The effect of phosphate on the heat resistance of spores of dairy isolates of Geobacillus stearothermophilus.

In this study, we show that phosphate decreases the spore heat resistance by accelerating the rate of loss of cations from spores. Heat resistance of spores of Geobacillus stearothermophilus A1, D1, P3 and ATCC 12980 were determined in distilled water containing varying concentrations (0.1, 1 and 2% w/v) of di­sodium phosphate. The average decimal reduction times (D value) for strains A1, D1, P3 and ATCC 12980 in distilled water were 5.8, 6.8, 5.7 and 9 min at 110 °C respectively. On the addition of 0.1, 1 and 2% w/v of di­sodium phosphate, the average D110 values of all the strains in distilled water were lowered by 50, 61 and 70% respectively. Addition of 0.05% w/v of Na-EDTA to distilled water resulted in lowering of the average D110 value of all the strains by 55%. Heat resistance of spores of A1, D1, P3 and ATCC 12980 was found to be associated with the Dipicolinic Acid (DPA) content whose concentrations were 0.25, 0.30, 0.27 and 1.6 pg per spore respectively. Analysis by atomic absorption spectroscopy revealed that the phosphate present in the heating medium causes excess release of calcium from spores with 2% w/v phosphate being highly effective, thus confirming the chelating effect of phosphate. This study provides insight into the heat resistance and the increased heat sensitivity of spores of G. stearothermophilus A1, D1 and P3 in the presence of phosphate, which can be used in the design of Cleaning in Place (CIP) systems involving phosphate based cleaning agents to combat biofilms and spores in the dairy industry.

Insights into the Geobacillus stearothermophilus species based on phylogenomic principles.

BACKGROUND: The 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. RESULTS: This 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. CONCLUSIONS: The 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.

Characterization of thermophilic bacilli from a milk powder processing plant.

AIMS: 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. METHODS AND RESULTS: Ten G. stearothermophilus strains and one Anoxybacillus species were isolated from a milk powder manufacturing plant. A microtitre plate assay was used to show that these strains differed in their abilities to form biofilms and produce spores. Scanning electron microscopy showed differences in the biofilm morphologies of three of the G. stearothermophilus strains. Biochemical profiling, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and fatty acid profiling further showed that they had distinct characteristics. CONCLUSIONS: These G. stearothermophilus strains, isolated from the same environment, showed differences in their ability to form biofilms and produce endospores. Based on the multiple characterization methods used in this study, these strains of G. stearothermophilus isolated from one manufacturing plant are diverse. SIGNIFICANCE AND IMPACT OF THE STUDY: Differences in the ability of G. stearothermophilus to form biofilms and produce spores may influence the cleaning method used to control the growth of thermophilic bacilli in a dairy processing environment.

Characteristics of three listeriaphages isolated from New Zealand seafood environments.

AIM: To isolate and characterize listeriaphages from seafood environments. METHODS AND  RESULTS: Listeriaphages (phages) isolated from seafood environments were distinguished by physical and biological techniques including restriction digestion of phage DNA. Three phages belonged to order Caudovirales and showed psychrotrophic characteristics. The phages had broad host ranges against 23 Listeria strains by productive infection or at least by adsorption. At 15 ± 1°C, adsorption rate constants of the three phages ranged from 8·93 × 10(-9) to 3·24 × 10(-11 ) ml min(-1) across different Listeria monocytogenes strains. In indicator hosts, the mean burst sizes of phages LiMN4L, LiMN4p and LiMN17 were c. 17, 17 and 11 plaque-forming units (PFU) per cell, respectively, at 15 ± 1°C. The respective latent periods were c. 270 min for phages LiMN4p and LiMN17, whereas for phage LiMN4L, it was c. 240 min. CONCLUSIONS: The three virulent psychrotrophic phages isolated from seafood-processing environments had broad host ranges and low productive replication. These characteristics suggest that the phages may be suitable as passive biocontrol agents against seafood-borne L. monocytogenes. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report on the isolation of autochthonous virulent listeriaphages from seafood-processing environments and information on single-step replication and adsorption characteristics of such listeriaphages.

Determination of the mode of action of enterolysin A, produced by Enterococcus faecalis B9510.

AIM: The current study aimed to visualize the damage caused by enterolysin A to the cells of sensitive strains and to find out cleavage site within the peptidoglycan moiety of bacterial cell walls. METHODS AND RESULTS: Enterolysin A produced by a local isolate, Enterococcus faecalis B9510 was found to rapidly kill cells of the sensitive strain Lactococcus lactis ssp. cremoris 2144 during 120 min of treatment as compared to the untreated control where no such effect was observed. Transmission electron microscopy of the enterolysin A-treated cells revealed leaking of the cytoplasmic contents ultimately resulting in complete lysis of cell walls. To find the cleavage site, purified cell walls of L. lactis ssp. cremoris 2144, Pediococcus pentosaceus 43201 and Lactobacillus delbrueckii ssp. bulgaricus ATCC 11842 were treated with enterolysin A, and liberated amino acids were derivatized for N and C terminals and analysed using thin layer chromatography on silica gel with isopropanol as solvent. The results showed that enterolysin A cleaves the peptide bonds at two locations within peptidoglycan subunits. The first location is between L-alanine and D-glutamic acid of the stem peptide and the other location is between L-lysine of the stem peptide and D-aspartic acid of the interpeptide bridge. CONCLUSIONS: Enterolysin A cleaves the peptide bonds within the stem peptide as well as in the interpeptide bridge of Gram-positive bacterial cell walls. This gives a possible reason for the broad spectrum of enterolysin A activity. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report identifying the cleavage site of enterolysin A within the cell walls of sensitive bacteria. This will help in identifying potential applications for enterolysin A.

Development of a chemically defined medium for the production of enterolysin A from Enterococcus faecalis B9510.

AIM: This study aimed to develop a simplified chemically defined medium that could sustain the growth and bacteriocin (enterolysin A) production by Enterococcus faecalis B9510. METHODS AND RESULTS: The nutritional requirements of E. faecalis B9510 in a chemically defined medium were determined by single omission experiments. It was observed that eight amino acids (arginine, glycine, histidine, isoleucine, leucine, methionine, tryptophan and valine), three B vitamins (nicotinic acid, Ca-pantothenic acid and pyridoxal) and magnesium sulphate were essential for growth. Based on this information, a Simplified Defined Medium (SDM) was formed consisting of 26 components. Comparison of SDM with M-17 showed that growth and bacteriocin production in SDM was similar to that in M-17. The bacteriocin from SDM was then purified by ultrafiltration. The retentate of ultrafiltration step was analysed by SDS-PAGE and the results showed a single active band in the gel, which was excised and analysed by mass spectrometry, which indicated that the active band was enterolysin A, a cell wall degrading bacteriocin. CONCLUSIONS: A simplified defined medium can be formulated for the growth and bacteriocin production by Enterococcus faecalis, whose efficiency is comparable with that of a complex commercial medium. SIGNIFICANCE AND IMPACT OF THE STUDY: The development of such a medium can be useful for bacteriocin production and subsequent purification in a simplified manner and, therefore, helpful in the identification of novel bacteriocins.

Spore germination of the psychrotolerant, red meat spoiler, Clostridium frigidicarnis.

AIMS: To determine germination triggers of Clostridium frigidicarnis, an important spoilage bacterium of chilled vacuum-packed meat. METHODS AND RESULTS: Germination of Cl. frigidicarnis spores in the presence of a range of potential nutrient and non-nutrient germinants was tested by monitoring the fall in optical density and by phase-contrast microscopy. The amino acid L-valine induced strong germination when paired with L-lactate in sodium phosphate under anaerobic conditions. Several other amino acids promoted germination when paired with L-lactate in sodium phosphate and the co-germinants NaHCO3 and L-cysteine. Heat activation, while not necessary for germination, increased the rate of germination. Spore germination was not observed when spores were incubated aerobically. CONCLUSIONS: Spores of psychrotolerant Cl. frigidicarnis germinated in the presence of L-valine in combination with L-lactate in sodium phosphate buffer under anaerobic conditions. SIGNIFICANCE AND IMPACT OF THE STUDY: Anaerobic conditions, L-valine and L-lactate, have been identified as triggering germination in Cl. frigidicarnis, and are all present in packs of fresh, vacuum-packaged, red meat. This new information adds to what is known about red meat spoilage by cold tolerant clostridia and can be used to develop intervention strategies to prevent meat spoilage.

Effect of NaOH (caustic wash) on the viability, surface characteristics and adhesion of spores of a Geobacillus sp. isolated from a milk powder production line.

AIM: To investigate the viability, surface characteristics and ability of spores of a Geobacillus sp. isolated from a milk powder production line to adhere to stainless steel surfaces before and after a caustic (NaOH) wash used in clean-in-place regimes. METHODS AND RESULTS: Exposing sessile spores to 1% NaOH at 65°C for 30min decreased spore viability by two orders of magnitude. The zeta potential of the caustic treated spores decreased from -20 to -32 mV and they became more hydrophobic. Transmission electron microscopy revealed that caustic treated spores contained breaks in their spore coat. Under flow conditions, caustic treated spores suspended in 0·1 mol l(-1) KCl were shown to attach to stainless steel in significantly greater numbers (4·6 log(10) CFU cm(-2) ) than untreated spores (3·6 log(10) CFU cm(-2) ). CONCLUSIONS: This research suggests that spores surviving a caustic wash will have a greater propensity to attach to stainless steel surfaces. SIGNIFICANCE OF STUDY: The practice of recycling caustic wash solutions may increase the risk of contaminating dairy processing surfaces with spores.

Characterization of spore surfaces from a Geobacillus sp. isolate by pH dependence of surface charge and infrared spectra.

AIMS: The surfaces of spores from a Geobacillus sp. isolated from a milk powder production line were examined to obtain fundamental information relevant to bacterial spore adhesion to materials. MATERIALS AND RESULTS: The surfaces of spores were characterized using transmission electron microscopy and infrared spectroscopy. Thin sections of spores stained with ruthenium red revealed an exosporium with a hair-like nap around the spores. Attenuated total reflection infrared spectra of the spores exposed to different pH solutions on a ZnSe prism revealed that pH-sensitive carboxyl and phosphodiester groups associated with proteins and polysaccharides contributed to the spore's negative charge which was revealed by our previous zeta potential measurements on the spores. Lowering the pH to the isoelectric point of spores resulted in an increase in intensity of all spectral bands, indicating that the spores moved closer to the zinc selenide (ZnSe) surface as the charged surface groups were neutralized and the spore surface polymers compressed. The attachment of spores to stainless steel was threefold higher at pH 3 compared with pH 7. CONCLUSIONS: This research showed that spore attachment to surfaces is influenced by electrostatic interactions, surface polymer conformation and associated steric interactions. SIGNIFICANCE AND IMPACT OF THE STUDY: The adhesion of thermophilic spores is largely controlled by functional groups of surface polymers and polymer conformation.

The role of surface charge and hydrophobicity in the attachment of Anoxybacillus flavithermus isolated from milk powder.

The aim of the present study was to investigate the attachment mechanisms that enable the thermophile Anoxybacillus flavithermus (B12) to attach to stainless-steel surfaces. Passing a B12 culture through a column of stainless-steel chips, collecting the first cells to pass through, re-culturing, and repeating the process six times, resulted in the isolation of a mutant, labeled X7, with tenfold reduced ability to attach to stainless steel as well as a reduced ability to attach to plastic. A comparison of bacterial cell-surface properties indicated that X7 was less hydrophobic than its parental strain B12. Cell-surface charge measurements also suggest that X7 had a lower net-negative surface charge. Disruption of extracellular polysaccharides and DNA appeared to have no effect on the attachment process. Removal of surface proteins caused a reduction in attachment of both B12 and X7, suggesting surface protein involvement in attachment.

Factors affecting the attachment of micro-organisms isolated from ultrafiltration and reverse osmosis membranes in dairy processing plants.

AIMS: To identify the types of micro-organisms involved in the formation of biofilms on dairy ultrafiltration and reverse osmosis membranes and investigate factors affecting the attachment of those isolates. METHODS AND RESULTS: Micro-organisms isolated from industrial membranes following standard cleaning were identified using the API culture identification system. Thirteen different isolates representing eight genera were isolated and their ability to attach to surfaces was compared using a microtitre plate assay. Three Klebsiella strains attached best, while mixed strains of Pseudomonas and Klebsiella attached better than individual strains. Whey enhanced the attachment of the isolates. The micro-organisms were characterized according to cell surface hydrophobicity using the microbial adhesion to hydrocarbon (MATH) test, and cell surface charge by measuring the zeta potential. These cell surface characteristics did not show a clear relationship with the attachment of our strains. CONCLUSIONS: A variety of different micro-organisms is associated with dairy ultrafiltration and reverse osmosis membranes after cleaning, suggesting several possible sources of contamination. The cleaning of these membranes may be inadequate. The attachment of the different isolates is highly variable and enhanced in the presence of whey. SIGNIFICANCE AND IMPACT OF THE STUDY: Knowledge of persistent microflora colonizing dairy membrane systems will help develop strategies to mitigate biofilm development in this environment, improving hygiene in membrane processing plants.

The formation of spores in biofilms of Anoxybacillus flavithermus.

AIMS: To examine the rate and the extent of spore formation in Anoxybacillus flavithermus biofilms and to test the effect of one key variable - temperature - on spore formation. METHODS AND RESULTS: A continuous flow laboratory reactor was used to grow biofilms of the typical dairy thermophile A. flavithermus (strain CM) in skim milk. The reactor was inoculated with either a washed culture or a spore suspension of A. flavithermus CM, and was run over an 8.5 h period at three different temperatures of 48, 55 and 60 degrees C. Change in impedance was used to determine the cell numbers in the milk and on the surface of the stainless steel reactor tubes. The biofilm developed at all three temperatures within 6-8 h. Spores formed at 55 and 60 degrees C and amounted to approx. 10-50% of the biofilm. No spores formed at 48 degrees C. CONCLUSIONS: The results suggest that both biofilm formation and spore formation of A. flavithermus can occur very rapidly and simultaneously. In addition, temperature variation has a considerable effect on the formation of spores. SIGNIFICANCE AND IMPACT OF THE STUDY: This information will provide direction for developing improved ways in which to manipulate conditions in milk powder manufacturing plants to control biofilms and spores of A. flavithermus.

Recovery of spores from thermophilic dairy bacilli and effects of their surface characteristics on attachment to different surfaces.

Spores from four Geobacillus spp. were isolated from a milk powder manufacturing line in New Zealand. Liquid sporulation media produced spore yields of approximately 10(7) spores ml(-1); spores were purified using a two-phase system created with polyethylene glycol 4000 and 3 M phosphate buffer. The zeta potentials of the spores from the four isolates ranged from -10 to -20 mV at neutral pH, with an isoelectric point between pH 3 and 4. Through contact angle measurements, spores were found to be hydrophilic and had relative hydrophobicity values of 10 to 40%, as measured by the microbial adhesion to hexadecane assay. The most hydrophilic spore isolate with the smallest negative charge attached in the highest numbers to Thermanox and stainless steel (1 x 10(4) spores cm(-2)), with fewer spores attaching to glass (3 x 10(3) spores cm(-2)). However, spores produced by the other three strains attached in similar numbers (P > 0.05) to all substrata (approximately 1 x 10(3) spores cm(-2)), indicating that there was no simple relationship between individual physicochemical interactions and spore adherence. Therefore, surface modifications which limit the attachment of one strain may not be effective for all stains, and control regimens need to be devised with reference to the characteristics of the particular strains of concern.

Evaluation of the effect of cleaning regimes on biofilms of thermophilic bacilli on stainless steel.

AIMS: To determine the mechanism for both the removal and inactivation of 18-h biofilms of a thermophilic Bacillus species that optimally grows at 55 degrees C on stainless steel. METHODS AND RESULTS: The cleaning strategies tested were based on biofilm biochemistry and physiology, and focused on the chemistry of the cleaners, the duration and temperature of the cleaning process and a combination of various cleaners. The success of the cleaning regimes was determined based on the removal of cells and organic debris and the elimination of viable cells. The results confirmed that a caustic (75 degrees C for 30 min) and acid (75 degrees C for 30 min) wash, relied upon heavily in most food processing industries for cleaning-in-place systems, was successful in removing these biofilms. However, any changes in the concentrations of these cleaners or the temperature of cleaning drastically affected the overall outcome. Alternative cleaning agents based on enzymatic or nonenzymatic breakdown of cellular proteins or polysaccharides, surfactant action, use of oxidative attack and free radicals varied in degrees of their success. Combining proteolytic action with surfactants increased wetability and therefore enhanced the cleaning efficiency. CONCLUSIONS: Several procedures, including caustic/acid and enzyme based cleaners, will be satisfactory, provided that the correct process parameters are observed i.e. concentration, time, temperature and kinetic energy (flow). Confirmation of these results should be carried out in a pilot plant through several use/clean cycles. SIGNIFICANCE AND IMPACT OF THE STUDY: Confidence in standard and alternative cleaning procedures for food manufacturing plant to prevent contamination with thermophilic bacilli that threaten product quality.

Physiology of biofilms of thermophilic bacilli-potential consequences for cleaning.

Thermophilic Bacillus species readily attached and grew on stainless steel surfaces, forming mature biofilms of >10(6.0) cells/cm2 in 6 h on a surface inoculated with the bacteria. Clean stainless steel exposed only to pasteurized skim milk at 55 degrees C developed a mature biofilm of >10(6.0) cells/cm2 within 18 h. When bacilli were inoculated onto the steel coupons, 18-h biofilms were 30 microm thick. Biofilm growth followed a repeatable pattern, with a reduction in the numbers of bacteria on the surface occurring after 30 h, followed by a recovery. This reduction in numbers was associated with the production of a substance that inhibited the growth of the bacteria. Variations in the environment, including pH and molarity, affected the viability of the cells. Chemicals that attack the polysaccharide matrix of the biofilm were particularly effective in killing and removing cells from the biofilm, demonstrating the importance of polysaccharides in the persistence of these biofilms. Treatment of either the biofilm or a clean stainless steel surface with lysozyme killed biofilm cells and prevented the attachment of any bacteria exposed to the surface. This suggests that lysozyme may have potential as an alternative control method for biofilms of these bacteria.

Comparison between Streptococcus macedonicus and Streptococcus waius strains and reclassification of Streptococcus waius (Flint et at. 1999) as Streptococcus macedonicus (Tsakalidou et al. 1998).

Two species of dairy streptococci, Streptococcus waius and Streptococcus macedonicus, were originally characterized by 16S-23S intergenic spacer sequence analysis, random amplified polymorphic DNA fingerprinting, PFGE analysis and DNA-DNA reassociation experiments. All genetic data suggested that S. waius strains belong to the previously described species S. macedonicus. Likewise, the phenotypic characterization showed that strains of S. macedonicus and S. waius were highly related and easily differentiated from the closest phylogenetic neighbour, Streptococcus bovis, principally by their failure to produce a blackening reaction in medium containing aesculin. The utilization of maltose and cellobiose by S. macedonicus/S. waius strains allowed their differentiation from the most studied dairy species, Streptococcus thermophilus. On the basis of genetic and phenotypic data S. macedonicus and S. waius species should be considered synonyms and S. macedonicus has the priority.

Factors influencing attachment of thermophilic bacilli to stainless steel.

AIMS: This project aimed to investigate the mechanism of attachment of the vegetative cells and spores of thermophilic bacilli to stainless steel with a view to devising strategies to limit biofilm development and survival. METHODS AND RESULTS: Spores and vegetative cells of bacterial isolates were exposed to protein denaturing agents (sodium dodecyl sulphate (SDS) and trypsin) and polysaccharide removing agents (sodium metaperiodate, trichloroacetic acid (TCA) and lysozyme). Treatment with sodium metaperiodate, TCA and lysozyme increased the number of vegetative cells attaching in many of the strains studied, while SDS and trypsin decreased attachment. Spores attached to stainless steel in greater numbers than vegetative cells, and the various treatments had less effect on this attachment than for vegetative cells. Viability of the cells or spores was not an important factor in attachment, as cells and spores rendered non-viable also attached to stainless steel in similar numbers. Coating the stainless steel with skim milk proteins decreased the attachment of both vegetative cells and spores. There was no correlation between the degree of attachment and the amount of extracellular polysaccharide (EPS) produced by each strain, surface hydrophobicity or zeta potential of vegetative cells or spores, though spores were found to be more hydrophobic than vegetative cells. CONCLUSIONS: The results suggest that biofilm formation by these thermophilic bacilli is probably a multifactorial process, and that cell-surface proteins play a very important role in the initial process of attachment during the formation of biofilms by these bacteria. SIGNIFICANCE AND IMPACT OF THE STUDY: This information will provide direction for developing improved cleaning systems to control biofilms of thermophilic bacilli in dairy manufacturing plants.

Rapid detection of Bacillus stearothermophilus using impedance-splitting.

An impedance splitting method was used to detect Bacillus stearothermophilus in suspension and attached to stainless steel surfaces. The effects of bacterial metabolism on the impedance of the culture medium and the ionic layers of the measuring electrodes were recorded using the BacTrac 4000 microorganism growth analyser. Impedance changes were measured at 55 degrees C. Seven of the eight media produced changes in the electrode impedance (E-value) and all media produced negligible changes in the impedance of the culture medium (M-value). Good correlations were obtained between cell numbers and the E-value measured over 18 h (r > 0.9) for the two strains of B. stearothermophilus tested in trypticase soy broth. The E-value correlations were used to estimate the numbers of both vegetative and spore forms of B. stearothermophilus as either planktonic or adhered cells. For the detection of B. stearothermophilus using impedance, only methods where the E-value impedance is recorded, can be used.

Functional grouping of thermophilic Bacillus strains using amplification profiles of the 16S-23S internal spacer region.

Molecular and biochemical assays were used to determine the identification of thermophilic bacilli isolated from New Zealand milk powder. One hundred and forty one isolates of thermophilic bacilli were classified into six species using biochemical profiles. Geobacillus stearothermophilus represented 56% of the isolates. All isolates were also analysed by randomly amplified polymorphic DNA (RAPD) analysis, with 45 types identified. Amplification of the 16S-23S rDNA internal spacer region produced two to eight amplification products per strain. The patterns from gel electrophoresis of the internal spacer region amplicons formed two major groupings suggesting the possibility of two distinct species. Partial sequences of 16S rDNA from representatives from each group were compared with sequences in GeneBank and were found to match the 16S rDNA sequences of B. flavothermus and G. thermoleovorans. Primers were designed for these species and used to screen an arbitrary selection of 59 of the dairy isolates. This enabled the identification of 28 isolates as B. flavothermus and 31 isolates as Geobacillus species and these appear to be the predominant isolates in the New Zealand milk powder samples examined. Comparison of the fragment pattern generated by amplification of the 16S-23S rDNA internal spacer region is a simple method to differentiate thermophilic Bacillus species associated with the dairy industry.

Streptococcus waius sp. nov., a thermophilic Streptococcus from a biofilm.

Thermophilic streptococci were isolated from biofilms on stainless steel samples exposed to pasteurized skimmed milk and from dairy products from a dairy manufacturing plant. The phenotypic characters of these isolates were distinct from those of other thermophilic streptococci of dairy origin (Streptococcus thermophilus and Streptococcus bovis). Genotypic data [restriction endonuclease analysis, ribotyping, random amplified polymorphic DNA (RAPD) profiles, DNA-DNA hybridization and G + C contents] support the classification of these isolates as a new species. The sequence of the 16S rRNA was compared with that of 29 species of streptococci and shown to be significantly different. The sequence of the 16S-23S rRNA intergenic spacer region also differed from published sequences of closely related species. A fluorescent in situ hybridization probe prepared to a specific part of the 16S rRNA gene sequence was able to distinguish the unknown isolates from reference isolates of S. thermophilus and S. bovis. It is proposed that these thermophilic streptococcal isolates from a dairy environment be classified in the genus Streptococcus as a new species, Streptococcus waius (from waiu, the New Zealand Maori word for milk). The type strain is 3/1T (= NZRCC 20100T).