Fate of Salmonella Typhimurium in laboratory-scale drinking water biofilms.

Investigations were carried out to evaluate and quantify colonization of laboratory-scale drinking water biofilms by a chromosomally green fluorescent protein (gfp)-tagged strain of Salmonella Typhimurium. Gfp encodes the green fluorescent protein and thus allows in situ detection of undisturbed cells and is ideally suited for monitoring Salmonella in biofilms. The fate and persistence of non-typhoidal Salmonella in simulated drinking water biofilms was investigated. The ability of Salmonella to form biofilms in monoculture and the fate and persistence of Salmonella in a mixed aquatic biofilm was examined. In monoculture S. Typhimurium formed loosely structured biofilms. Salmonella colonized established multi-species drinking water biofilms within 24 hours, forming micro-colonies within the biofilm. S. Typhimurium was also released at high levels from the drinking water-associated biofilm into the water passing through the system. This indicated that Salmonella could enter into, survive and grow within, and be released from a drinking water biofilm. The ability of Salmonella to survive and persist in a drinking water biofilm, and be released at high levels into the flow for recolonization elsewhere, indicates the potential for a persistent health risk to consumers once a network becomes contaminated with this bacterium.

Cloning and characterization of a 4-hydroxyphenylacetate 3-hydroxylase from the thermophile Geobacillus sp. PA-9.

A 4-hydroxyphenylacetic acid (4-HPA) hydroxylase-encoding gene, on a 2.7-kb genomic DNA fragment, was cloned from the thermophile Geobacillus sp. PA-9. The Geobacillus sp. PA-9 4-HPA hydroxylase gene, designated hpaH, encodes a protein of 494 amino acids with a predicted molecular mass of 56.269 Da. The deduced amino-acid sequence of the hpaH gene product displayed <30% amino-acid sequence identity with the larger monooxygenase components of the previously characterized two-component 4-HPA 3-hydroxylases from Escherichia coli W and Klebsiella pneumoniae M5a1. A second oxidoreductase component was not present on the 2.7-kb genomic DNA fragment. The deduced amino-acid sequence of a second C-terminal truncated open reading frame, designated hpaI, exhibited homology to extradiol oxygenases and displayed the highest amino-acid sequence identity (43%) with the 3,4-dihydroxyphenylacetate 2,3-dioxygenase of Arthrobacter globiformis, encoded by mndD. These results, along with catalytic activity observed in crude intracellular extracts prepared from Escherichia coli cells expressing hpaH, is in support of a role for hpaH in the 4-HPA degradative pathway of Geobacillus sp. PA-9.

Prevalence of bacterial pathogens in biofilms of drinking water distribution systems.

Water for human consumption is required to be free from any bacteria that might pose a health risk. The presence of biofilms in the drinking water distribution system may play a role in the presence of potential pathogens in the drinking water supply. Ninety-five biofilm samples from various parts of South Africa were tested for the presence of Escherichia coli, Aeromonas, Pseudomonas, Salmonella, Shigella and Vibrio spp. Members of these genera were quantified by the three-tube most probable number (MPN) approach using enrichment broths and plating on selective agars. The heterotrophic culturable counts were determined for both the planktonic and biofilm phases of the samples. Biofilm density varied between 10 and 1.9 x 10(9) colony forming units cm(-2). The 16S rRNA identity of the putative pathogenic isolates revealed that high numbers of Aeromonas, Pseudomonas, Klebsiella and Enterobacter were present, but no putative Salmonella and Shigella could be confirmed. None of the Pseudomonas isolates belonged to the pathogenic Pseudomonas aeruginosa or Pseudomonas mendocina while the Aeromonas isolates showed relatedness to known pathogenic members of this group.

Lectin binding profile of the small intestine of five-week-old pigs in response to the use of chlortetracycline as a growth promotant and under gnotobiotic conditions.

Antibiotics have traditionally been used for growth promotion in the pork industry; however, their use in animal feed has recently been limited because of human health concerns. The intestinal microbiota plays an important role in mediating many physiological functions such as digestion and animal growth. It was hypothesized that use of antibiotics as growth promotants and subsequent variations in intestinal microbiota induce significant changes in the intestinal glycoconjugate composition, which ultimately affects animal growth and disease susceptibility. The aim of this study was to characterize the lectin binding profiles of the ileum of weanling pigs in response to the absence of intestinal microbiota and to the use of the antibiotic chlortetracycline as growth promotant. Eighteen half-sib piglets obtained by cesarean section were divided into 3 treatment groups (n = 6) and maintained as control, antibiotic-fed, and gnotobiotic piglets until 5 wk of age. The glycoconjugate composition of the ileal tissues was examined by lectin histochemistry. Lycopersicon esculentum lectin, Jacalin, Pisum sativum agglutinin, Lens culinaris agglutinin (LCA), and Sambucus nigra lectin showed significant differences (P < 0.05) in binding intensities on the dome and villous epithelium between the treatment groups. Griffonia simplicifolia lectin I, Glycine maxi agglutinin, and Arachis hypogea agglutinin exhibited differences (P < 0.05) between treatment groups in lectin binding on goblet cells. Triticum vulgaris agglutinin, Pisum sativum agglutinin, and LCA showed significant differences (P < 0.05) in binding intensities on dome, corona, and follicular regions of the ileum among treatment groups of animals. Overall, ileal tissues from gnotobiotic piglets expressed significantly weaker (P < 0.05) lectin binding for many lectins compared with control and antibiotic groups. This suggests that the intestinal microbiota plays an important role in the expression of sugar moieties in the intestine. Lectins LCA, Phaseolus vulgaris Leucoagglutinin, and Maackia amurensis lectin II showed significant differences (P < 0.05) in lectin bindings between control and antibiotic-fed piglets. This indicates that chlortetracycline as a growth promotant induces biologically relevant changes in the lectin binding profile of the ileum. These findings will help in further understanding the role of the gut microbiota and the mechanisms of action of antibiotics as growth promotants in pigs.

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.

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.

Diversity of nontuberculoid Mycobacterium species in biofilms of urban and semiurban drinking water distribution systems.

Nontuberculous mycobacteria (NTM) are ubiquitous and have been isolated from a variety of environmental sources, including water. Various NTM were isolated from biofilms in drinking water distribution systems in two urban and two semiurban areas in South Africa. Most of the isolates belonged to opportunistic pathogenic species of the NTM group, but none belonged to the Mycobacterium avium complex.

The occurrence of campylobacters in water sources in South Africa.

Campylobacter spp., mainly C. jejuni and C. coli, are recognized as significant human bacterial pathogens, being responsible for increasing numbers of gastroenteritis cases worldwide. Several reports have indicated that environmental waters are potential reservoirs and transmitting vehicles for these bacteria. The purpose of this study was thus to examine the occurrence of campylobacters in drinking and environmental water sources of South Africa, a country with a warmer climate and higher microbial pollution levels than those previously addressed in the Northern Hemisphere where similar investigations have been undertaken. Various types of water samples (five drinking water, four ground water, 11 surface water and four raw sewage) were collected from different parts of South Africa. Detection was by enrichment in Bolton broth prior to plating on both selective mCCDA or through a 0.6microm membrane filter on non-selective blood agar isolation media. Out of 100 initially selected Campylobacter-like isolates, only 22 did not grow aerobically and were subsequently identified as Campylobacter spp. by biochemical tests. However, the results obtained by 16S rRNA sequence analysis indicated that only three of these strains (13.6%) were Campylobacter jejuni and the remaining 19 strains were identified as Arcobacter butzleri. The spread of Arcobacter via water warrants further investigation, especially in view of the higher levels of detection and pathogenic nature of these bacteria.

Mechanisms contributing to hypochlorous acid resistance of a Salmonella isolate from a poultry-processing plant.

AIMS: We have recently reported the isolation of Salmonella that have acquired tolerance to hypochlorous acid (HOCl) (Mokgatla et al. 1998). The aim of this work was to investigate possible protective mechanisms involved in the increased tolerance to HOCl of a selected resistant strain. METHODS AND RESULTS: One resistant (Salmonella 104) and one sensitive (Salmonella 81) isolate in exponential phase were exposed to HOCl at a final active concentration of 28 mg l(-1). Cultures were assayed for superoxide dismutase and catalase activity, as well as for four membrane-bound dehydrogenases (malate, lactate, glutamate and glucose-6-phosphate dehydrogenase). The degree of single-strand breaks in genomic DNA was analysed and lipopolysaccharide profiles determined. The resistant Salmonella isolate differed from the sensitive isolate in a number of ways. It responded within 10 min of exposure by producing catalase and decreasing the activity levels of four membrane-bound dehydrogenases. This combination would lead to lower levels of hydroxyl radicals and singlet oxygen, moieties thought to be integrally involved in the antibacterial action of HOCl. Furthermore, the resistant strain did not display the same degree of DNA damage as did the sensitive strain. CONCLUSIONS: Strain 104 is believed to grow in the presence of 28 mg l(-1) HOCl by protecting itself against HOCl by decreasing the levels of species that could react with HOCl to generate toxic reactive oxygen radicals and by improved DNA damage repair mechanisms. SIGNIFICANCE AND IMPACT OF THE STUDY: The occurrence of Salmonella able to grow in the presence of 28 mg l(-1) HOCl is of relevance to the food-processing and drinking water treatment industries as these strains would survive sanitation regimes.

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. METHODS AND RESULTS: Isolates were identified as B. cereus by 16S rDNA sequencing. External and internal cell pH and buffering capacity data of a representative strain, Bacillus DL5, were compared to B. cereus ATCC 10702. Results indicated that a buffering system was induced when the pH of the growth medium increased to above pH 10, which was effective up to pH 12 and presumably cell wall associated. Volume measurements and confocal scanning laser microscope images of Bacillus DL5 cells showed that cells exhibited more pronounced stress symptoms when exposed to pH 10 than at pHs above 10. Long-term exposure of Bacillus DL5 to pH 10 or 10.5 indicated that cells grew in planktonic form and formed biofilms at both pHs. CONCLUSIONS: Bacillus DL5 was a neutrophile with a growth pH range similar to B. cereus ATCC 10702, but tolerated alkaline pH. This may be a general trait of the B. cereus species rather than a specific phenomenon of isolates from alkaline ecosystems. SIGNIFICANCE AND IMPACT OF THE STUDY: Other neutrophilic B. cereus isolates may exhibit similar responses to alkaline conditions as the isolates studied here. These results may have important implications for dairy manufacturers.

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. METHODS AND RESULTS: Surfaces were sampled daily before and after sanitizer treatment and cells and spores dislodged and enumerated by standard methods. Duplicate surfaces were prepared for confocal scanning laser microscopy (CSLM) and scanning electron microscopy. Higher counts of Ps. fluorescens M2 were obtained in single species biofilms, microcolonies stained green (viable) in CSLM images and were closely packed on attachment surfaces. By contrast, higher counts of B. cereus DL5 were obtained in binary biofilms, microcolonies stained green in CSLM images, but were more spread out. Lower spore counts were obtained for B. cereus DL5 in binary biofilms. The survival of Ps. fluorescens M2 cells after exposure to chlorine dioxide was apparently enhanced by the presence of B. cereus DL5 in binary biofilms. By contrast, B. cereus DL5 showed increased susceptibility to sanitizer treatment in the presence of Ps. fluorescens M2. CONCLUSIONS: Co-cultured bacteria in biofilms influence each other with respect to attachment capabilities and sanitizer resistance/susceptibility. SIGNIFICANCE AND IMPACT OF THE STUDY: Binary biofilms endemic in food-processing industries can survive sanitation regimes and may represent reservoirs of product contamination leading to subsequent spoilage and/or food safety risks.

The use of glass wool as an attachment surface for studying phenotypic changes in Pseudomonas aeruginosa biofilms by two-dimensional gel electrophoresis.

Two-dimensional polyacrylamide gel electrophoresis was used to demonstrate phenotypic differences between Pseudomonas aeruginosa biofilm cells and the planktonic counterpart cells under defined culture conditions. Glass wool was used as a substratum for cell attachment as it affords a large surface-to-volume ratio (1 g with a mean diameter of 15 microns = 1300 cm2), supports the growth of biofilms, allows for free movement of cells between the inter-strand spaces, and it facilitates the exchange of nutrients and oxygen. It also allows for the separation of the biofilm biomass from the surrounding surface influenced planktonic (SIP) cells for further characterization. Comparative analysis of the respective proteomes indicated striking differences in the protein patterns of planktonic, biofilm and SIP cells. We selected 41 proteins, the levels of which varied in a significant and reproducible way in the respective protein profiles. In the biofilm cells, a general up-regulation of the spots was seen, but in SIP cells expression of these spots were generally down-regulated. Altogether six unique proteins were seen in the planktonic cells, while the biofilm and SIP cells contained five and two unique proteins, respectively. Glass wool, therefore, appears to be an ideal attachment surface for the study of biofilm development.

A sensitive seminested PCR method for the detection of Shigella in spiked environmental water samples.

A rapid seminested polymerase chain reaction (PCR) method for the specific, sensitive detection of virulent Shigella spp. in spiked environmental water samples was developed. A set of primers specific for the invasion plasmid antigen gene (ipaH) of virulent Shigella spp. and enteroinvasive Escherichia coli produced a 620-bp fragment that was used as template for the seminested primer pair delineating a 401-bp fragment. By using agarose gel electrophoresis for detection of the seminested PCR-amplified products, a detection limit of 1.6 x 10(3) cfu S. flexneri was obtained with amplification reactions from crude bacterial lysates. The PCR procedure coupled with an enrichment culture incubated for 6 h detected as few as 1.6 S. flexneri organisms in pure culture. Treated sewage, ground, surface and drinking water samples collected from various sources were seeded with S. flexneri and incubated in GN broth for 6 h before detection by seminested PCR. A detection limit lower than 14 cfu/ml was achieved in some water samples. The results indicate that the described seminested PCR has the advantage of a rapid turnaround time and it fulfills the requirements of sensitivity and specificity for use in an environmental laboratory.

Novel method for the proteomic investigation of a dairy-associated Bacillus cereus biofilm.

The biofilm proteome of a dairy-associated Bacillus cereus strain (B. cereus 5) was investigated. Biofilm biomass of sufficient concentration for 2D-PAGE was obtained by growing the culture in the presence of glass wool. B. cereus 5 readily attached to the glass wool and biofilms formed within 18 h. The biofilm proteome of whole-cell proteins revealed that 10 proteins were synthesized as a result of surface attachment of which four were unique to the biofilm profile. Seven proteins appeared to be absent in the biofilm profile. The altered proteomes indicated that changes took place in the regulation of protein expression when B. cereus 5 cells attached to surfaces.

Detection of toxigenic Vibrio cholerae from environmental water samples by an enrichment broth cultivation-pit-stop semi-nested PCR procedure.

A pit-stop semi-nested PCR assay for the detection of toxigenic Vibrio cholerae in environmental water samples was developed and its performance evaluated. The PCR technique amplifies sequences within the cholera toxin operon specific for toxigenic V. cholerae. The PCR procedure coupled with an enrichment culture detected as few as four V. cholerae organisms in pure culture. Treated sewage, surface, ground and drinking water samples were seeded with V. cholerae and following enrichment, a detection limit of as few as 1 V. cholerae cfu ml(-1) was obtained with amplification reactions from crude bacterial lysates. The proposed method, which includes a combination of enrichment, rapid sample preparation and a pit-stop semi-nested PCR, could be applicable in the rapid detection of toxigenic V. cholerae in environmental water samples.

The response of a bacterial biofilm community in a simulated industrial cooling water system to treatment with an anionic dispersant.

The effect of a dispersant on the microbial community in a simulated open recirculating cooling water system was determined by continuous operation of the system over two consecutive periods of 196 and 252 d, respectively. An open recirculating cooling water system feeding a modified Robbin's Device with synthetic cooling water to simulate the environment of an industrial cooling water system was set up. Planktonic and biofilm (mild steel and Nylon(R)) samples were taken weekly in 1997 (196-d period) and fortnightly in 1998 (252-d period). Each biofilm was scraped off and diluted in 10-ml 1 x phosphate-buffered saline (PBS). Serial dilutions were performed and plated onto R2A agar (pH 8.0) to obtain the predominant culturable bacteria. The diversity was determined by allocating groups according to colony morphology, diameter and colour. Diversity was calculated according to the Shannon-Weaver Index. During the first run (1997), dispersant was added on day 57 to a final concentration of 15 mg l-1 for 49 d, stopped for 49 d and dosed at 30 mg l-1 for 41 d. The second run entailed adding dispersant to a final concentration of 30 mg l-1 on day 98 for 70 d, stopping dosing for 56 d and resuming dosing at 30 mg l-1 for another 28 d. The 2-year evaluation period demonstrated that the biofilm-removing action of the dispersant decreased to a point where it was not effective at all. Our results showed that the synthetic dispersant evaluated was only effective initially, but was ineffective in controlling biofouling on Nylon, and to a lesser degree on mild steel at the recommended (15 mg l-1) as well as at double the recommended concentration in the long term. The release of cells from biofilms observed when dispersant dosing was terminated, supports the notion that a community attaching in the presence of the surface active agent was selected for. The decreased efficacy may therefore be due to a selection of strains able to remain attached and/or attach in the presence of the dispersant as demonstrated by shifts in the biofilm communities on both Nylon and mild steel.

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.

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).

Electro-chemically activated water in dental unit water lines.

OBJECTIVE: To investigate the effect of electro-chemically activated water on biofilm contamination in dental unit water lines. DESIGN: Thirteen dental units fitted with independent water systems and used for 12 years with distilled water were divided into two groups, A and B. At the start, one week later, and again four weeks later, the bacterial counts in water from all units were determined. Also specimens of tubing were taken from the units at the beginning and at the end of the study for SEM investigation. In Group A distilled water was replaced with electrochemically activated water (a Russian invention), and used continuously for the duration of the study. In group B, distilled water was used as before, until confirmed to be contaminated. For ethical reasons group B was treated, one week into the study with conventional disinfectants. SETTING: The project was carried out in a clinic of a department of periodontology of a faculty of dentistry during 1998. RESULTS: Both groups showed a marked reduction in bacterial counts. Under SEM Group A showed a total elimination of the biofilm and Group B a partial removal. CONCLUSIONS: Distilled water was ineffective in controlling bacterial counts and biofilm. Electrochemically activated water was effective for this purpose.