C. J. Thomas

Significance of toxin-coregulated pili as protective antigens of vibrio cholerae in the infant mouse model

The infant mouse cholera model has been used to evaluate the relative importance of toxin-coregulated pili (TCP) as protective antigens of Vibrio cholerae 01. Electron microscopic and immunoblotting analyses revealed that, under the cultural conditions examined, TCP were only expressed by strains of classical biotype. Antibodies to TCP were sufficient to confer protection against two such strains, and were more efficient if the challenge vibrios were cultured for TCP expression. In contrast, such antibodies did not protect mice against challenge with any of four strains of El Tor biotype. Since two of the latter have previously been shown to possess non-lipopolysaccharide protective antigens, these results suggest that TCP are not the only such antigen in this model.

Genes for biosynthesis and assembly of CS3 pili of CFA/II enterotoxigenic Escherichia coli: novel regulation of pilus production by bypassing an amber codon

The complete nucleotide sequence of the 4746bp HindIII fragment encoding the genes for the biosynthesis and assembly of CS3 pili has been determined. By site‐directed mutagenesis in conjunction with analysis of the plasmid‐encoded proteins in minicells, the actual reading frames for the various products have been determined. This demonstrated that the genes for four of the proteins (63 kD, 48 kD, 33 kD, and 20kD in size) are encoded entirely within the same open reading frame as a fifth protein (104kD). However, for synthesis of this latter protein, suppression or readthrough of an internal amber codon is required. Termination at this codon is also necessary for synthesis of the former proteins. Two further proteins are also encoded within the HindIII fragment: a 27 kD precursor of a periplasmic protein and the 17.5kD precursor of the major CS3 fimbrial subunit.

The toxin-coregulated pilus (TCP) of Vibrio cholerae: molecular cloning of genes involved in pilus biosynthesis and evaluation of TCP as a protective antigen in the infant mouse model

A serum containing antibodies to non-lipopolysaccharide (non-LPS) protective antigens of Vibrio cholerae has been used, after extensive absorption, to facilitate the cloning of genes involved in the synthesis of toxin-coregulated pili (TCP). A gene bank was constructed from V. cholerae Z17561 DNA using a mobilizable cosmid vector in Escherichia coli, and subsequently transferred by conjugation into V. cholerae O17. This strain does not produce TCP in vitro and lacks non-LPS protective antigens. Eight positive clones were isolated, and of these, four produced TCP as determined by electron microscopic and immunoblotting analyses. TCP-positive O17 clones were 70-fold more virulent than TCP-negative clones or O17 in the infant mouse cholera model. Only the former could remove protective antibodies from the clone-probing serum by absorption. As a corollary, serum containing antibodies to TCP protected mice from challenge with TCP-positive clones, but not with TCP-negative clones or O17. Our data indicate that TCP can function as both a virulence determinant and a protective antigen in the infant mouse model.

Temperate phages in Salmonella enterica serovar Typhimurium: Implications for epidemiology

Salmonella enterica serovar Typhimurium is the most common Salmonella serovar isolated from humans in Australia. The most common definitive phage types (DT) include 9, 64 and 135. Induction of lysogenic phages from DT 64 with mitomycin C followed by cesium chloride gradient purification, resulted in separation of two populations of phage particles. DNA extracted from these particles that was digested with SmaI showed two distinct patterns of banding. Transmission electron microscopy showed that both phage particles belong to the podovirus family of the C1 morphotype. One of the phages, ST64T is capable of mediating both generalized transduction and bacteriophage type conversion. Crude phage lysate induced from S. Typhimurium DT 64 was capable of phage type conversion. S. Typhimurium DT 9 was converted to DT 64 and DT 135 was converted to DT 16. S. Typhimurium DT 41 was also converted to DT 29. Amplified-fragment length polymorphism revealed differences between the original isolates and the convertants. Phage type conversion raises the question of the stability of the bacterial phage types in natural settings and the possibility of its occurrence during an outbreak scenario.

The Listeria monocytogenes gene ctpA encodes a putative P-type ATPase involved in copper transport.

A Tn917 transposon derivative was used to construct a lacZ transcriptional fusion mutant in Listeria monocytogenes DRDC8 that displayed increased β-galactosidase activity in response to cation stress. A 4.3 kb fragment of L. monocytogenes chromosomal DNA flanking the lacZ fusion was cloned and sequenced. A 1962 bp open reading frame was identified, and designated ctpA. Analysis of the deduced 653 amino acid sequence revealed significant similarity to the family of ATP-dependent enzymes involved in copper transport in prokaryotes and eukaryotes. CtpA is distinctive by virtue of an N-terminal truncation in the domain responsible for cation binding. Growth of ctpA insertion mutants was restricted by the copper-chelating agent 8-hydroxyquinoline. DNA/RNA hybridisation studies revealed that levels of ctpA mRNA were increased following growth in media containing low and high copper concentrations. These results suggest the isolation of a region of DNA that encodes a novel copper-transporting system in L. monocytogenes.

Effect of Water Uptake by Poultry Tissues on Contamination by Bacteria During Immersion in Bacterial Suspensions

Effects of water-induced changes in poultry tissue microtopography on numbers of bacteria retained by pieces of tissue immersed in saline suspensions of test organisms were examined. Skin and muscle fascia, not previously exposed to water, retained more bacteria following extended dips in these suspensions compared to a control 15-s dip. Nonmotile bacteria were retained equally as well as motile test strains. Scanning electron microscopy revealed significant changes in tissue microtopography occurred during the course of the immersion experiments. Also shown by this technique was bacteria neither attached nor accumulated at any specific site on the surface of the tissue sample examined under the experimental conditions used. These results suggested contamination of poultry tissues by bacteria during immersion in aqueous fluids, was related to changes in tissue microtopography.

Viability of Listeria monocytogenes in co-culture with Acanthamoeba spp.

Listeria monocytogenes is a human pathogen, ubiquitous in the environment, and can grow and survive under a wide range of environmental conditions. It contaminates foods via raw materials or food-processing environments. However, the current knowledge of its ecology and, in particular, the mode of environmental survival and transmission of this intracellular pathogen remains limited. Research has shown that several intracellular pathogens are able to survive or replicate within free-living amoebae. To examine the viability of L. monocytogenes in interaction with Acanthamoeba spp., bacteria were co-cultured with three freshly isolated amoebae, namely Acanthamoeba polyphaga, Acanthamoeba castellanii and Acanthamoeba lenticulata. The survival of bacteria and amoebae was determined using culture techniques and microscopy. Under the experimental conditions used, all amoebae were able to eliminate bacteria irrespective of the hly gene. Bacteria did not survive or replicate within amoeba cells. However, extra-amoebic bacteria grew saprophytically on materials released from amoebae, which may play an important role in the survival of bacteria under extreme environmental conditions.

Pilot-scale extraction of PHB from recombinant E. coli by homogenization and centrifugation

A new method of poly-β-hydroxybutyrate (PHB) extraction from recombinant E. coli is proposed, using homogenization and centrifugation coupled with sodium hypochlorite treatment. The size of PHB granules and cell debris in homogenates was characterised as a function of the number of homogenization passes. Simulation was used to develop the PHB and cell debris fractionation system, enabling numerical examination of the effects of repeated homogenization and centrifuge-feedrate variation. The simulation provided a good prediction of experimental performance. Sodium hypochlorite treatment was necessary to optimise PHB fractionation. A PHB recovery of 80% at a purity of 96.5% was obtained with the final optimised process. Protein and DNA contained in the resultant product were negligible. The developed process holds promise for significantly reducing the recovery cost associated with PHB manufacture.

Molecular cloning and expression in Escherichia coli K-12 of the O101 rfb region from E. coli B41 (O101:K99/F41) and the genetic relationship to other O101 rfb loci.

The gene cluster (rfb region) which determines the synthesis of O101 lipopolysaccharide (LPS) O‐antigen was cloned from the Escherichia coli O101:K99:F41 reference strain B41 to give plasmid pPM1301. The smallest subclones represented by pPM1305 and pPM 1330expressed O‐antigen in E. coli K‐12 similar to (but not identical to) B41, as judged by immunogold electron microscopy and silver staining of LPS separated by sodium dodecyl sulphate‐polyacrylamide gel electrophoresis (SDS‐PAGE). At least six proteins were detected by minicell analysis of proteins encoded by pPM1305, which suggests that O‐antigen synthesis is genetically complex.

Thermal inactivation kinetics of three vegetative bacteria as influenced by combined temperature and pH in a liquid medium

The kinetics of thermal inactivation as affected by combined temperature and liquid pH for three vegetative bacteria— Escherichia coli (ATCC 25922), Listeria monocytogenes (SLCC 5764) and Pseudomonas fluorescens (172)— have been studied using published bench-scale data and additional experimentally determined data from the heating of samples in ampoules of thin-walled glass. These bacteria represent common micro-organisms known to grow in solid and liquid foods. Up to six levels of temperature (52, 54, 56, 58, 60 and 62° C) in combination with up to eight levels of pH (4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0 and 7.5) with exposure times ranging from 10 seconds to five minutes, were used in experimental designs to cover the wide biokinetic range of interest. The carrier liquid, a 2 kg m −3 mucilage of Carbopol ® 934 was selected because of the stability of its viscosity over the range of temperature and pH values, and its resistance to bacterial growth. The viscosity of this mucilage closely simulates that of a range of liquid foods. The effect of pH on the rate of thermal inactivation was significant for all three bacteria, especially at the lower exposure temperatures. However, because survivor data showed tailing with longer exposure times, the widely held assumption of first-order kinetics for thermal inactivation of vegetative bacteria is not supported. Concave-up tails appear in the data for P. fluorescens and E. coli and both concave-up and concave-down tails appear in the data for L. monocytogenes . Taken together, data for all three bacteria support the necessity for a model formulation for non-linear survivor kinetics as influenced by combined exposure temperature and liquid pH.