The antimicrobial peptide aureocin A53 as an alternative agent for biopreservation of dairy products.

AIMS: The aim of this study was to investigate the potential of aureocin A53, a staphylococcal antimicrobial peptide, for improving food safety. METHODS AND RESULTS: The antimicrobial activity of aureocin A53 against strains of Listeria monocytogenes isolated from food was tested and the bacteriocin proved to be bactericidal and bacteriolytic against the listerial strains. Aureocin A53 was neither toxic to eukaryotic cell lines nor haemolytic against sheep erythrocytes. It also exhibited a remarkable stability during storage at different temperatures and sensitivity to both simulated gastric juice and bile salts. When the antibacterial activity of aureocin A53 (256 AU ml(-1) ) was tested in skimmed milk artificially inoculated with a L. monocytogenes strain (1·0 × 10(4)  CFU ml(-1) ) isolated from food, during storage at 4°C, the bacteriocin reduced the viable counts by 7·7-log10 units up to 7 days of incubation, when compared with the controls not treated with the bacteriocin. CONCLUSIONS: Aureocin A53 exhibited several features considered important for biopreservation and remained fully active in a food matrix. SIGNIFICANCE AND IMPACT OF THE STUDY: Taken together, the results confirmed that aureocin A53 has potential to be used as a food preservative, representing an alternative to the use of nisin in biopreservation of dairy products.

Nisin and lysostaphin activity against preformed biofilm of Staphylococcus aureus involved in bovine mastitis.

AIMS: The biofilm produced by Staphylococcus aureus isolates involved in clinical or subclinical bovine mastitis and the activity of nisin and lysostaphin against the preformed biofilm produced by these strains were investigated. METHODS AND RESULTS: Eighteen strains were tested and all produced biofilm. Eight strains with distinct biofilm composition were selected for the antimicrobial activity assays. The minimal inhibitory concentration of each bacteriocin was determined against the planktonic cells and ranged from 15·6 to 500 µg ml(-1) for nisin, and from 3·9 to 50 µg ml(-1) , for lysostaphin. Lysostaphin treatment (0·4 µg ml(-1) ) for 4 h caused a strong Staph. aureus 4181 biofilm detachment and death of the majority of the sessile cells, while nisin treatment (100 µg ml(-1) ) for the same time caused only a great reduction in cell viability. Additionally, combination of both bacteriocins for 4 h resulted in significant death of the sessile cells but no biofilm detachment. CONCLUSIONS: The treatment with lysostaphin alone or in combination with nisin was effective in killing most biofilm sessile cells. SIGNIFICANCE AND IMPACT OF THE STUDY: The action of lysostaphin, either alone or in combination with nisin, against established staphylococcal biofilm may represent an alternative to bovine mastitis control. However, the duration of the treatment should be considered for its application so that the best effectiveness can be achieved.

Staphylococcus haemolyticus as an important hospital pathogen and carrier of methicillin resistance genes.

Phenotypic and molecular methods were used to characterize the antibiotic resistance of 64 clinical isolates of Staphylococcus haemolyticus. By PCR of the mecA gene, 87% were found to be methicillin resistant. Approximately 55% harbored staphylococcal cassette chromosome mec element (SCCmec) type V, and only one SCCmec type IV. Many isolates (75%) displayed multiresistance, and pulsotype analysis showed a high diversity.

Staphylococcal antimicrobial peptides: relevant properties and potential biotechnological applications.

Bacteriocins are bacterial antimicrobial peptides with bactericidal activity against other bacteria. Staphylococcins are bacteriocins produced by staphylococci, which are Gram-positive bacteria with medical and veterinary importance. Most bacteriocins produced by staphylococci are either lantibiotics (e.g., Pep5, epidermin, epilancin K7, epicidin 280, staphylococcin C55/BacR1, and nukacin ISK-1) or class II bacteriocins (e.g., aureocins A70 and 53). Only one staphylococcin belonging to class III, lysostaphin, has been described so far. Production of staphylococcins is a self-protection mechanism that helps staphylococci to survive in their natural habitats. However, since these substances generally have a broad spectrum of activity, inhibiting several human and animal pathogens, they have potential biotechnological applications either as food preservatives or therapeutic agents. Due to the increasing consumer awareness of the risks derived not only from food-borne pathogens, but also from the artificial chemical preservatives used to control them, the interest in the discovery of natural food preservatives has increased considerably. The emergence and dissemination of antibiotic resistance among human and animal pathogens and their association with the use of antibiotics constitute a serious problem worldwide requiring effective measures for controlling their spread. Staphylococcins may be used, solely or in combination with other chemical agents, to avoid food contamination or spoilage and to prevent or treat bacterial infectious diseases. The use of combinations of antimicrobials is common in the clinical setting and expands the spectrum of organisms that can be targeted, prevents the emergence of resistant organisms, decreases toxicity by allowing lower doses of both agents and can result in synergistic inhibition.

Peptide-like substances as antimicrobial barriers to Corynebacterium sp. adhesion to silicone catheters.

AIMS: To show medical application of antimicrobial peptides such as Pep5 and epidermin in inhibiting adhesion of Corynebacterium spp. to silicone catheters. METHODS AND RESULTS: The inhibitory activity of crude preparations of Pep5 and epidermin was tested on Corynebacterium spp. isolated from catheter-related infections. The addition of these substances at 640 AU ml(-1) to a cell suspension of Corynebacterium sp. 633544 resulted in a decrease of 3 log cycles in the number of viable cells over a period of 12 h. When Pep5 and epidermin were added to in vitro catheter colonization experiments, there was a decrease of 1 log unit (P < 0.01) in the cell number of Corynebacterium spp. adhered to silicone catheters. Scanning electron microscopy revealed that antimicrobial-treated catheters presented zones with absence of adhered cells, and some parts of the catheter presented aggregates suggesting damaged cells. CONCLUSIONS: The crude preparations of Pep5 and epidermin were able to inhibit Corynebacterium sp. 633544 isolated from catheter-related infection. The capability of Pep5 and epidermin to inhibit catheter colonization may indicate their usefulness as a barrier to block or to reduce the bacteremia by Corynebacterium spp. SIGNIFICANCE AND IMPACT OF THE STUDY: Peptide-like antimicrobial substances used to reduce bacterial attachment to medical devices may represent a novel strategy to control catheter-related infections.

Bacteriocins as alternative agents for control of multiresistant staphylococcal strains.

AIMS: To investigate the activity of seven staphylococcins, bacteriocins produced by staphylococci, against multiresistant Staphylococcus aureus and coagulase-negative staphylococci (CNS) involved in human infections. METHODS AND RESULTS: Four bacteriocins produced by Staph. epidermidis (Pep5, epidermin, epilancin K7 and epicidin 280) and three produced by Staph. aureus (aureocins A70, A53 and 215FN) were tested. Sixteen Staph. aureus strains, including a representative strain of the endemic Brazilian methicillin-resistant clone (MRSA), and 57 CNS strains were used as indicators. Among the staphylococcins used, Pep5 was able to inhibit 77.2% of the CNS strains and 87.5% of the Staph. aureus strains tested, including the Brazilian MRSA endemic clone, responsible for a large number of hospital-acquired infections in Brazil. On the other hand, aureocin A53 and epidermin presented a high antagonistic activity only against the Staph. aureus strains, being able to inhibit, respectively, 87.5% and 81.3% of them, including also the Brazilian MRSA endemic clone. The remaining bacteriocins inhibited only a low percentage of the nosocomial staphylococcal strains tested. CONCLUSIONS: Aureocin A53 and epidermin have potential applications against MRSA, whereas Pep5 seems to be an attractive agent against both MRSA and CNS, including mupirocin-resistant strains and the Brazilian endemic clone of MRSA, which is also found disseminated in other countries. SIGNIFICANCE AND IMPACT OF THE STUDY: Bacteriocins may represent alternative agents to control important nosocomial pathogens.

Antimicrobial resistance and plasmid profiles of Campylobacter jejuni and Campylobacter coli from human and animal sources.

AIMS: The purpose of this study was to determine the susceptibility of Campylobacter jejuni and Campylobacter coli isolates to antimicrobial agents and to investigate the presence of plasmid DNA. METHODS AND RESULTS: A total of 15 clinical isolates from children faeces, and 29 animal isolates of Campylobacter jejuni (n=22) and Campylobacter coli (n=22) were tested for susceptibility to 9 antimicrobial agents using a disc diffusion method, and screened for the presence of plasmid DNA by agarose gel electrophoresis. Of the 44 isolates, 56.8% were resistant to sulphonamide, 25% to norfloxacin, 18.2% to erythromicin, ciprofloxacin and ampicillin, and 13.6% to tetracycline. All isolates were susceptible to gentamicin, chloramphenicol and cefotaxime. Plasmids were detected in one Camp. jejuni (4.54%) strain isolated from sheep and in six (27.27%) Camp. coli strains isolated from rhesus monkey(3), swine(2), and poultry(1) with sizes ranging from 3.4 to 50 kb. CONCLUSIONS: The majority of the human isolates were susceptible to antibiotics commonly used for the treatment of campylobacteriosis. SIGNIFICANCE AND IMPACT OF THE STUDY: The origin and spread of Campylobacter resistance to antibiotics are discussed, with particular respect to the current situation in Brazil.

Expression of the major heat shock proteins DnaK and GroEL in Streptococcus pyogenes: a comparison to Enterococcus faecalis and Staphylococcus aureus.

One of the outstanding problems in the field of heat shock response has been to elucidate the mechanism underlying the induction of heat shock proteins (HSPs). In this work, we initiate an analysis of the expression of heat shock groEL and dnaK genes and their promoters in S. pyogenes. The synthesis of total cellular proteins was studied upon transfer of a log-phase culture from 37 degrees C to 42 degrees C by performing 5-min pulse-labeling experiments with (35)S-Met. The heat shock responses in the pathogenic Gram-positive cocci, Enterococcus faecalis and Staphylococcus aureus, were also analyzed.

Production of bacteriocin by Bacteriodes fragilis and partial characterization.

The ability of Bacteroides fragilis strains, isolated from various sources, to produce bacteriocin was evaluated. All strains isolated from intestinal infections were producers in high levels and less susceptible to the others. Strains from other origins were found to produce bacteriocin at a medium level and they were variably susceptible. Some properties of one bacteriocin produced by the Bact. fragilis 079298-3 strain were analysed, providing evidence of its protein nature, with stability over a wide range of pH and retained inhibitory activity after heating. This variability seems to suggest that bacteriocin typing is a good method for this species.

Molecular characterization and transfer among Staphylococcus strains of a plasmid conferring high-level resistance to mupirocin.

In this work, mupirocin resistance was correlated with the presence of plasmids in methicillin-resistant Staphylococcus aureus (MRSA) strains isolated in the Rio de Janeiro Federal University Hospital in Brazil, where topical mupirocin has been used extensively since 1990. Of 19 strains studied, those exhibiting high-level resistance carried a large and relaxable plasmid of about 35 kb. Mupirocin-sensitive derivatives, obtained by growth at 42 C of a strain exhibiting high-level resistance, were devoid of the large plasmid, which was designated pMG1. Mupirocin resistance was transferred to strain RN8411 during overnight filter-matings at low frequencies (7.0 x 10(-9)/donor). The pMG1 plasmid was shown to be responsible for high-level mupirocin resistance in our isolates and to be incompatible with pGO1. Hybridization experiments suggested that mupirocin resistance in pMG1 is due to the presence of the ileS-2 gene. The pMG1 plasmid was successfully and bidirectionally transferred from Staphylococcus aureus to Staphylococcus epidermidis, suggesting that the latter may be a reservoir of this resistance plasmid. No transfer was detected to Staphylococcus haemolyticus. The development of self-transferable high-level mupirocin resistance should be considered when using mupirocin to control the spread of MRSA in hospitals.

Inhibitory activity of Paenibacillus polymyxa SCE2 against human pathogenic micro-organisms.

Paenibacillus polymyxa strain SCE2 was shown to inhibit the growth of different potential human pathogenic bacterial strains and fungi in vitro. To determine the genetic characterization of this antimicrobial substance, strain SCE2 was transformed with plasmid pTV32(Ts), a delivery vector for Tn917-lac. After transposition, four mutants were shown to have lost their capability to inhibit Micrococcus sp. and Staphylococcus aureus RN450, but they continued to inhibit the growth of Corynebacterium fimi NCTC7547 and Escherichia coli HB101. Hybridization experiments using the DNA of the four mutants digested with different endonucleases and pTV32(Ts) as a probe showed that the place of insertion of Tn917-lac in the chromosome was the same in mutants 4 and 36 and in mutants 31 and 59, but different between these pairs. It is thought possible that more than one antimicrobial substance is being produced by strain SCE2.

Staphylococcal strains involved in bovine mastitis are inhibited by Staphylococcus aureus antimicrobial peptides.

The inhibitory activity of five bacteriocin (Bac)-producer strains of Staphylococcus aureus was tested against bacteria pathogenic for cattle. Sixty-five epidemiologically unrelated strains of Staph. aureus involved in bovine mastitis were used as indicators in an agar diffusion test. Bacteriocins produced by four strains could inhibit only a limited number of test organisms. However, all 65 indicator strains proved to be susceptible to the combined action of both bacteriocins encoded by pRJ9, a Bac plasmid found in strain A53. Therefore, the bacteriocins produced by this strain may represent new antimicrobial peptides with potential applications in the prevention and treatment of bovine mastitis.

Genetic analysis of the bacteriocin-encoding plasmids pRJ6 and pRJ9 of Staphylococcus aureus by transposon mutagenesis and cloning of genes involved in bacteriocin production.

pRJ6 and pRJ9, small Staphylococcus aureus plasmids which code for bacteriocins, exhibited a bactericidal activity against several lactic acid bacteria and strains of Listeria monocytogenes, an important food-borne pathogen. Filter-mating experiments using plasmid derivatives tagged with either Tn551 or Tn917-lac showed that pRJ6, but not pRJ9, could be mobilized by staphylococcal conjugative plasmids. Transposon mutagenesis of both plasmids was also performed. The bacteriocin and immunity structural genes of pRJ6 are part of the same operon, which is located around co-ordinate 4.0, being transcribed from right to left. However, gene cloning experiments using a staphylococcal vector showed some evidence for the involvement of additional functions of pRJ6 in bacteriocin expression. One function involved in pRJ6 mobilization mapped around co-ordinate 5.2, and it appears to be transcribed from left to right. The bactericidal action exerted by strains harbouring pRJ9 appears to reflect the activity of at least two bacteriocins, whose combined action results in a broader spectrum of activity and in a higher antagonistic activity. Gene cloning experiments also supported these assumptions.

Regulation of transfer of the Enterococcus faecalis pheromone-responding plasmid pAD1: temperature-sensitive transfer mutants and identification of a new regulatory determinant, traD.

The enterococcal, conjugative, cytolysin plasmid pAD1 confers a mating response to the peptide sex pheromone cAD1 secreted by plasmid-free strains of Enterococcus faecalis. Cells carrying pAM714, a pAD1::Tn917 derivative with wild-type conjugation properties, were mutagenized with ethyl methanesulfonate to obtain variants that were induced (in the absence of pheromone) to transfer plasmid DNA upon shifting from 32 to 42 degrees C. Of 31 such mutants generated, the results of analyses of 7 are presented in detail. All seven strains were thermosensitive in the E. faecalis host FA2-2; colony morphology, clumping, and DNA transfer correlated well with each other at the two temperatures. In the nonisogenic host E. faecalis OG1X, however, only one derivative (pAM2725) exhibited correlation of all three traits at both temperatures. Three (pAM2700, pAM2703, and pAM2717) clumped and had colonies characteristic of pheromone-induced cells at 32 degrees C but transferred plasmid DNA at a higher frequency only at the elevated temperature. The other three (pAM2708, pAM2709, and pAM2712) were derepressed at both temperatures for all three characteristics. Four of the mutations, including that of pAM2725, mapped within the traA determinant, whereas two mapped identically in a previously unnoted open reading frame (designated traD) putatively encoding a short (23-amino-acid) peptide downstream of the inhibitor peptide determinant iad and in the opposite orientation. One mutant could not be located in the regions sequenced. Studies showed that the traA and traD mutations could be complemented in trans with a DNA fragment carrying the corresponding regions.

Incompatibility and molecular relationships between small bacteriocinogenic plasmids of Staphylococcus aureus.

The sequence relations between small bacteriocinogenic plasmids (pRJ6, pRJ9, pRJ10 and pRJ11) of Staphylococcus aureus were investigated by comparing restriction maps and by hybridization. Plasmids pRJ10 and pRJ11 showed identical restriction maps, similar to that of pRJ9. The restriction map of pRJ6 differed from those of pRJ9 and pRJ10/pRJ11. Both groups of plasmids were shown to share a region of homology of at least 2.6 kb. The incompatibility relationships between them were also investigated by using plasmid derivatives tagged with transposon Tn551. Plasmids pRJ6 and pRJ9 proved to belong to different incompatibility groups.

pRJ5: a naturally occurring Staphylococcus aureus plasmid expressing constitutive macrolide-lincosamide-streptogramin B resistance contains a tandem duplication in the leader region of the ermC gene.

The 2.55 kb Staphylococcus aureus plasmid, pRJ5, confers constitutive resistance to macrolide-lincosamide-streptogramin B (MLS) antibiotics. pRJ5 is nearly identical to the inducible MLS resistance plasmid pT48, and has homology with the S. aureus plasmids pE194 and pSN2. The HindIII-C and/or Hind-B fragments were required for stable maintenance of the plasmid and probably carry palA. Plasmids pRJ5 and pT48 were shown to belong to the same incompatibility group, Inc12 (L). DNA sequencing showed that pRJ5 contains a 28 bp direct tandem duplication in the leader/attenuator region of ermC. This is likely to change the secondary structure of the methylase mRNA, allowing constitutive expression of ermC. The type of mutation found on plasmid pRJ5 is different from those observed in similar 2.5 kb constitutive MLS-resistance plasmids isolated from other Gram-positive bacteria, including staphylococci.

Cloning of the replication region on the bacteriocinogenic plasmid pRJ9 of Staphylococcus aureus.

A 5.8-kb ClaI fragment of pRJ9, a bacteriocinogenic plasmid of Staphylococcus aureus, was cloned in the unique ClaI site of pRJ5. The recombinant plasmid obtained, pRJ23, failed to confer bacteriocin production and immunity to bacteriocin on host cells. The cloned fragment was shown to contain the complete replicon of pRJ9. Attempts to clone the 4.4-kb ClaI fragment of pRJ9 were unsuccessful, apparently due to the inactivation of the basic replicon of the cloning vector. Therefore, plasmid pRJ5 cut at its ClaI site appears to be a suitable vector for cloning replication regions of plasmids that can replicate in S. aureus.

Identification of structural nitrogen-fixation (nif) genes in Bacillus polymyxa and Bacillus macerans.

Some naturally occurring Nif(+) and Nif(-)uperscript> strains of Bacillus polymyxa and Bacillus macerans were tested for DNA homology to the Klebsiella pneumoniae nif genes by Southern hybridization. Genomic DNA from all Nif(+) strains carried homologous sequences only to structural nif genes. The homology detected was limited to nifH and nifD. The hybridization pattern observed suggested that both genes are contiguous on the chromosome of the Bacillus strains. No homology was found between the genomic DNA from Nif(-)uperscript> strains and the K. pneumoniae nif genes.

Distinct groups of plasmids correlated with bacteriocin production in Staphylococcus aureus.

The genetic basis of bacteriocin (Bac) production by six strains of Staphylococcus aureus was examined. Gene transfer experiments (in which the plasmids were tagged with the erythromycin resistance transposon Tn551) and plasmid-elimination experiments by growth at 43 degrees C associated bacteriocin production with a particular plasmid in each strain. The Bac plasmids could be separated into two distinct groups: the first comprised plasmids larger than 40 kb, which did not specify immunity to bacteriocins; the second comprised small plasmids (8.0-10.4 kb) which also specified immunity to bacteriocins. The sequence relations among the small plasmids (pRJ6, pRJ9, pRJ10 and pRJ11) were investigated by comparing restriction enzyme digest patterns and by hybridization. Plasmids pRJ10 and pRJ11 were indistinguishable and very closely related to plasmid pRJ9. Plasmid pRJ6, although different from the others, shared regions of sequence homology with them. No homology was found between plasmids pRJ6 or pRJ9 and the large Bac plasmids.

Transposon Tn554 encodes three products required for transposition.

Tn554 is a high-frequency, site-specific, transposable element having integrative properties resembling those of lysogenic bacteriophages. Nucleotide sequence analysis indicates that Tn554 has three transposition genes, designated tnpA, tnpB and tnpC. Mutations in each of these were complemented efficiently in trans by clones containing internal fragments of Tn554; thus the products of these genes function in trans. Elements carrying deletions of the Tn554 termini could not be complemented. The product of tnpC is not absolutely required for transposition, since deletion mutations encompassing 80% of tnpC, as well as frameshift mutations located near the amino terminus of tnpC, transposed at frequencies as high as 2% of that observed with wild-type Tn554. However, such mutations affected the orientation of insertion. With wild-type Tn554 insertion occurs in a single orientation regardless of the orientation of the donor. In tnpC mutants insertion orientation was dictated by the orientation of Tn554 in the donor molecule. A mutant lacking the carboxy-terminal 59 residues of tnpB also exhibited altered insertion orientation. Thus it appears that the tnpC gene product is required for correct orientation of the element upon insertion and that this protein may interact with the carboxy-terminal portion of the tnpB gene product.