Alexander von Holy

Proteomic analysis reveals differential protein expression by Bacillus cereus during biofilm formation.

ABSTRACT Bacillus cereus, a dairy-associated toxigenic bacterium, readily forms biofilms on various surfaces and was used to gain a better understanding of biofilm development by gram-positive aerobic rods. B. cereus DL5 was shown to readily adapt to an attached mode of growth, with dense biofilm structures developing within 18 h after inoculation when glass wool was used as a surface. Two-dimensional gel electrophoresis (2DE) revealed distinct and reproducible phenotypic differences between 2- and 18-h-old biofilm and planktonic cells (grown both in the presence and in the absence of glass wool). Whereas the 2-h-old biofilm proteome indicated expression of 15 unique proteins, the 18-h-old biofilm proteome contained 7 uniquely expressed proteins. Differences between the microcolony (2-h) proteome and the more developed biofilm (18-h) proteome were largely due to up- and down-regulation of the expression of a multitude of proteins. Selected protein spots excised from 2DE gels were subjected to N-terminal sequencing and identified with high confidence. Among the proteins were catabolic ornithine carbamoyltransferase and l-lactate dehydrogenase. Interestingly, increased levels of YhbH, a member of the sigma 54 modulation protein family which is strongly induced in response to environmental stresses and energy depletion via both σB and σH, could be observed within 2 h in both attached cells and planktonic cultures growing in the presence of glass wool, indicating that this protein plays an important role in regulation of the biofilm phenotype. Distinct band differences were also found between the extracellular proteins of 18-h-old cultures grown in the presence and in the absence of glass wool.

Microbiological quality and safety of ready-to-eat street-vended foods in Johannesburg, South Africa.

Fifty-one ready-to-eat street foods, 18 dishwater, and 18 surface swab samples were collected from six vendors in Johannesburg, South Africa. Food temperatures were recorded at the time of sampling. Standard methods were used to determine aerobic plate counts (APCs), spore counts (SCs), and Enterobacteriaceae counts (ECs) for food samples as well as coliform counts (CCs) for water and swab samples. In addition, Petrifilm Escherichia coli count (PC) plates were used for the enumeration of coliforms in food, water, and swab samples. The presence of selected foodborne pathogens in the food samples as well as the presence of nonpathogenic E. coli 1 (in food and water samples) was also tested for. Predominant colonies isolated from APC plates were characterized to the genus level. Holding temperatures for cooked meats and gravies ranged from 42.0 to 94.0 degrees C, and those for uncooked salads ranged from 29.0 to 39.0 degrees C. Mean APC values of 3.4 (+/-0.4) log CFU/g, 4.0 (+/-1.2) log CFU/ml, and 2.1 (+/-0.4) log CFU/25 cm2 were obtained for food, water, and swab samples, respectively. Mean SC values of 1.6 (+/-0.2) log CFU/g and 1.5 (+/-0.3) log CFU/25 cm2 were obtained for food and swab samples, respectively. A mean EC value of 2.0 (+/-0.4) log CFU/g for food samples and mean CC values of 2.5 (+/-0.3) log CFU/ml and 1.3 (+/-0.3) log CFU/25 cm2 for water and swab samples, respectively, were determined. Mean PC values of 1.6 (+/-0.1) log CFU/g, 1.9 (+/-0.6) log CFU/ml, and 1.4 (+/-0.4) log CFU/25 cm2 were determined for food, water, and swab samples, respectively. Bacillus cereus was detected in 22%, Clostridium perfringens in 16%, Salmonella spp. in 2%, and E. coli (non-O157:H+) in 2% of the 51 food samples. E. coli was found in 14 water samples (78%) and in 3 food samples (6%). Campylobacter spp., Listeria monocytogenes, Staphylococcus aureus, Vibrio cholerae, and Yersinia enterocolitica were also tested for in the food samples, but they were not detected. The 340 isolates obtained from APC plates for food, water, and swab samples were predominantly Bacillus spp., Micrococcus spp., and Staphylococcus spp. for all three sample types. It was concluded that the foods analyzed in this study were of acceptable quality and safety.

Microbiological hazard identification and exposure assessment of street food vending in Johannesburg, South Africa

One hundred and thirty-two samples of beef, chicken, salad and gravy were collected from two street vendors over eleven replicate surveys to assess microbiological safety and quality. For each food type samples were collected during preparation and holding. Dish water was also collected and food preparation surfaces swabbed during preparation and display. Standard methods were used to determine aerobic plate counts, Enterobacteriaceae counts, coliform counts and spore counts. Six hundred and seventy-five predominant colonies were isolated from aerobic plate counts of all samples and characterised. The incidence of selected foodborne bacterial pathogens and non-pathogenic E. coli 1 was also determined. In most cases mean bacterial counts of the raw materials were significantly higher (P < 0.05) than those of corresponding cooked foods. No significant differences (P > 0.05) in all count types were observed between food samples collected during cooking and those collected during holding. In addition, no significant differences (P > 0.05) in all count types were observed between prepared salads and their raw materials. Mean bacterial counts of water and swab samples collected from vendor 1 were lower than those of water and swab samples collected from vendor 2.The predominant populations isolated from the aerobic plate counts were Bacillus spp., Staphylococcus spp., Enterobacteriaceae and Alcaligenes spp. Bacillus cereus was detected in 17%, Clostridium perfringens in 1%, Staphylococcus aureus in 3% and Vibrio metchnikovii in 2% of the food samples. Campylobacter jejuni, Listeria monocytogenes, and Escherichia coli O157:H7 were not detected. Non-pathogenic E. coli 1 was detected in 13% of food samples, in 86 and 36% of dish water samples collected from vendors 1 and 2, respectively, and in 36% of surface swab samples from vendor 2.

Characterization and determination of origin of lactic acid bacteria from a sorghum-based fermented weaning food by analysis of soluble proteins and amplified fragment length polymorphism fingerprinting.

ABSTRACT The group that includes the lactic acid bacteria is one of the most diverse groups of bacteria known, and these organisms have been characterized extensively by using different techniques. In this study, 180 lactic acid bacterial strains isolated from sorghum powder (44 strains) and from corresponding fermented (93 strains) and cooked fermented (43 strains) porridge samples that were prepared in 15 households were characterized by using biochemical and physiological methods, as well as by analyzing the electrophoretic profiles of total soluble proteins. A total of 58 of the 180 strains wereLactobacillus plantarum strains, 47 were Leuconostoc mesenteroides strains, 25 were Lactobacillus sake-Lactobacillus curvatus strains, 17 were Pediococcus pentosaceus strains, 13 were Pediococcus acidilacticistrains, and 7 were Lactococcus lactis strains. L. plantarum and L. mesenteroides strains were the dominant strains during the fermentation process and were recovered from 87 and 73% of the households, respectively. The potential origins of these groups of lactic acid bacteria were assessed by amplified fragment length polymorphism fingerprint analysis.

Multiple bacteriocin production by Leuconostoc mesenteroides TA33a and other Leuconostoc/Weissella strains.

Abstract.Leuconostoc (Lc.) mesenteroides TA33a produced three bacteriocins with different inhibitory activity spectra. Bacteriocins were purified by adsorption/desorption from producer cells and reverse phase high-performance liquid chromatography. Leucocin C-TA33a, a novel bacteriocin with a predicted molecular mass of 4598 Da, inhibited Listeria and other lactic acid bacteria (LAB). Leucocin B-TA33a has a predicted molecular mass of 3466 Da, with activity against Leuconostoc/Weissella (W.) strains, and appears similar to mesenterocin 52B and dextranicin 24, while leucocin A-TA33a, which also inhibited Listeria and other LAB strains, is identical to leucocin A-UAL 187. A survey of other known bacteriocin-producing Leuconostoc/Weissella strains for the presence of the three different bacteriocins revealed that production of leucocin A-, B- and C-type bacteriocins was widespread. Lc. carnosum LA54a, W. paramesenteroides LA7a, and Lc. gelidum UAL 187-22 produced all three bacteriocins, whereas W. paramesenteroides OX and Lc. carnosum TA11a produced only leucocin A- and B-type bacteriocins.

Practical aspects of biofouling control in industrial water systems

Abstract Both water consumption and discharge in industrial water systems are currently minimised. The circulation of such water results in concentration of dissolved and suspended substances, promoting the growth of waterborne microbes, biofouling and subsequent macrofouling of the system and concomitant microbially induced corrosion. A number of reviews have been published on the mechanisms of microbially induced corrosion and the organisms involved. The subject of biofilm formation has also been well covered in the literature. A lack of information on the community structure and physiology is, however, apparent. Many advances have, nevertheless, been made in biofouling control. Probably the most important is the shift in emphasis from planktonic bacterial monitoring to sessile bacterial monitoring. This led to the introduction of a variety of different sessile monitoring techniques. Much experience has since been obtained on the use and limitations of these techniques and, to date, one of the main problem areas remaining is the monitoring of biofouling. Research has also indicated the problem of microbial resistance to nonoxidising biocides. This has suggested that some of these compounds may be mutagens. From an environmental point of view, it has become very important to verify this. This has also indicated the need to develop biocides which do not induce resistance in micro-organisms, and to investigate whether oxidising biocides are also capable of inducing resistance in micro-organisms. Recent studies have indicated that biofilm ecosystems respond to stress (i.e. biocides) in ways similar to macro-ecosystems. Generally, there is a decline in species diversity and a selection of more tolerant isolates. These developments have placed the spotlight on alternative technologies, like biodispersants, which have shown potential as biofouling control agents, and which should be investigated further. Physical control measures are currently still limited to pigging, although a number of other technologies show promise. Although fluid dynamics and their effect on biofouling control programmes have been well reported in the literature, it remains an aspect which is neglected by industry in terms of practical applications.

Microbial populations associated with commercially produced South African sorghum beer as determined by conventional and Petrifilm™ plating

Microbial populations of 46 commercially produced sorghum beer samples from retail outlets in Johannesburg, South Africa, were enumerated and characterized. Aerobic plate counts, lactic acid bacteria counts and yeast counts were performed by conventional and Petrifilm plating. Conventional methods yielded yeast counts of 7.84 log CFU/ml, lactic acid bacteria counts of 6.44 log CFU/ml and aerobic plate counts of 5.96 log CFU/ml. In comparison, Petrifilm counts were 7.85 log CFU/ml for yeasts, 5.31 log CFU/ml for lactic acid bacteria and 5.34 log CFU/ml for aerobic bacteria. Characterization of 419 predominant bacterial isolates from Standard One Nutrient Agar, MRS Agar and corresponding Petrifilm plates yielded 88.0% lactic acid bacteria, 8.4% Bacillus species, 2.9% Micrococcus species and 0.7% Gram negative bacteria. Composition of predominant lactic acid bacteria populations from Standard One Nutrient Agar and both types of Petrifilm plates showed marginal differences. Increased proportions of heterofermentative lactic acid bacteria were, however, isolated from conventional MRS Agar compared to the modified Petrifilm product which represented the equivalent to MRS Agar.

Bacteriocins of Leuconostocs isolated from meat

Several bacteriocin-producing Leuconostoc strains have been isolated from meat and identified as Leuconostoc gelidum UAL 187, Leuconostoc paramesenteroides-La7a, Leuconostoc carnosum-Ta11a and Leuconostoc carnosum-La54a. All strains produce bacteriocins that are active against Listeria monocytogenes and other lactic acid bacteria of concern in meat spoilage. All of the bacteriocins studied are heat stable in acidic environments and are inactivated by a range of proteolytic enzymes but not by catalase or lysozyme. Most are detected early in the growth cycle and are produced at refrigeration temperatures and in a pH range of 4.0-7.0. Leucocin A-UAL187, produced by Leuconostoc gelidium UAL 187, is a small peptide (MW 3930) translated as a 61 amino acid prepeptide consisting of a 24 amino acid leader region and 37 amino acid active bacteriocin that is secreted. A probe designed from a region of the leucocin gene has been used to locate the bacteriocin genes in the other strains (La7a, La54a and Ta11a). Strong hybridization signals were detected from 8.9 MDa, 32 MDa and 8.9 MDa plasmids in strains La7a, La54a and Ta11a, respectively. The bacteriocin structural gene from Leuconostoc carnosum-Ta11a (leucocin B-Ta11a) has been cloned and sequenced and the bacteriocin shows 100% homology to leucocin A-UAL187; however, the prepeptide differs in six residues. The mature extracellular bacteriocin from strain UAL 187 was purified and characterized by precipitation, gel filtration, hydrophobic interaction chromatography followed by RP-HPLC and amino-terminal sequencing, whilst those of the other strains are in the process of being purified and characterized using similar techniques.(ABSTRACT TRUNCATED AT 250 WORDS)

Genotypic Analysis of Escherichia coli Strains from Poultry Carcasses and Their Susceptibilities to Antimicrobial Agents

ABSTRACT Plasmid profiling and amplified fragment length polymorphism (AFLP) analysis were used to genotype 50 Escherichia coli strains from poultry carcasses. Thirty different plasmid profiles were evident, and clustering of the AFLP data showed that they were a distinctly heterogeneous group of strains. Susceptibility testing against five antimicrobial agents used in the South African poultry industry showed all strains to be susceptible to danofloxacin and colistin, while the majority (96%) were resistant to two tetracyclines.

Molecular Diversity and Characterization of Tetracycline-Resistant Staphylococcus aureus Isolates from a Poultry Processing Plant

ABSTRACT DNA fingerprinting and molecular characterization showed that the tetracycline-resistant Staphylococcus aureus population of a South African poultry processing plant comprised one or possibly several tet(K)-containing endemic clones that contaminated chicken and machinery surfaces at all sampled processing stages. The tet(K) gene was transferable by filter mating to S. aureus recipient 80CR5 and was located on a pT181-like plasmid.