A. von Holy

Differential efficacy of a chlorine dioxide-containing sanitizer against single species and binary biofilms of a dairy-associated Bacillus cereus and a Pseudomonas fluorescens isolate

Aims: Daily exposure to 100 p.p.m. chlorine dioxide of single species and binary biofilms of dairy‐associated Bacillus cereus DL5 and Pseudomonas fluorescens M2, attached to stainless steel surfaces in a laboratory flow system, was studied.

Different responses of planktonic and attached Bacillus subtilis and Pseudomonas fluorescens to sanitizer treatment

Three commercial sanitizers containing iodophor (I), peracetic acid/ hydrogen peroxide (PAH), or chlorhexidine gluconate (CG) were evaluated in vitro against planktonic and sessile Bacillus subtilis or Pseudomonas fluorescens cells grown in Standard One Nutrient Broth. Sessile cells were attached to stainless steel or polyurethane test surfaces. Planktonic and attached cells of both bacteria were enumerated by plate counts after sanitizer treatment for 1, 3, or 5 min. Sessile cells were dislodged from test surfaces by shaking them with beads. Cell morphologies were monitored by scanning electron microscopy (SEM). Attached B. subtilis and P. fluorescens cells on both surface types were less susceptible to all three sanitizers than their planktonic counterparts. PAH, I, and CG were equally effective against planktonic P. fluorescens cells, which were reduced by 99.999% after 1, 3, and 5 min exposure. PAH was the only sanitizer effective against attached P. fluorescens cells on both surface types; it reduced counts by < or = 99.9% after 1, 3, and 5 min exposure. PAH was also the most effective sanitizer against planktonic B. subtilis cells, reducing counts by 99.9% after 1, 3, and 5 min. Sessile B. subtilis cells on both surface types were the least susceptible to all sanitizers; counts were reduced by only 99.5% or less after exposure to PAH for 5 min. SEM revealed that planktonic and attached cells of both bacteria exhibited symptoms of surface roughness, indentations, and shape distortions after treatment with any of the sanitizers.

Quantification and characterization of microbial populations associated with spoiled, vacuum-packed Vienna sausages

Abstract Blowing, souring and exudate formation in 52 terminally spoiled samples of vacuum-packed, smoked Vienna sausages was associated with high numbers of lactic acid bacteria. These bacteria predominated at levels of 107–108 g−1 of product (approximately 109 in exudate samples), while numbers in ten unspoiled samples were only 103–104 g−1. Enterobacteriaceae, yeasts, enterococci and staphylococci never exceeded 103 g−1 in either unspoiled or spoiled samples. Characterization of the predominant organisms indicated that populations in spoiled samples were dominated by homofermentative lactobacilli (58·0%) and leuconostocs (36·3%). These groups proliferated mainly at the expense of heterofermentative lactobacilli. The latter accounted for 30·2% of isolates from unspoiled samples, while leuconostocs and homofermentative lactobacilli comprised 64·6% of the total isolates.

Thermotolerance of meat spoilage lactic acid bacteria and their inactivation in vacuum-packaged vienna sausages

Heat resistance of three meat spoilage lactic acid bacteria was determined in vitro. D-values at 57, 60 and 63 degrees C were 52.9, 39.3 and 32.5 s for Lactobacillus sake, 34.9, 31.3 and 20.2 s for Leuconostoc mesenteroides and 22.5, 15.6 and 14.4 s for Lactobacillus curvatus, respectively. The three lactic acid bacteria were heat sensitive, as one log reductions in numbers were achieved at 57 degrees C in less than 60 s. Z-values could not be accurately determined as D-values did not change by a factor of 10 over the temperature range studied. In-package pasteurization processes were calculated using the highest in vitro D-value and applied to vacuum-packaged vienna sausages. Microbiological shelf life (time for lactic acid bacteria count to reach 5 x 10(6) CFU/g) increased from 7 days for non-pasteurized samples to 67, 99 and 119 days for samples of the three pasteurization treatments at 8 degrees C storage. Enterobacteriaceae were detected at levels of log 4.0 CFU/g in non-pasteurized samples, but were reduced to < log 1.0 CFU/g in pasteurized samples. The incidence of listeriae in non-pasteurized samples was low as only one Listeria innocua strain was isolated. No Listeria spp. were isolated from pasteurized samples. Numbers of Clostridium isolates increased from one in non-pasteurized samples to 25 in pasteurized samples. Increasing incidences of clostridia, and the presence of C. perfringens in pasteurized samples indicated that in-package pasteurization could compromise product safety.

Physiology of dairy-associated Bacillus spp. over a wide pH range.

Bacillus species isolated from alkaline wash solutions used for cleaning in place in South African dairy factories have been suggested to contaminate product contact surfaces of dairy processing equipment and result in post-pasteurization spoilage of milk and milk products. Growth and attachment of such Bacillus isolates under alkaline and acidic conditions have not been previously described. Therefore, the in vitro growth temperature and pH ranges, attachment abilities and hydrophobicity, and enzyme production capabilities of four Bacillus isolates (tentatively identified as B. subtilis115, B. pumilus122, B. licheniformis137 and B. cereus144) previously isolated from the alkaline wash solutions in a South African dairy were examined. Growth pH ranges were determined in buffered Standard One-like Nutrient Broth and in unbuffered 1% Milk Medium at pH values ranging from 3 to 12. Growth and attachment to stainless steel surfaces and production of protease and lipase enzymes were determined in 1% Milk Medium at pH 4, 7 and 10. Colony hydrophobicity of each isolate by the Direction of Spreading Method (DOS) was also determined at pH 4, 7 and 10. In addition, Arrhenius plots were used to examine the growth temperature ranges of the isolates. All isolates grew at pH values ranging from 4.5 to 9.5 in buffered Standard One-like Nutrient Broth, and from pH 4 to 10 in 1% Milk Medium. All isolates also attached to stainless steel at pH 4, 7 and 10 in 1% Milk Medium. Generally the attachment of B. subtilis115, B. pumilus122 and B. lichenformis137 to stainless steel surfaces was enhanced at pH 4 and 10, compared to pH 7. By contrast, the best attachment of B. cereus144 cells to stainless steel surfaces was at pH 7. Planktonic and attached cells of all isolates produced proteolytic enzymes at pH 7 and 10, but not at pH 4. Similarly, planktonic and attached cells of B. subtilis115, B. pumilus122 and B. licheniformis137 produced lipolytic enzymes at pH 7 and 10, and weak lipolysis was observed at pH 4. The Bacillus cereus144 isolate showed no lipolytic activity at pH 10. All isolates exhibited low hydrophobic properties at all pH values even though attachment to stainless steel at the same pH values occurred. None of the isolates grew below 11 degrees C or above 56 degrees C, and optimum growth temperatures were in the high mesophilic range (36-44 degrees C).

Bacterial populations associated with a sorghum-based fermented weaning cereal

Microbiological surveys, to determine the quality and safety, were conducted on 45 sorghum samples comprising dry powders (n = 15) and corresponding fermented (n = 15) and cooked fermented porridge (n = 15) samples collected from households in an informal settlement of the Gauteng Province of South Africa. Mean aerobic plate counts, Gram-negative counts and bacterial spore counts of sorghum powder samples decreased in fermented and cooked fermented porridge samples. However, mean lactic acid bacteria counts increased in fermented porridge samples, but decreased slightly in cooked fermented porridge samples. The mean pH value of sorghum powder samples decreased in fermented and cooked fermented porridge, respectively. Bacillus (B.) cereus was detected in all 15 sorghum powder samples, while Escherichia (E.) coli was detected in 53%, Clostridium perfringens in 27%, Listeria monocytogenes in 13% and Aeromonas spp., Salmonella spp., Staphylococcus aureus, Shigella spp. and Yersinia spp., each in 7% of sorghum powder samples. Of the fermented porridge samples, 40% contained B. cereus and 7% contained E. coli. None of the pathogens tested for were detected in cooked fermented porridge samples. B. cereus (53%), B. subtilis (21%), B. thuringiensis (13%), B. licheniformis (10%) and B. coagulans (3%) were identified from 120 isolates randomly selected from spore count plates of the highest dilution showing growth.

Characterization of leucocin B-Ta11a: a bacteriocin fromLeuconostoc carnosum Ta11a isolated from meat.

Leuconostoc (Lc.)carnosum Ta11a, isolated from vacuum-packaged processed meats, produced a bacteriocin designated leucocin B-Ta11a. The crude bacteriocin was heat stable and sensitive to proteolytic enzymes, but not to catalase, lysozyme, or chloroform. It was active againstListeria monocytogenes and several lactic acid bacteria. Leucocin B-Ta11a was optimally produced at 25°C in MRS broth at an initial pH of 6.0 or 6.5 An 8.9-MDa plasmid inLeuconostoc carnosum Ta11a hybridized to a 36-mer oligonucleotide probe (JF-1) that was homologous to leucocin A-UAL187. A 4.9-kbSau3A fragment from a partial digest of the 8.9-MDa plasmid was cloned into pUC118. The 8.1-kb recombinant plasmid (pJF8.1) was used for sequencing and revealed the presence of two open reading frames (ORFs). ORF1 codes for a protein of 61 amino acids comprising a 37-amino-acid bacteriocin that was determined to be the leucocin B-Ta11a structural gene by virtue of its homology to leucocin A-UAL 187 (Hastings et al. 1991. J. Bacteriol 173: 7491–7500). The 24-amino-acid N-terminal extension, however, differs from that of leucocin A-UAL187 by seven residues. The predicted protein of the ORF2 has 113 amino acids and is identical with the amino acid sequence of the cognate ORF of the leucocin A-UAL 187 operon.

Sequence and structural relationships of leucocins A-, B- and C-TA33a from Leuconostoc mesenteroides TA33a

Amino acid sequences of two of the three bacteriocins from Leuconostoc mesenteroides TA33a were determined and their sequence-structure relationships investigated. Leucocin B-TA33a consists of 31 amino acid residues, with a molecular mass of 3466 Da. Leucocin B-TA33a does not belong to the pediocin family of bacteriocins, but shares 62% homology with mesenterocin 52B. A partial sequence of 36 amino acids of leucocin C-TA33a (4598 Da) was determined. Leucocin C-TA33a belongs to the class II bacteriocins having the consensus YGNGV motif. The third bacteriocin, leucocin A-TA33a, is identical to leucocin A-UAL 187. Circular dichroism spectra of the leucocins in aqueous solution and micellar SDS indicated that they undergo a structural transition when in a membrane-mimicking environment. Theoretical predictions from circular dichroism data suggest that leucocins A-, B- and C-TA33a adopt a beta-structure (48%) in membrane-mimicking environments. Sequence alignments and secondary structure predictions for the N-terminus of leucocins A- and C-TA33a predicted that Cys-9 and Cys-14 are connected by a disulfide bridge and form two beta-strands.

Biogenic amine formation by poultry‐associated spoilage and pathogenic bacteria

The production of biogenic amines by 50 poultry‐associated bacterial strains (25 Pseudomonas, 13 Salmonella and 12 Listeria) was investigated on amine agar plates containing lysine, histidine, ornithine, phenylalanine, tryptophan and tyrosine. Seventy‐four per cent of all the strains produced cadaverine and putrescine, while phenylethylamine, histamine, tyramine and tryptamine were produced by 72, 56, 34 and 24% of strains, respectively. Different patterns of biogenic amine production amongst the three bacterial genera tested were apparent as well as amongst strains of the same genus. This study highlighted a high incidence of biogenic amine‐producing bacterial strains associated with poultry.

Microbiological survey of street-vended salad and gravy in Johannesburg city, South Africa

Abstract Salad (55) and gravy (55) samples were collected over a period of 4 months from 16 street vendors located in central Johannesburg and working under conditions perceived as unsuitable for the preparation and selling of ready-to-eat foods. Standard methods were used to determine aerobic plate counts (APC), gram-negative counts (GC) and spore counts (SC). Predominant colonies (362) were isolated from APC plates of the highest dilution showing growth of all samples and characterised to genus level. The incidence of Bacillus cereus and Clostridium perfringens in 35 salad and 35 gravy samples was also determined. Mean APC and SC values of salad samples were significantly higher ( P P >0.05) were, however, observed between mean GC values of salad and gravy samples. The 362 predominant isolates recovered from APC plates of salad (190) and gravy (172) samples were mostly Bacillus spp. and Micrococcaceae. B. cereus was not detected in any of the samples analysed and C. perfringens was detected in 1 gravy and 1 salad sample. It was concluded that the quality and safety of salads and gravies analysed in this study was acceptable despite the observed unhygienic food handling practices and unsanitary environmental conditions under which the vendors operated.