Production, purification, sequencing and activity spectra of mutacins D-123.1 and F-59.1.

BACKGROUND: The increase in bacterial resistance to antibiotics impels the development of new anti-bacterial substances. Mutacins (bacteriocins) are small antibacterial peptides produced by Streptococcus mutans showing activity against bacterial pathogens. The objective of the study was to produce and characterise additional mutacins in order to find new useful antibacterial substances. RESULTS: Mutacin F-59.1 was produced in liquid media by S. mutans 59.1 while production of mutacin D-123.1 by S. mutans 123.1 was obtained in semi-solid media. Mutacins were purified by hydrophobic chromatography. The amino acid sequences of the mutacins were obtained by Edman degradation and their molecular mass was determined by mass spectrometry. Mutacin F-59.1 consists of 25 amino acids, containing the YGNGV consensus sequence of pediocin-like bacteriocins with a molecular mass calculated at 2719 Da. Mutacin D-123.1 has an identical molecular mass (2364 Da) with the same first 9 amino acids as mutacin I. Mutacins D-123.1 and F-59.1 have wide activity spectra inhibiting human and food-borne pathogens. The lantibiotic mutacin D-123.1 possesses a broader activity spectrum than mutacin F-59.1 against the bacterial strains tested. CONCLUSION: Mutacin F-59.1 is the first pediocin-like bacteriocin identified and characterised that is produced by Streptococcus mutans. Mutacin D-123.1 appears to be identical to mutacin I previously identified in different strains of S. mutans.

[Streptococcus mutans and oral streptococci in dental plaque]. / Streptococcus mutans et les streptocoques buccaux dans la plaque dentaire.

The human oral microbial biota represents a highly diverse biofilm. Twenty-five species of oral streptococci inhabit the human oral cavity and represent about 20 % of the total oral bacteria. Taxonomy of these bacteria is complex and remains provisional. Oral streptococci encompass friends and foes bacteria. Each species has developed specific properties for colonizing the different oral sites subjected to constantly changing conditions, for competing against competitors, and for resisting external agressions (host immune system, physico-chemical shocks, and mechanical frictions). Imbalance in the indigenous microbial biota generates oral diseases, and under proper conditions, commensal streptococci can switch to opportunistic pathogens that initiate disease in and damage to the host. The group of "mutans streptococci" was described as the most important bacteria related to the formation of dental caries. Streptococcus mutans, although naturally present among the human oral microbiota, is the microbial species most strongly associated with carious lesions. This minireview describes the oral streptococci ecology and their biofilm life style by focusing on the mutans group, mainly S. mutans. Virulence traits, interactions in the biofilm, and influence of S. mutans in dental caries etiology are discussed.

Streptococcus salivarius mutants defective in mannose phosphotransferase systems show reduced sensitivity to mutacins I-T9 and R-3B.

Twenty-four mutacin-producing Streptococcus mutans strains were screened for their propensity to produce class II one-peptide bacteriocin using a deferred antagonism assay. Streptococcus salivarius and 3 mutants defective in their mannose phosphotransferase systems (mannose-PTS) were used as sensitive strains to identify which mannose-PTS could act as the docking site for class II one-peptide bacteriocin activity. We observed that only 2 strains of S. mutans, T9 and 3B, potentially produce class II one-peptide bacteriocin, namely mutacins I-T9 and R-3B, but with no preference for any mannose-PTS complex as a target.

Relationship between African dust carried in the Atlantic trade winds and surges in pediatric asthma attendances in the Caribbean.

Asthma is epidemic in developed and developing countries including those in the Caribbean where it is widely believed that African dust, transported in high concentrations in the Trade Winds every year, is a major causative factor. The link between asthma and dust in the Caribbean is based largely on anecdotal evidence that associates sharp increases in the occurrence of asthma symptoms with hazy conditions often caused by dust. Here we report on a 2-year study of the relationship between the daily concentrations of dust measured in on-shore Trade Winds at Barbados and pediatric asthma attendance rates at Queen Elizabeth Hospital (QEH). We looked for large increases in QEH daily attendances in relation to daily dust concentrations as previously suggested by anecdotal observations. We could not find any obvious relationship although there may be more subtle linkages between dust and asthma. Our measurements show, however, that the concentration of dust in the size range under 2.5 microm diameter is sufficiently high as to challenge United States Environmental Protection Agency air quality standards for respirable particles. Thus, African dust may constitute a health threat of a different nature, producing symptoms less obvious than those of asthma.

An investigation of sources of Campylobacter in a poultry production and packing operation in Barbados.

Chicken meat is frequently contaminated with Campylobacter jejuni and is thought to be the major source of organisms causing human Campylobacter enteritis. Genotypic similarities between Campylobacter isolates from chicken meat at retail outlets and patients with gastroenteritis in Barbados suggested that it is a vehicle for infection of humans on the island and prompted this investigation of transmission of Campylobacter in a local poultry operation. Campylobacter testing was conducted at the hatchery, on the broiler farm and in the processing plant for two consecutive production cycles. The genetic relatedness of Campylobacter isolates was determined by RAPD typing with primer OPA 11. Hatchery samples and week-old chicks were negative for Campylobacter. Flocks became colonized as early as three weeks after introduction to the farm. Ten distinct RAPD genotypes were identified among isolates. Some genotypes were similar and may be of clonal origin. There was no evidence of vertical transmission of Campylobacter. The results suggest that the broiler flock was infected from more than one source in the farm environment.

Bacteriocin-like inhibitory substances from Campylobacter spp.

Twenty-five Campylobacter isolates were screened for production of antimicrobial substances using a deferred antagonism assay. Sixteen isolates showed activity against either Staphylococcus aureus, Salmonella enterica serovar Enteritidis or Candida albicans. The inhibitory activity was sensitive to treatment with pronase E, trypsin and pepsin, suggesting that the antimicrobial compound(s) are proteinaceous. Activity spectra of isolates included S. aureus, Micrococcus luteus, Streptococcus sp., Bacillus subtilis, a drug-resistant clinical isolate of S. aureus and one isolate of C. albicans. Producing isolates showed cross-immunity and inhibitory activity was only observed on solid media. The findings of this study suggest that Campylobacter produces proteinaceous inhibitory substances.

Isolation and genetic characterization of avian influenza viruses and a Newcastle disease virus from wild birds in Barbados: 2003-2004.

Zoonotic transmission of an H5N1 avian influenza A virus to humans in 2003-present has generated increased public health and scientific interest in the prevalence and variability of influenza A viruses in wild birds and their potential threat to human health. Migratory waterfowl and shorebirds are regarded as the primordial reservoir of all influenza A viral subtypes and have been repeatedly implicated in avian influenza outbreaks in domestic poultry and swine. All of the 16 hemagglutinin and nine neuraminidase influenza subtypes have been isolated from wild birds, but waterfowl of the order Anseriformes are the most commonly infected. Using 9-to-11-day-old embryonating chicken egg culture, virus isolation attempts were conducted on 168 cloacal swabs from various resident, imported, and migratory bird species in Barbados during the months of July to October of 2003 and 2004. Hemagglutination assay and reverse transcription-polymerase chain reaction were used to screen all allantoic fluids for the presence of hemagglutinating agents and influenza A virus. Hemagglutination positive-influenza negative samples were also tested for Newcastle disease virus (NDV), which is also found in waterfowl. Two influenza A viruses and one NDV were isolated from Anseriformes (40/168), with isolation rates of 5.0% (2/40) and 2.5% (1/40), respectively, for influenza A and NDV. Sequence analysis of the influenza A virus isolates showed them to be H4N3 viruses that clustered with other North American avian influenza viruses. This is the first report of the presence of influenza A virus and NDV in wild birds in the English-speaking Caribbean.

Mutacin H-29B is identical to mutacin II (J-T8).

BACKGROUND: Streptococcus mutans produces bacteriocins named mutacins. Studies of mutacins have always been hampered by the difficulties in obtaining active liquid preparations of these substances. Some of them were found to be lantibiotics, defined as bacterial ribosomally synthesised lanthionine-containing peptides with antimicrobial activity. The goal of this study was to produce and characterize a new mutacin from S. mutans strain 29B, as it shows a promising activity spectrum against current human pathogens. RESULTS: Mutacin H-29B, produced by S. mutans strain 29B, was purified by successive hydrophobic chromatography from a liquid preparation consisting of cheese whey permeate (6% w/v) supplemented with yeast extract (2%) and CaCO3 (1%). Edman degradation revealed 24 amino acids identical to those of mutacin II (also known as J-T8). The molecular mass of the purified peptide was evaluated at 3246.08 +/- 0.1 Da by MALDI-TOF MS. CONCLUSION: A simple procedure for production and purification of mutacins along with its characterization is presented. Our results show that the amino acid sequence of mutacin H-29B is identical to the already known mutacin II (J-T8) over the first 24 residues. S. mutans strains of widely different origins may thus produce very similar bacteriocins.

Human Campylobacter-associated enteritis on the Caribbean island of Barbados.

A longitudinal study of the incidence of Campylobacter enteritis in Barbados was undertaken from January 2000 to August 2003. Diarrheal stools received by the central public health laboratory were cultured for Campylobacter. The number of reported Campylobacter cases exceeded those of Shigella but were less than those of Salmonella, and increased steadily with each year. Isolates from stools were mainly C. jejuni (63.6%) and C. coli (31.8%). The highest isolation rate was found in children 1-4 years of age (40.8%), followed by infants less than 1 year of age (16.9%) and those 5-9 years of age (11.3%). The number of reported cases was higher in March, from June to August, and in November and December. There was no correlation between incidence and either rainfall, temperature, or humidity. Further epidemiologic investigation of this disease is needed to evaluate risk factors for Campylobacter infection and determine routes of transmission in Barbados.

Identification of Streptococcus iniae by commercial bacterial identification systems.

The fish pathogen Streptococcus iniae cannot be identified by most commercial bacterial identification systems. The results presented here indicate that over 70% of our S. iniae isolates have been identified using the Biolog(R) GP microplate panels and Microlog(R) database. The isolates were confirmed as S. iniae by specific PCR methods and have been found to conform to the result obtained with the type strain S. iniae ATCC 29178.

In vivo activity of mutacin B-Ny266.

OBJECTIVES: The objective of this study was to assess the in vivo activity of mutacin B-Ny266 (a bacteriocin produced by Streptococcus mutans) in order to eventually use it as an antibiotic. METHODS: Intraperitoneal infection was induced with a methicillin-susceptible Staphylococcus aureus strain in mice. Some of the mice were simultaneously injected intraperitoneally with mutacin B-Ny266, some with the vehicle only and some with vancomycin. RESULTS: While there was 70 and 100% mortality in the control groups of mice, no mortality was observed in the mice injected with vancomycin or mutacin B-Ny266. CONCLUSIONS: The results presented here show, for the first time, the in vivo efficacy of a mutacin (B-Ny266) against an experimental intraperitoneal infection by S. aureus in a mouse model.

Pet dogs and chicken meat as reservoirs of Campylobacter spp. in Barbados.

Campylobacter spp. are the second most common pathogen isolated from stools of patients with gastroenteritis in Barbados. The aim of this study was to identify reservoirs of Campylobacter and the likely source(s) of human infection. Fecal specimens from 596 animals and 311 samples of animal food products were analyzed for the presence of Campylobacter spp. by standard culture techniques. Isolates were characterized by conventional phenotypic tests, confirmed by latex agglutination and PCR with genus-specific primers, and identified by the use of species-specific primers. High isolation rates were obtained for chickens (94.2%), pigs (90.5%), dogs (46.9%), cats (37.3%), and wild birds (39.3%). Campylobacter was also recovered from monkeys (17.1%) and sheep (4.2%) but not from cows. Chicken meat was frequently contaminated with Campylobacter (58.4%), but its recovery from other animal food products was rare. Campylobacter jejuni was the most commonly identified species in humans (63.6%), chickens (86.6%), dogs (51.5%), and chicken meat (79.8%). Porcine isolates were predominantly C. coli (98.4%), while cats harbored mainly C. upsaliensis and C. helveticus. Wild birds alone carried urease-positive thermophilic campylobacters. C. jejuni and C. coli isolates from different sources were compared with isolates from humans by randomly amplified polymorphic DNA typing with the primers OPA 11 and HLWL 85. Genotyping revealed similarities between isolates from chicken meat and those from humans and could not distinguish between two clinical isolates and four canine strains. Our results suggest that dogs are significant reservoirs of Campylobacter and contribute to human enteric infections and that chicken meat is a likely vehicle for the transmission of campylobacters to humans.

Improved methods for mutacin detection and production.

Studies of mutacins have always been hampered by the difficulties in obtaining active liquid preparations of these substances. In order to be commercially produced, good mutacin yields have to be obtained, preferably in inexpensive media. The results presented here indicate that mutacins can be produced in supplemented cheese whey permeate. The influence of carbon and nitrogen supplements on mutacin production varied according to the producer strain. The use of CaCO3 as a buffer in batch cultures resulted in improved yields of mutacin in the supernatants. Antimicrobial activity assays were improve by acidification of the diluent (pH 2) and were less variable in peptone water (0.5%). The culture medium consisting of cheese whey permeate (6% w/v), yeast extract (2% w/v) and CaCO3 (1% w/v) was found to be an inexpensive medium for the efficient production of mutacins.

Replacement of trifluoroacetic acid with HCl in the hydrophobic purification steps of pediocin PA-1: a structural effect.

Trifluoroacetic acid (TFA) is a purification contaminant associated with pediocin PA-1 that interferes with Fourier transform infrared spectroscopy structural analysis. As revealed by circular dichroism, its presence affects the structural folding of pediocin. Consequently, we propose a new pediocin PA-1 purification procedure using HCl instead of TFA in all of the hydrophobic steps. This procedural change does not affect the purification yield or the amount of pediocin PA-1 purified. Furthermore, removing HCl, as opposed to TFA, after purification is an easier procedure to carry out. In fact, the removal of TFA requires more experimentation and results in protein loss. Thus, HCl is a good alternative to TFA in pediocin PA-1 purification and can be extended to the purification of other proteins. We also show that TFA-induced structural modifications do not significantly affect the antimicrobial activity of pediocin PA-1.

Diversity of Streptococcus mutans bacteriocins as confirmed by DNA analysis using specific molecular probes.

Mutacin-producing strains have been classified into 24 groups (designated by letters A to X) by similarity in activity spectra and cross-immunity. Similarity in primary structure among these groups can be revealed using DNA hybridization. The amino acid sequences of four mutacins (B-Ny266, 1140/mutacin III and mutacin II) were used to design two DNA probes in order to detect similar genes among groups of Streptococcus mutans strains demonstrating inhibitory activity. In addition to the appropriate parent strain, each probe hybridized with the total DNA from only two out of the 24 mutacin group type strains. Thus, the remaining 18 groups of strains produce mutacins that differ from the mutacins sequenced to date. In order to explore the similarity between genes coding for mutacins B-Ny266 and JH1140, the group B specific probe was utilized to detect a DNA fragment of 1.9 kb in the genome of S. mutans strain Ny266. The sequence of the cloned fragment codes for three open reading frames (lanA, lanA' and lanB) similar to those of strains JH1140 and UA787. The gene lanA' is strongly similar to the structural gene lanA (67%), but only one RNA transcript of about 300 bases was detected by Northern hybridization using the lanA-lanA' probe. Transcription of lanA alone was verified by RT-PCR.