Zoocin A and lauricidin in combination reduce Streptococcus mutans growth in a multispecies biofilm.

Dental caries is the most prevalent human infection. It is a multifactorial disease in which the microbial composition of dental plaque plays a major role in the development of clinical symptoms. The bacteria most often implicated in the development of caries are that group of streptococci referred to as the mutans streptococci, in particular Streptococcus mutans and Streptococcus sobrinus. One approach to the prevention of caries is to reduce the numbers of mutans streptococci in plaque to a level insufficient to support demineralization of the tooth. In this study, zoocin A, a peptidoglycan hydrolase, combined with lauricidin, a cell membrane active lipid, was shown over a 72 h period to selectively suppress the growth of S. mutans in a triple species biofilm. Growth of the non-target species Streptococcus oralis and Actinomyces viscosus was not inhibited. In treated systems the amount of extracellular polysaccharide matrix produced was much reduced as determined by use of fluorescein isothiocyanate conjugated wheat germ agglutinin. The pH of treated biofilms remained above neutral as opposed to a value of 4.3 in untreated controls. We conclude that use of antimicrobial compounds that specifically target cariogenic bacteria should be further explored.

Inhibition of foodborne bacteria by the lactoperoxidase system in a beef cube system.

Biopreservatives are being developed to inhibit the growth of foodborne pathogens and thus improve food safety. The lactoperoxidase system (LPS) is a naturally occurring system that has potential for use as an antimicrobial agent in foods. Growth of single strains of the pathogens Staphylococcus aureus, Listeria monocytogenes, Escherichia coli O157:H7, Salmonella enterica subsp. enterica serovar Typhimurium, Yersinia enterocolitica, Pseudomonas aeruginosa and beef microflora were assessed on LPS-treated meat surfaces in an experimental system. Beef cubes inoculated with approximately 10(4) cfu cm(-2) of bacteria were treated with the LPS and incubated at 37 degrees C for 24 h, 12 degrees C for 7 days or in a chilling regime: 12 to -1 degrees C over 1 week and held at -1 degrees C for 4 weeks. Treatment with LPS was more effective at storage temperatures non-permissive for rapid bacterial growth with strong inhibition of growth achieved on LPS-treated cubes at 12 degrees C and reduction in pathogen viable counts at chilling temperatures. At chilling temperatures, the LPS inhibited the growth of native pseudomonads but did not prevent the development of native lactic acid bacteria.

Development of a method to quantify in vitro the synergistic activity of "natural" antimicrobials.

Despite numerous papers being published on the use of hurdle technology to control food-borne pathogens or spoilage organisms, there is no commonly accepted methodology to quantify the level of synergistic activity. This paper describes a method to quantify in vitro the synergistic activity of antibacterial agents against bacteria. Initially, a microtiter plate growth assay was used to determine the inhibitory concentrations of four "natural" antimicrobials (nisin, lauricidin, totarol, and the lactoperoxidase system (LPS)) against a panel of eight bacteria. Using the same microtiter system, the impact of various combinations of antimicrobials was assessed. The degree of synergy was based on the analysis of three criteria: (1) increase in lag phase, (2) reduction in culture density after 24 h, (3) and residual viability at 24 h. Only the lactoperoxidase system was active against all the Gram-positive and Gram-negative bacteria tested. Nisin, lauricidin, and totarol were only effective against the Gram-positive bacteria. The method successfully identified three combinations (nisin-lauricidin, LPS-nisin, and LPS-lauricidin) previously reported to have synergistic activity and highlighted the synergistic activity of two novel combinations (nisin-totarol and LPS-totarol). The development of a quick and reliable method to identify and quantify synergistic activity is a useful screening tool to establish preservative techniques that could have potential antimicrobial synergy in food-based systems.

Different bacteriocin activities of Streptococcus mutans reflect distinct phylogenetic lineages.

Bacteriocins produced by mutans streptococci are known as mutacins. In this study 16 broadly active mutacin-producing Streptococcus mutans strains from New Zealand, North America and Europe were classified into four groups (A-D) on the basis of differences in their activity in deferred antagonism tests against either the homologous producer strain (to test for presence of self-immunity) or indicator strains Staphylococcus aureus 46 and Enterococcus faecium TE1. Two of the strains included in the study (UA140 and UA96) were representatives of the group I and II mutacin producer strains previously described by Caufield and co-workers. One of the New Zealand isolates of group A (S. mutans strain N) appeared to produce inhibitory activity similar to that of the group I prototype strain UA140. Four other New Zealand isolates of group B (S. mutans strains M19, M34, B34 and D14) had mutacin II-like activity. The group B mutacin producers differed from the group A mutacin producers in their additional activity against Staph. aureus 46. Seven S. mutans strains (M46, B46, B57, M12, M28, B28 and 13M) were distinguished from the group A and group B mutacin producers in that they inhibited E. faecium TE1. These were called group C mutacin producers. Strains H7 and H23 resembled the group C strains in their action on both indicator strains TE1 and 46. However, these two strains failed to exhibit immunity to their own inhibitory products in the deferred antagonism test and were separately classified as group D mutacin producers. Phylogenetic analysis of the strains by several genotypic and phenotypic characteristics revealed that the mutacin groups were associated with distinct evolutionary lineages of S. mutans.

Functional characterization of domains found within a lytic enzyme produced by Streptococcus equi subsp. zooepidemicus.

Zoocin A is a lysostaphin-like streptococcolytic enzyme produced by Streptococcus equi subsp. zooepidemicus 4881 that specifically targets the cell walls of some closely related species. On the basis of sequence homology it was suggested that zoocin A was a domain-structured enzyme with the N-terminal domain responsible for catalysis (CAT) and the C-terminal domain for target recognition (SBD). Polypeptides corresponding to zoocin A (rZooA) and each of the putative domains (rCAT and rSBD) were prepared by use of recombinant technology. The biological activities of each was compared by use of a dye-release assay and a cell-binding assay. Cell wall hydrolysis was shown to be a function of CAT and target recognition a function of the SBD. Expression of the zoocin A immunity factor gene produced cell walls resistant to hydrolysis by either rZooA or its component domains, and with reduced capacity to bind rZooA and rSBD.

Inhibition of bacterial foodborne pathogens by the lactoperoxidase system in combination with monolaurin.

The lactoperoxidase system (LPS) and monolaurin (ML) are potential natural antimicrobial agents for use in foods. The LPS is considered to have greatest activity against Gram-negative bacteria while ML is usually considered to have greatest activity against Gram-positive bacteria. An LPS-ML combination system (utilizing lactoperoxidase (LPX) in the range 5-200 mg kg(-1) and ML in the range 50-1,000 ppm) inhibited growth of Escherichia coli O157:H7 and Staphylococcus aureus. Growth of S. aureus was inhibited more strongly in broth than in milk, in milk than in ground beef A similar pattern was observed for E. coli O157:H7, though enhanced inhibition by LPS-ML systems over that obtained in comparable LPS only systems was not observed in ground beef The inhibitory action of the LPS in combination with other lipids was also examined, with progressively weaker inhibition observed in combinations including palmitoleic acid, monopalmitolein, lauric acid, caprylic acid, and sodium lauryl sulphate.

Diverse activity spectra of bacteriocin-like inhibitory substances having activity against mutans streptococci.

Mutans streptococci (MS) are known to be causative agents of dental caries. It has been suggested that these cariogenic bacteria could be eliminated from dental plaque by application of bacteriocins or bacteriocin-like inhibitory substances (BLIS). In the present study 272 bacterial strains of the genera Streptococcus, Enterococcus and Staphylococcus were tested for their production of BLIS activity against MS by use of a deferred antagonism test on agar media supplemented with either whole blood or yeast extract. Although only 14.3% of the test strains displayed anti-MS activity, the inhibitory agents produced by these strains were characterised by considerable diversity in the range of their inhibitory action against both MS and other common oral streptococcal species. It is suggested that combinations of relatively specifically targeted anti-MS BLIS may have potential application to the prevention of dental caries.

Dental caries and the microbial ecology of dental plaque: a review of recent advances.

Our understanding of the microbial ecology of dental plaque has rapidly grown with recent developments in the techniques of molecular biology. In particular, knowledge of the mechanisms underlying the acquisition, establishment, pathogenicity, and evolution of the group of organisms responsible for dental caries--the mutans streptococci--has expanded to the point that we can now contemplate new opportunities for caries prevention. These advances reinforce developing concepts of dental plaque as an interdependent, interacting community of specialised organisms with an ability to rapidly adapt conferred by gene structures that facilitate the expeditious modular rearrangement of protein components.

Streptococcus mutans strain N produces a novel low molecular mass non-lantibiotic bacteriocin.

Streptococcus mutans strain N was shown to have bacteriocin production and immunity characteristics consistent with those of Group I mutacin-producing strains of S. mutans. The bacteriocin mutacin N was purified from agar cultures of S. mutans strain N using XAD andp6 reversed phase chromatography. The molecular mass of mutacin N was 4806 Da and the entire 49 amino acid sequence was determined by N-terminal sequencing. Database searches indicate that mutacin N is a novel bacteriocin, but with some homology to the protein IIC domain of a hypothetical sugar-phosphotransferase enzyme from Acholeplasma florum.

Dental caries is a preventable infectious disease.

Dental caries is the most common infectious disease affecting humans. The principal causative agents are a group of streptococcal species collectively referred to as the mutans streptococci of which Streptococcus mutans and Streptococcus sobrinus are the most important agents of human caries. This review outlines what is currently known about these ubiquitous pathogens and discusses novel methods for elimination of these bacteria from dental plaque.

Zoocin A immunity factor: a femA-like gene found in a group C streptococcus.

A 6.8-kb fragment of Streptococcus equi subsp. zooepidemicus 4881 DNA containing the zoocin A gene (zooA) was cloned in Escherichia coli and sequenced. We have identified a gene we call zoocin A immunity factor (zif), which protects the producer cell from the otherwise lethal action of its own product. Transformation of Streptococcus gordonii DL1 with zooA and zif changed its phenotypic character from a non-zoocin A producing-zoocin A sensitive cell to a zoocin A producing-zoocin A resistant cell. zif has sequence homology to femA (factor essential for methicillin resistance) and lif (lysostaphin immunity factor). No differences were observed in amino acid or amino sugar compositions of peptidoglycan purified from zoocin A sensitive vs. zoocin A immune cells.

Cloning and sequence analysis of zooA, a Streptococcus zooepidemicus gene encoding a bacteriocin-like inhibitory substance having a domain structure similar to that of lysostaphin.

The nucleotide sequence has been determined for zooA, a gene encoding the bacteriocin-like inhibitory substance zoocin A in Streptococcus zooepidemicus strain 4881. The zooA gene product corresponds to the 285-amino acid (aa) zoocin A pre-peptide from which a leader sequence is cleaved to form the 262-aa biologically active molecule of estimated molecular mass 27,877 Da. Expression of zooA in a Gram-negative host was shown by the extracellular release from Escherichia coli, containing cloned zooA, of a biologically active peptide having an identical range of anti-bacterial activity to that of zoocin A, purified from S. zooepidemicus strain 4881. Data base searches revealed sequences having homologies with known muralytic proteins produced by both Gram-positive and Gram-negative bacteria and indicate a 'mix and match' blending of domain-type structures, the C-terminal putative receptor-recognition region of the molecule being joined by a threonine-proline-rich linker to an N-terminal putative catalytic region having homology with several known endopeptidases, including lysostaphin.

Mode of action of a lysostaphin-like bacteriolytic agent produced by Streptococcus zooepidemicus 4881.

Electron microscopy of zoocin A-treated sensitive streptococcus cells revealed cytoplasmic disruption and ultimately complete rupture of the cell wall. Culture viability and optical density were shown to decrease rapidly and simultaneously in Streptococcus pyogenes FF22 but less quickly in the relatively more resistant Streptococcus mutans 10449. Zoocin A was shown to cleave hexaglycine in a colorimetric cell-free microtiter assay system, and it is concluded that the killing action of zoocin A, like that of lysostaphin, is most probably the result of direct cleavage of the peptidoglycan cross-links in the cell wall. The relationship between sensitivity to zoocin A and the peptidoglycan cross-linkage structure of Streptococcus zooepidemicus, Lactococcus spp., S. pyogenes, Streptococcus gordonii, Streptococcus oralis, S. mutans, and Streptococcus rattus has been evaluated.

Persistently infected cultures as a source of hepatitis A virus.

Primary African green monkey kidney, continuous African green monkey kidney cell line BS-C-1, and buffalo green monkey kidney cultures were infected with a uniform inoculum of hepatitis A virus (HAV). Although both the cell line BS-C-1 and primary African green monkey kidney cultures produced useful amounts of virus, HAV was detected earlier and in greater quantities in primary African green monkey kidney cultures. A persistently infected primary African green monkey kidney culture was developed. The influence of incubation time (4 to 40 days) and concentration (2 to 15%) of fetal calf serum in the maintenance medium on production of HAV by this culture was examined. An incubation period of 24 to 28 days was found to be optimal; reducing this period led to decreased yields of HAV. No significant difference in the amount of HAV produced was observed with differing concentrations of fetal calf serum. Three different methods of extraction and the effect of multiple extractions on the recovery of HAV from cell lysates were examined. Sonication was a critical factor. Two extractions yielded more than 90% recoverable virus. Yields in excess of 10(11) physical particles of HAV per 850-cm2 roller bottle were routine. The total yield could be increased by concentrating the HAV present in spent maintenance medium by using bentonite or organic flocculation.