The aim of this study was to assess the effectiveness of carcass washing systems in 4 large broiler-processing plants in removing Campylobacter species. Washing systems evaluated included combinations of inside/outside carcass washers and homemade cabinet washers. Processing aids evaluated were trisodium phosphate (TSP) and acidified sodium chlorite (ASC). The washer systems consisted of 1 to 3 carcass washers and used from 2.16 to 9.73 L of water per carcass. The washer systems used chlorinated water with 25 to 35 ppm of total chlorine. These washer systems on average reduced Campylobacter populations by log 0.5 cfu/mL from log 4.8 cfu/mL to log 4.3 cfu/mL. Washer systems with TSP or ASC reduced Campylobacter populations on average by an additional log 1.03 to log 1.26, respectively. Total average reductions in Campylobacter populations across the washer system and chill tank were log 0.76 cfu/mL. Washer systems that included antimicrobial systems had total average reductions in Campylobacter populations of log 1.53 cfu/mL. These results suggest that carcass washer systems consisting of multiple washers provide minimal reductions in Campylobacter populations found on poultry in processing plants. A more effective treatment of reducing Campylobacter populations is ASC or TSP treatment; however, these reductions, although significant, will not eliminate the organism from raw poultry.

Animals↗

PubMed2004

Natural atypical Listeria innocua strains with Listeria monocytogenes pathogenicity island 1 genes.

Identification of bona fide Listeria isolates into the six species of the genus normally requires only a few tests. Aberrant isolates do occur, but even then only one or two extra confirmatory tests are generally needed for identification to species level. We have discovered a hemolytic-positive, rhamnose and xylose fermentation-negative Listeria strain with surprising recalcitrance to identification to the species level due to contradictory results in standard confirmatory tests. The issue had to be resolved by using total DNA-DNA hybridization testing and then confirmed by further specific PCR-based tests including a Listeria microarray assay. The results show that this isolate is indeed a novel one. Its discovery provides the first fully documented instance of a hemolytic Listeria innocua strain. This species, by definition, is typically nonhemolytic. The L. innocua isolate contains all the members of the PrfA-regulated virulence gene cluster (Listeria pathogenicity island 1) of L. monocytogenes. It is avirulent in the mouse pathogenicity test. Avirulence is likely at least partly due to the absence of the L. monocytogenes-specific allele of iap, as well as the absence of inlA, inlB, inlC, and daaA. At least two of the virulence cluster genes, hly and plcA, which encode the L. monocytogenes hemolysin (listeriolysin O) and inositol-specific phospholipase C, respectively, are phenotypically expressed in this L. innocua strain. The detection by PCR assays of specific L. innocua genes (lin0198, lin0372, lin0419, lin0558, lin1068, lin1073, lin1074, lin2454, and lin2693) and noncoding intergenic regions (lin0454-lin0455 and nadA-lin2134) in the strain is consistent with its L. innocua DNA-DNA hybridization identity. Additional distinctly different hemolytic L. innocua strains were also studied.

Animals↗

PubMed2003

An improved cloning vector for construction of gene replacements in Listeria monocytogenes.

Listeria monocytogenes is a gram-positive, facultative intracellular bacterium implicated in severe food-borne illness (listeriosis) in humans. The construction of well-defined gene replacements in the genome of L. monocytogenes has been instrumental to several genetic studies of the virulence and other attributes of the organism. Construction of such mutations by currently available procedures, however, tends to be labor intensive, and gene replacement mutants are sometimes difficult to recover due to lack of direct selection for the construct. In this study we describe the construction and use of plasmid vector pGF-EM, which can be conjugatively transferred from Escherichia coli S17-1 to L. monocytogenes and which provides the genetic means for direct selection of gene replacements.

ATP-Dependent Proteases↗

PubMed2001

Survival of clinical and poultry-derived isolates of Campylobacter jejuni at a low temperature (4 degrees C).

Campylobacter jejuni is a leading cause of bacterial gastroenteritis in humans, and contamination of poultry has been implicated in illness. The bacteria are fastidious in terms of their temperature requirements, being unable to grow below ca. 31 degrees C, but have been found to be physiologically active at lower temperatures and to tolerate exposure to low temperatures in a strain-dependent manner. In this study, 19 field isolates of C. jejuni (10 of clinical and 9 of poultry origin) were studied for their ability to tolerate prolonged exposure to low temperature (4 degrees C). Although substantial variability was found among different strains, clinical isolates tended to be significantly more likely to remain viable following cold exposure than poultry-derived strains. In contrast, the relative degree of tolerance of the bacteria to freezing at -20 degrees C and freeze-thawing was strain specific but independent of strain source (poultry versus clinical) and degree of cold (4 degrees C) tolerance.

Animals↗

PubMed2001

A novel serotype-specific gene cassette (gltA-gltB) is required for expression of teichoic acid-associated surface antigens in Listeria monocytogenes of serotype 4b.

Listeria monocytogenes serotype 4b strains account for about 40% of sporadic cases and many epidemics of listeriosis. Mutations in a chromosomal locus resulted in loss of reactivity with all three monoclonal antibodies (MAbs) which were specific to serotype 4b and the closely related serotypes 4d and 4e. Here we show that this locus contains a serotype 4b-4d-4e-specific gene cassette (3,071 bp) which consists of two genes, gltA and gltB, and is flanked by palindromic sequences (51 and 44 nucleotides). Complete loss of reactivity with the three serotype-specific MAbs resulted from insertional inactivation of either gltA or gltB. The gltA and gltB mutants were characterized by loss and severe reduction, respectively, of glucose in the teichoic acid, whereas galactose, the other serotype-specific sugar substituent in the teichoic acid, was not affected. Within L. monocytogenes, only strains of serotypes 4b, 4d, and 4e harbored the gltA-gltB cassette, whereas coding sequences on either side of the cassette were conserved among all serotypes. Comparative genomic analysis of a serotype 1/2b strain showed that the 3,071-bp gltA-gltB cassette was replaced by a much shorter (528-bp) and unrelated region, flanked by inverted repeats similar to their counterparts in serotype 4b. These findings indicate that in the evolution of different serotypes of L. monocytogenes, this site in the genome has become occupied by serotype-specific sequences which, in the case of serotype 4b, are essential for expression of serotype-specific surface antigens and presence of glucose substituents in the teichoic acids in the cell wall.

Amino Acid Sequence↗

PubMed2000

Use of non-porous reversed-phase high-performance liquid chromatography for protein profiling and isolation of proteins induced by temperature variations for Siberian permafrost bacteria with identification by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and capillary electrophoresis-electrospray ionization mass spectrometry.

Non-porous reversed-phase high-performance liquid chromatography (NP-RP-HPLC) has been used to separate and isolate proteins from whole cell lysates of ED 7-3, a bacterium from the buried Siberian permafrost sediment. The proteins collected from the liquid eluent of this separation were then analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and capillary electrophoresis-electrospray ionization mass spectrometry (CE-ESI-MS). In order to study the differences in expression of cold-shock proteins (CSPs) at different growth temperatures, cultures of the ED 7-3 strain were prepared at 4 degrees C and 25 degrees C. The goals of this work were twofold: firstly, to identify the presence of CSPs and other proteins that are highly expressed at 4 degrees C but not at 25 degrees C; and secondly, to isolate these proteins for MALDI-TOF-MS and CE-ESI-MS identification. In this initial work, distinct protein profiles were observed for these cultures as a function of temperature. Fraction collection from the eluent of NP-RP-HPLC of some of the highly expressed proteins was performed and the proteins were mass analyzed for molecular mass. Peptide maps of the proteins were generated by tryptic digestion and were analyzed by CE-ESI-MS and MALDI-TOF-MS for database identification of the expressed proteins.

Amino Acid Sequence↗

PubMed2000

Low-temperature recovery strategies for the isolation of bacteria from ancient permafrost sediments.

Permafrost represents a unique ecosystem that has allowed the prolonged survival of certain bacterial lineages at subzero temperatures. To better understand the permafrost microbial community, it is important to identify isolation protocols that optimize the recovery of genetically diverse bacterial lineages. We have investigated the impact of different low-temperature isolation protocols on recovery of aerobic bacteria from northeast Siberian permafrost of variable geologic origin and frozen for 5000 to 3 million years. Low-nutrient media enhanced the quantitative recovery of bacteria, whereas the isolation of diverse morphotypes was maximized on rich media. Cold enrichments done directly in natural, undisturbed permafrost led not only to recovery of increased numbers of bacteria but also to isolation of genotypes not recovered by means of liquid low-temperature enrichments. On the other hand, direct plating and growth at 4 degrees C also led to recovery of diverse genotypes, some of which were not recovered following enrichment. Strains recovered from different permafrost samples were predominantly oligotrophic and non-spore-forming but were otherwise variable from each other in terms of a number of bacteriological characteristics. Our data suggest that a combination of isolation protocols from different permafrost samples should be used to establish a culture-based survey of the different bacterial lineages in permafrost.

Bacteria↗

PubMed2000

A sheep in wolf's clothing: Listeria innocua strains with teichoic acid-associated surface antigens and genes characteristic of Listeria monocytogenes serogroup 4.

Listeria monocytogenes serotype 4b has been implicated in numerous food-borne epidemics and in a substantial fraction of sporadic listeriosis. A unique lineage of the nonpathogenic species Listeria innocua was found to express teichoic acid-associated surface antigens that were otherwise expressed only by L. monocytogenes of serotype 4b and the rare serotypes 4d and 4e. These L. innocua strains were also found to harbor sequences homologous to the gene gtcA, which has been shown to be essential for teichoic acid glycosylation in L. monocytogenes serotype 4b. Transposon mutagenesis and genetic studies revealed that the gtcA gene identified in this lineage of L. innocua was functional in serotype 4b-like glycosylation of the teichoic acids of these organisms. The genomic organization of the gtcA region was conserved between this lineage of L. innocua and L. monocytogenes serotype 4b. Our data are in agreement with the hypothesis that, in this lineage of L. innocua, gtcA was acquired by lateral transfer from L. monocytogenes serogroup 4. The high degree of nucleotide sequence conservation in the gtcA sequences suggests that such transfer was relatively recent. Transfer events of this type may alter the surface antigenic properties of L. innocua and may eventually lead to evolution of novel pathogenic lineages through additional acquisition of genes from virulent listeriae.

Amino Acid Sequence↗

PubMed2000

Absence of serotype-specific surface antigen and altered teichoic acid glycosylation among epidemic-associated strains of Listeria monocytogenes.

Outbreaks of food-borne listeriosis have often involved strains of serotype 4b. Examination of multiple isolates from three different outbreaks revealed that ca. 11 to 29% of each epidemic population consisted of strains which were negative with the serotype-specific monoclonal antibody c74.22, lacked galactose from the teichoic acid of the cell wall, and were resistant to the serotype 4b-specific phage 2671.

Antibodies, Monoclonal↗

PubMed1999

Transposon-induced mutations in two loci of Listeria monocytogenes serotype 1/2a result in phage resistance and lack of N-acetylglucosamine in the teichoic acid of the cell wall.

Teichoic acid-associated N-acetylglucosamine and rhamnose have been shown to serve as phage receptors in Listeria monocytogenes serotype 1/2a. We generated and characterized two single-copy Tn916DeltaE mutants which were resistant to phage A118 and several other serotype 1/2a-specific phages. In one mutant the insertion was immediately upstream of the recently identified ptsHI locus, which encodes two proteins of the phosphoenolpyruvate-dependent carbohydrate uptake system, whereas in the other the insertion was immediately upstream of an operon whose most distal gene was clpC, involved in stress responses and virulence. Transduction experiments confirmed the association of the phage-resistant phenotype of these mutants with the transposon insertion. Phage A118 resistance of the mutants could be attributed to inability of the phage to adsorb onto the mutant cells, and biochemical analysis of cell wall composition showed that the teichoic acids of both mutants were deficient in N-acetylglucosamine. Rhamnose and other teichoic acid and cell wall components were not affected.

Acetylglucosamine↗

PubMed1999

Cell wall teichoic acid glycosylation in Listeria monocytogenes serotype 4b requires gtcA, a novel, serogroup-specific gene.

We have identified a novel gene, gtcA, involved in the decoration of cell wall teichoic acid of Listeria monocytogenes serotype 4b with galactose and glucose. Insertional inactivation of gtcA brought about loss of reactivity with the serotype 4b-specific monoclonal antibody c74.22 and was accompanied by a complete lack of galactose and a marked reduction in the amounts of glucose on teichoic acid. Interestingly, the composition of membrane-associated lipoteichoic acid was not affected. Complementation of the mutants with the cloned gtcA in trans restored galactose and glucose on teichoic acid to wild-type levels. The complemented strains also recovered reactivity with c74.22. Within L. monocytogenes, sequences homologous to gtcA were found in all serogroup 4 isolates but not in strains of any other serotypes. In serotype 4b, gtcA appears to be the first member of a bicistronic operon which includes a gene with homology to Bacillus subtilis rpmE, encoding ribosomal protein L31. In contrast to gtcA, the latter gene appears conserved among all screened serotypes of L. monocytogenes.

Amino Acid Sequence↗

PubMed1999

Insertional inactivation of the Listeria monocytogenes cheYA operon abolishes response to oxygen gradients and reduces the number of flagella.

The nucleotide sequence of a region downstream of the Listeria monocytogenes flagellin gene, flaA, revealed two putative chemotaxis genes, cheY and cheA. These genes have been shown to be transcribed as a bicistronic unit. In this study Tn916 delta E mutagenesis was used to generate two mutants, PF10 and PF16, which contain transposon inserts in the promoter region of this operon. These mutants were motile in liquid, but had reduced flagellin expression and were unable to burrow or swarm on soft agar plates. Complementation of the single transposon-copy mutant PF16 with cloned cheY and cheA in trans partially restored microaerotaxis and swarming on soft agar. The complemented strain did not exhibit any increase in flagellin production. Both PF10 and PF16 appear deficient in their ability to attach to the mouse fibroblast cell line 3T3.

Animals↗

PubMed1997

DNA fragments from regions involved in surface antigen expression specifically identify Listeria monocytogenes serovar 4 and a subset thereof: cluster IIB (serotypes 4b, 4d, and 4e).

Listeria monocytogenes serotype 4b has frequently been implicated in sporadic as well as epidemic listeriosis. On the basis of pulsed-field fingerprinting, serotype 4b strains, along with strains of serotypes 4d and 4e, constitute one genomic cluster (IIB). We have identified two genomic regions essential for the expression of surface antigens which previously were shown to be specific to cluster IIB strains. A DNA probe of 1.1 kb derived from one of the regions (probe 1) hybridized only with strains of serotypes 4b, 4d, and 4e in Southern blots and dot blots. A different DNA probe of 0.3 kb (probe 2), derived from the other region, hybridized with all serovar 4 strains (serotypes 4b, 4a, 4c, 4d, and 4e). All other L. monocytogenes serotypes were negative with probe 1 or 2. Use of probe 1 in Southern blots of EcoRI-digested genomic DNA revealed a restriction fragment length polymorphism in serotype 4b strains, with the hybridizing EcoRI fragments being 4.5 kb (strains of the epidemic clone) and either 4.5 or 5.0 kb (all other serotype 4b strains). Although the probes hybridized with a special group of Listeria innocua strains which also expressed the surface antigens, the latter could be readily distinguished by the size of the hybridizing EcoRI fragment with probe 1 (ca. 2.2 kb). These data suggest that the combined use of these probes with L. monocytogenes can readily and specifically identify cluster IIB strains as well as the entire serovar 4 complex.

Antibodies, Monoclonal↗

PubMed1997

Host-mediated modification of Sau3AI restriction in Listeria monocytogenes: prevalence in epidemic-associated strains.

Most major food-related outbreaks of listeriosis have been traced to a cluster of genetically related strains of serovar 4b (epidemic clone). In spite of numerous searches, distinct bacteriologic or virulence-related features unique to these strains have eluded identification, although a restriction fragment length polymorphism (RFLP) characteristic of the epidemic clone has previously been described (W. Zheng and S. Kathariou, Appl. Environ. Microbiol. 61:4310-4314, 1995). We found that DNAs from 75 strains which were derived from three separate outbreaks and which had the epidemic clone-specific RFLP were also invariably resistant to digestion by Sau3AI and other restriction endonucleases sensitive to cytosine methylation at 5' GATC 3' sites. This modification of Sau3AI restriction was host mediated, as it did not persist when DNA was cloned and propagated in Escherichia coli, and was uncommon among other Listeria strains. Epidemic-associated strains with this modification were resistant to infection by phage propagated in a serotype 4b strain which was not known to be involved in an epidemic and which lacked the epidemic clone-specific RFLP. Screening for susceptibility to MboI digestion revealed that these epidemic strains lacked methylation of adenines at GATC sites. This type of modification was rare among Listeria strains and was found in only three (of eight screened) strains of serovar 1/2b, possibly representing one clonal lineage.

Adenine↗