D. Lindsay

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.

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).

Biofilm-Spore Response in Bacillus cereus and Bacillus subtilis during Nutrient Limitation

This study aimed to trace the dynamics of biofilm formation by vegetative cells and endospores of Bacillus cereus DL5 and Bacillus subtilis 168. Counts of B. cereus DL5 and B. subtilis 168 vegetative cells and spores either attached to glass wool or, correspondingly, planktonic cells were determined by standard plate-counting methods. Results from this study highlighted the biofilm-forming potential of both spores and vegetative cells of two different Bacillus species. It was shown that once Bacillus spores had attached to a surface, the spores germinated under favorable (B. cereus DL5) and even unfavorable (B. subtilis 168) nutrient conditions, resulting in biofilms containing both spores and vegetative populations. Furthermore, it was suggested that vegetative B. cereus DL5 cells exhibited a low propensity for spore formation in attached and planktonic growth forms in nutrient-limited growth medium. By contrast, vegetative B. subtilis 168 cells readily formed spores in planktonic and attached microcosms when exposed to nutrient-limited growth conditions. Sporulation in attached Bacillus populations is an important practical consideration for many food industries, such as dairy processing, where bacilli are routinely isolated from populations attached to processing-equipment surfaces.

Carbon : nitrogen : phosphorus ratios influence biofilm formation by Enterobacter cloacae and Citrobacter freundii

Aims:  To test the effects of C : N : P ratio modification of a well‐known nutrient medium formulation, the Endo formulation on biofilm formation by Enterobacter cloacae Ecl and Citrobacter freundii Cf1 in both single‐species and binary species biofilms.

What food safety professionals should know about bacterial biofilms

Purpose – This paper seeks to highlight the importance of bacterial associations with surfaces, with particular reference to food processing.Design/methodology/approach – A historical and interdisciplinary review of recent literature combined with research on biofilms on surfaces was conducted.Findings – The association of micro‐organisms with surfaces is the prevailing microbial lifestyle and bacterial biofilms may represent reservoirs for the spread of antimicrobial‐resistance genes.Originality/value – This paper is a condensed summary of relevant information on the discipline of bacterial biofilms as a whole, with special reference to food processing and safety.

Spore Formation in Bacillus subtilis Biofilms

Spore formation by a Bacillus strain (Bacillus subtilis SpoIVFB-GFP) engineered with a green fluorescent protein (GFP) fused to a polytopic membrane protein (SpoIVF) that fluoresces during sporulation was observed. Biofilms of B. subtilis SpoIVFB-GFP containing ca. 8 log CFU/ml vegetative cells and spores below the lower detection limit (i.e., <1 log CFU/ ml) were allowed to develop on glass wool (37 degrees C). These biofilms were subsequently exposed to nutrient limitation to stimulate spore formation, which was monitored for fluorescence by confocal scanning laser microscopy. Sporulation in corresponding planktonic cells was also monitored for comparative purposes. Planktonic B. subtilis SpoIVFB-GFP cells began fluorescing after 5 h, while B. subtilis SpoIVFB-GFP biofilm cells began fluorescing after 30 h. Results suggested that an existing biofilm of vegetative B. subtilis cells may be stimulated to form spores when exposed to conditions of nutrient limitation. From a practical point of view, it may be suggested that a window of time does exist before sporulation occurs in attached Bacillus biofilms highlighting the need for shorter operating runs between cleaning and sanitation of food-processing equipment surfaces.

Adaptation of a neutrophilic dairy‐associated Bacillus cereus isolate to alkaline pH

Aims: This study identified and studied the response of five Bacillus strains, isolated from alkaline cleaning in place (CIP) solutions, to alkaline conditions.

Potential cross‐contamination of the ready‐to‐eat dried meat product, biltong

Purpose – The aim of this paper is to evaluate the hygiene of surfaces that come into direct contact with the ready to eat dried meat product, biltong, at point‐of‐sale in three different retailers in Johannesburg, South Africa, by investigating the presence of indicator organisms.Design/methodology/approach – Samples were collected and plated in duplicate for aerobic plate, total Enterobacteriaceae, coliforms and Escherichia coli counts using standard methods. Typical E. coli colonies on Rapid E. coli 2 Agar™ were selected and further identified using 16S rDNA molecular sequencing methods.Findings – Bacterial counts associated with biltong product ranged between 6–7 Log CFU/g, while counts on cutting utensils ranged between 5–6 Log CFU/cm2. Overall, the lowest counts were associated with display cabinets (2–6 Log CFU/cm2). Predominant populations were often similar between biltong product and various surface samples, indicating potential cross‐contamination. Results from 16S rDNA sequence analysis showed...

Cytotoxicity of alkaline-tolerant dairy-associated Bacillus spp.

The cytotoxicity of five Bacillus spp. isolated from alkaline cleaning solutions in South African dairies was evaluated against McCoy mouse cells using a 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT)‐based assay, confocal scanning laser microscopy and scanning electron microscopy. According to the MTT‐based assay, two of the Bacillus isolates (Bacillus licheniformis 5 and B. pumilus 122) were cytotoxic to McCoy cells and the cytotoxic components were heat labile. Confocal scanning laser microscopy combined with fluorescent staining using propidium iodide and fluorescein diacetate indicated that cytotoxic effects occurred within 3 h, appeared to be membrane active and resulted in cell necrosis. Scanning electron microscopy showed that McCoy cells exposed to the cytotoxic components exhibited morphological damage.