Denise Lindsay

Thermophilic bacilli and their importance in dairy processing.

The thermophilic bacilli, such as Anoxybacillus flavithermus and Geobacillus spp., are an important group of contaminants in the dairy industry. Although these bacilli are generally not pathogenic, their presence in dairy products is an indicator of poor hygiene and high numbers are unacceptable to customers. In addition, their growth may result in milk product defects caused by the production of acids or enzymes, potentially leading to off-flavours. Dairy thermophiles are usually selected for by the conditions during dairy manufacture. These bacteria are able to grow in sections of dairy manufacturing plants where temperatures reach 40-65°C. Furthermore, because they are spore formers, they are difficult to eliminate. In addition, they exhibit a wide temperature growth range, exhibit a fast growth rate (generation time of approximately 15-20 min) and tend to readily form biofilms. Many strategies have been tested to remove, prevent and/or delay the formation of thermophilic bacilli biofilms in dairy manufacture, but with limited success. This is, in part, because little is known about the structure and composition of thermophilic bacilli biofilms in general and, more specifically, in milk processing environments. Therefore, new cleaning regimes often do not target the problem optimally. A greater understanding of the structure of thermophilic biofilms within the context of the milk processing environment and their link with spore formation is needed to develop better control measures. This review discusses the characteristics and food spoilage potential, enumeration and identification methods for the thermophilic bacilli, as well as their importance to dairy manufacture, with an emphasis on biofilm development and spore formation.

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.

Review of Shiga-toxin-producing Escherichia coli (STEC) and their significance in dairy production

The involvement of the pathogenic Shiga-toxin-producing Escherichia coli (STEC; also called verocytotoxic-producing E. coli or VTEC) in sporadic cases and disease outbreaks is presently increasing. Infrequent cases are due to ingestion of milk and dairy products. As ruminants are healthy carriers of STEC and most dairy products may provide these bacteria with favourable conditions for their growth, milk and dairy products are a potential source of STEC. But not all STEC serotypes are pathogens; only relatively small numbers in the entire family of STEC are pathogenic. This review focuses on the recent advances in understanding of STEC and their significance in milk and dairy products. It is intended to gather the information that is needed to understand how these bacteria are described, detected and characterised, how they contaminate milk and grow in dairy products, and how the dairy industry can prevent them from affecting the consumer.

Proteolysis produced within biofilms of bacterial isolates from raw milk tankers

In this study, six bacterial isolates that produced thermo-resistant enzymes isolated from the internal surfaces of raw milk tankers were evaluated for their ability to produce proteolysis within either single culture biofilms or co-culture biofilms. Biofilms were formed in an in vitro model system that simulated the upper internal surface of a raw milk tanker during a typical summers day of milk collection in New Zealand. The bacterial isolates were further evaluated for their ability to form biofilms at 25, 30 and 37°C. Mutual and competitive effects were observed in some of the co-culture biofilms, with all isolates being able to form biofilms in either single culture or co-culture at the three temperatures. The proteolysis was also evaluated in both biofilms and corresponding planktonic cultures. The proteolysis per cell decreased as the temperature of incubation (20-37°C) increased. Furthermore, mutualistic interactions in terms of proteolysis were observed when cultures were grown as co-culture biofilms. This is the first study to show that proteolytic enzymes can be produced in biofilms on the internal surfaces of raw milk tankers. This has important implications for the cleaning and the temperature control of raw milk transport tankers.

Microbiology of raw milk in New Zealand

The results of this study demonstrate the occurrence of the non-spore-forming pathogens, Staphylococcus aureus, Escherichia coli (total count and O157:H7), Listeria, Campylobacter and Salmonella, in New Zealands raw milk supply. Samples of raw milk were collected monthly within five major dairying regions over one year. Each month, samples from five randomly selected farm vats in each region were collected for analysis (297 samples in total). Methods based on plate count techniques were used to enumerate S. aureus and E. coli. Enrichment methods in combination with a modified most probable number detection method were used to monitor samples for the presence of E. coli O157:H7, Listeria, Campylobacter and Salmonella. Salmonella was not detected in this study, and Campylobacter was isolated once (0.34%). E. coli was present at <100 cfu/ml in 99% of samples and exceeded 10(3)cfu/ml in 0.7% of samples. E. coli O157:H7 was not detected whereas non-pathogenic E. coli O157 strains (i.e. lacking genes for stx1, stx2, eae and Hly A) were detected in 1% of samples. S. aureus was not detected (<1 cfu/ml) in 21% of samples; levels were >1 but <100 cfu/ml in 60% of samples and on one occasion (0.34%) S. aureus exceeded 10(4)cfu/ml. L. monocytogenes was isolated from 0.68% of samples and L. innocua was present in 4% of samples. The results demonstrate that raw milk sampled from farm vats in New Zealand, as in other countries, inevitably contains recognised pathogens and, hence, control by pasteurisation or an equivalent treatment of raw milk remains paramount. Even so, the prevalence of most of these pathogens was lower than those reported in many of the studies performed in other countries.

Lipolysis within single culture and co-culture biofilms of dairy origin

Bacteria in raw milk can produce heat-stable lipases, which survive pasteurisation and subsequently reduce the shelf life of dairy products because of their ability to break down the milk fat and increase rancidity. In this study, four bacteria, originating from the surfaces of raw milk transport tankers, and a known lipase-producing bacterium were evaluated for their ability to produce lipolysis in planktonic and biofilm cultures. Lipolysis was determined using two separate assays that measured hydrolysis of the ester p-nitrophenol palmitate (pnpp) and the lipid tributyrin. The hydrolysis of pnpp per CFU within biofilms and planktonic cultures ranged from 0.01 to 8.35 and 0.01 to 0.07 nU/CFU respectively. The amount of butyric acid released from hydrolysis of tributyrin per CFU within biofilms and planktonic cultures ranged from 0.1 to 1110.3 and 0.1 to 0.3 ng/CFU, respectively. The hydrolysis of pnpp and tributyrin per CFU within biofilms was at least 10 times higher compared with the corresponding planktonic cultures. This is the first study to show that lipolysis occurs within biofilms of bacteria that were originally isolated from the surfaces of raw milk tankers. This is relevant to the dairy industry, highlighting the importance of eliminating biofilms on milk tanker surfaces as a source of heat-stable lipases.

Characterization of thermophilic bacilli from a milk powder processing plant

To determine whether strains of Geobacillus stearothermophilus isolated from a milk powder manufacturing plant were different in their ability to form biofilms and produce spores. In addition, this study evaluated whether there were other physiological characteristics that could differentiate these strains.

Preconditioning with cations increases the attachment of Anoxybacillus flavithermus and Geobacillus species to stainless steel.

ABSTRACT Preconditioning of Anoxybacillus flavithermus E16 and Geobacillus sp. strain F75 with cations prior to attachment often significantly increased (P ≤ 0.05) the number of viable cells that attached to stainless steel (by up to 1.5 log CFU/cm2) compared with unconditioned bacteria. It is proposed that the transition of A. flavithermus and Geobacillus spp. from milk formulations to stainless steel product contact surfaces in milk powder manufacturing plants is mediated predominantly by bacterial physiological factors (e.g., surface-exposed adhesins) rather than the concentrations of cations in milk formulations surrounding bacteria.

Biofilm formation by spore-forming bacteria in food processing environments.

Abstract: Endospore-forming bacteria present a unique challenge in biofilm studies, as well as to food and beverage manufacturers within processing stages. This chapter discusses the mesophilic endospore formers, such as Bacillus species, with respect to attachment of spores to surfaces and subsequent biofilm development. In addition, many manufacturing processes utilize processing temperatures which are suitable for the growth of thermophilic endospore-forming bacteria, such as Geobacillus and Anoxybacillus strains, increasing concerns of contamination of product. This chapter also highlights the biofilm-forming ability coupled with spore development in these strains. From results obtained to date, biofilm-spore development in themophilic strains seems to be very different from that of mesophilic Bacillus isolates. Future studies will further elucidate the spore-biofilm interaction and allow for better control of such biofilms within industrial settings.

Insights into the Geobacillus stearothermophilus species based on phylogenomic principles

BackgroundThe genus Geobacillus comprises bacteria that are Gram positive, thermophilic spore-formers, which are found in a variety of environments from hot-springs, cool soils, to food manufacturing plants, including dairy manufacturing plants. Despite considerable interest in the use of Geobacillus spp. for biotechnological applications, the taxonomy of this genus is unclear, in part because of differences in DNA-DNA hybridization (DDH) similarity values between studies. In addition, it is also difficult to use phenotypic characteristics to define a bacterial species. For example, G. stearothermophilus was traditionally defined as a species that does not utilise lactose, but the ability of dairy strains of G. stearothermophilus to use lactose has now been well established.ResultsThis study compared the genome sequences of 63 Geobacillus isolates and showed that based on two different genomic approaches (core genome comparisons and average nucleotide identity) the Geobacillus genus could be divided into sixteen taxa for those Geobacillus strains that have genome sequences available thus far. In addition, using Geobacillus stearothermophilus as an example, we show that inclusion of the accessory genome, as well as phenotypic characteristics, is not suitable for defining this species. For example, this is the first study to provide evidence of dairy adaptation in G. stearothermophilus - a phenotypic feature not typically considered standard in this species - by identifying the presence of a putative lac operon in four dairy strains.ConclusionsThe traditional polyphasic approach of combining both genotypic and phenotypic characteristics to define a bacterial species could not be used for G. stearothermophilus where many phenotypic characteristics vary within this taxon. Further evidence of this discordant use of phenotypic traits was provided by analysis of the accessory genome, where the dairy strains contained a putative lac operon. Based on the findings from this study, we recommend that novel bacterial species should be defined using a core genome approach.