Antimicrobial substances produced by Staphylococcus aureus strains isolated from cattle in Brazil

Among 46 isolates of Staphylococcus aureus obtained from cattle in the State of Paraíba, Brazil, four were shown to produce antimicrobial substances (AMS). The two best AMS producers carried single plasmids of about 8·0 kbp and 50 kbp, respectively, which were designated pRJ34 and pRJ35. Curing experiments and molecular analysis associated the AMS production with the presence of these plasmids in the cells. The biochemical properties exhibited by the AMS suggested that they might be bacteriocins (Bac). The bacteriocin encoded by pRJ34 showed properties identical to those of the bacteriocins encoded by other small staphylococcal Bac plasmids. However, the bacteriocin encoded by the large plasmid pRJ35 has shown some properties which distinguish it from the other bacteriocins of Staph. aureus described so far, suggesting it may be a new member of the staphylococcal bacteriocin family.     

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.     

Characterization of a novel bacteriocin-encoding plasmid found in clinical isolates of Staphylococcus aureus

Plasmids specifying bacteriocin production and immunity to its action were found in three clinical isolates of Staphylococcus aureus obtained in different hospitals located in Rio de Janeiro. These plasmids (pRJ28, pRJ29 and pRJ30) of 8.0 kb were found to generate identical restriction fragment patterns upon digestion with several enzymes, although the range of strains susceptible to the respective bacteriocin varied among the producer strains, when different Gram-positive bacteria were used as indicators. pRJ29 was then chosen for further characterization in order to compare it with pRJ6 and pRJ9, two small bacteriocin-encoding plasmids previously described in strains isolated from food. pRJ29 was found to code for a bacteriocin with chemical properties (sensitivity to proteases, heat resistance, activity under anaerobiosis, and estimated molecular weight) similar to those of pRJ6-encoded bacteriocin, conferring cross-immunity to it. However, its restriction map differed from those of pRJ6 and pRJ9. These studies together with hybridization, incompatibility, and mobilization analyses using a derivative of pRJ29 tagged with Tn917-lac suggest that pRJ29 is a mosaic composed of genetic determinants found on pRJ6 and pRJ9, and that IS 257 was not involved in the recombination events which gave rise to pRJ29.     

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

Abstract A 5.8-kb Cla I fragment of pRJ9, a bacteriocinogenic plasmid of Sphylococcus aureus , was cloned in the unique Cla I 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 Cla I fragment of pRJ9 were unsuccessful, apparently due to the inactivation of the basic replicon of the cloning vector. Therefore, plasmid pRJ5 cut at its Cla I site appears to be a suitable vector for cloning replication regions of plasmids that cab replicate in S. aureus .     

Biochemical characterisation and genetic analysis of aureocin A53, a new,atypical bacteriocin from Staphylococcus aureus

Aureocin A53 is produced by Staphylococcus aureus A53. It is encoded on a 10.4 kb plasmid, pRJ9, and is active against Listeria monocytogenes. Aureocin A53 is a highly cationic 51-residue peptide containing ten lysine and five tryptophan residues. Aureocin A53 was purified to homogeneity by hydrophobic-interaction, cation-exchange, and reverse-phase chromatography. MALDI-TOF mass spectrometry yielded a molecular mass of 6012.5 Da, which was 28 Da higher than predicted from the structural gene sequence of the bacteriocin. The mass increment resulted from an N-formylmethionine residue, indicating that the aureocin A53 is synthesised and secreted without a typical bacteriocin leader sequence or sec-dependent signal peptide. The structural identity of aureocin A53 was verified by Edman sequencing after de-blocking with cyanogen bromide and extensive mass spectrometry analysis of enzymatically and laser-generated fragments. The complete sequence of pRJ9 was determined and none of the open reading frames identified in the vicinity of the structural gene aucA showed similarity to genes that are typically found in bacteriocin gene clusters. Thus, neither a dedicated protease or transporter, nor modifying enzymes and regulatory elements seemed to be involved in the production of aureocin A53. Further unique features that distinguish aureocin A53 from other peptide bacteriocins include remarkable protease stability and a defined, rigid structure in aqueous solution.     

Analysis of the genetic region encoding a novel rhizobiocin from Rhizobium leguminosarum bv. viciae strain 306

Cross-testing of a number of strains of Rhizobium leguminosarum for bacteriocin production revealed that strain 306 produced at least two distinct bacteriocins. Further analysis involving plasmid transfer to Agrobacterium and other hosts demonstrated that there were bacteriocin determinants on plasmids pRle306b and pRle306c, as well as a third bacteriocin. The bacteriocin encoded by pRle306b was indistinguishable from the bacteriocin encoded by strain 248, whereas the bacteriocin encoded by plasmid pRle306c had a distinctive spectrum of activity against susceptible strains, as well as different physical properties from other bacteriocins that we have studied in our lab. Two mutants altered in production of the pRle306c bacteriocin were generated by transposon Tn5 mutagenesis, and the DNA flanking the transposon inserts in these mutants was cloned and characterized. DNA sequence analysis suggested that the pRle306c bacteriocin was a large protein belonging to the RTX family, and that a type I secretion system involving an ABC type transporter was required for export of the bacteriocin. A mutant unable to produce this bacteriocin was unaltered in its competitive properties, both in broth and in nodulation assays, suggesting that the bacteriocin may not play a major role in determining the ecological success of this strain.     

Revealing the latent mobilization capability of the staphylococcal bacteriocinogenic plasmid pRJ9

Plasmid pRJ9 is a non-self-mobilizable bacteriocinogenic plasmid from Staphylococcus aureus. Despite this feature, DNA sequencing and RT-PCR experiments showed that it presents a Mob region with three genes (mobCAB), transcribed as an operon. In silico analysis of the Mob proteins encoded by pRJ9 showed that they present all the conserved functional features reported until present as being essential for plasmid mobilization. Moreover, they showed a high identity to Mob proteins encoded by mobilizable plasmids from Staphylococcus spp., especially to those encoded by plasmid pRJ6, which presents four mob genes (mobCDAB). A putative oriT region was also found upstream of the pRJ9 mob operon. pRJ9 could only be successfully mobilized by pGO1 when pRJ6 was present in the same strain. Further experiments showed that the pRJ9 oriT can be recognized by the pRJ6 Mob proteins, confirming its functionality. As pRJ9 does not possess a mobD gene while pRJ6 does, the absence of this gene was believed to be responsible for its lack of mobilization. However, conjugation experiments with a donor strain carrying also mobD cloned into an S. aureus vector showed that pRJ9 does not become mobilized even in the presence of the protein MobD encoded by pRJ6. Therefore, the reasons for pRJ9 failure to be mobilized are presently unknown.     

Characterization of a Staphylococcus aureus Bacteriocin

The bacteriocin produced by a strain of Staphylococcus aureus has been isolated and designated staphylococcin (414), and a study was made of its chemical, physical, and biological properties. The staphylococcin is released in appreciable quantities after breakage of the cells and can be purified through differential centrifugation and column chromatography. In the native state, it appears to be a lipoprotein-carbohydrate complex with a molecular weight in excess of 200,000. The complex can be dissociated by sodium dodecyl sulfate into smaller subunits which retain activity. The gross chemical and physical properties of the bacteriocin closely resemble those ascribed to certain preparations of cell membranes. Staphylococcin (414) is not a lytic enzyme like lysostaphin and does not have the same spectrum of activity. Like other bacteriocins from gram-positive microorganisms, it does not inhibit any gram-negative bacteria, but does inhibit several other genera.     

Spontaneous bacteriocin resistance in Listeria monocytogenes as a susceptibility screen for identifying different mechanisms of resistance and modes of action by bacteriocins of lactic acid bacteria

A practical system was devised for grouping bacteriocins of lactic acid bacteria (LAB) based on mode of action as determined by changes in inhibitory activity to spontaneously-acquired bacteriocin resistance (Bac^R). Wild type Listeria monocytogenes 39-2 was sensitive to five bacteriocins produced by 3 genera of LAB: pediocin PA-1 and pediocin Bac3 (Pediococcus), lacticin FS97 and lacticin FS56 (Lactococcus), and curvaticin FS47 (Lactobacillus). A spontaneous Bac^R derivative of L. monocytogenes 39-2 obtained by selective recovery against lacticin FS56 provided complete resistance to the bacteriocin made by Lactococcus lactis FS56. The lacticin FS56-resistant strain of L. monocyotgenes 39-2 was also cross-resistant to curvaticin FS47 and pediocin PA-1, but not to lacticin FS97 or pediocin Bac3. The same pattern of cross-resistance was also observed with Bac^R isolates obtained with L. monocytogenes Scott A-2. A spontaneous mutation that renders a strain cross-resistant to different bacteriocins indicates that they share a common mechanism of resistance due to similar modes of action of the bacteriocins. Spontaneous resistance was acquired to other bacteriocins (in aggregate) by following the same procedure against which the Bac^R strain was still sensitive. In subsequent challenge assays, mixtures of bacteriocins of different modes of action provided greater inhibition than mixtures of bacteriocins of the same mode of action (as determined by our screening method). This study identifies a methodical approach to classify bacteriocins into functional groups based on mechanism of resistance (i.e., mode of action) that could be used for identifying the best mixture of bacteriocins for use as biopreservatives.     

Genetic analysis of bacteriocin 43 of vancomycin-resistant Enterococcus faecium

A total of 636 vancomycin-resistant Enterococcus faecium (VRE) isolates obtained between 1994 and 1999 from the Medical School Hospital of the University of Michigan were tested for bacteriocin production. Of the 277 (44%) bacteriocinogenic strains, 21 were active against E. faecalis, E. faecium, E. hirae, E. durans, and Listeria monocytogenes. Of those 21 strains, a representative bacteriocin of strain VRE82, designated bacteriocin 43, was found to be encoded on mobilizable plasmid pDT1 (6.2 kbp). Nine open reading frames (ORFs), ORF1 to ORF9, were presented on pDT1 and were oriented in the same direction. The bacteriocin 43 locus (bac43) consists of the bacteriocin gene bacA (ORF1) and the immunity gene bacB (ORF2). The deduced bacA product is 74 amino acids in length with a putative signal peptide of 30 amino acids at the N terminus. The bacB gene encodes a deduced 95-amino-acid protein without a signal sequence. The predicted mature BacA protein (44 amino acids) showed sequence homology with the membrane-active class IIa bacteriocins of lactic acid bacteria and showed 86% homology with bacteriocin 31 from E. faecalis YI717 and 98% homology with bacteriocin RC714. Southern analysis with a bac43 probe of each plasmid DNA from the 21 strains showed hybridization to a specific fragment corresponding to the 6.2-kbp EcoRI fragment, suggesting that the strains harbored the pDT1-like plasmid (6.2 kb) which encoded the bacteriocin 43-type bacteriocin. The bac43 determinant was not identified among non-VRE clinical isolates.     

Bacteriocinogenic activity of lactobacilli from fermented sausages

During the screening of the inhibitory activity of 254 strains of lactobacilli isolated from fermented sausages at different times of ripening, 22% of the strains showed inhibition that was not related to acid or hydrogen peroxide, towards one or more indicator strains. Not all the strains were capable of secreting the inhibitory compound in the supernatant fluid. The characterization of the inhibitory compound from three strains showed that they were bacteriocins with a bactericidal mode of action and a molecular weight exceeding 10000 Da. Lactobacillus plantarum CTC 305, CTC 306 and Lact. sake CTC 372 inhibited Listeria monocytogenes. Lactobacillus sake CTC372 was cured of two plasmids of 84.8 kbp and 41.3 kbp, losing the production and the immunity of a bacteriocin as well as the ability to ferment lactose.     

Aminocyclitol resistance in Staphylococcus aureus: presence of plasmids and aminocyclitol-modifying enzymes

Aminocyclitol resistance in Staphylococcus aureus has been investigated by the analysis of the plasmids and aminocyclitol-modifying enzymes present in several clinical staphylococcal isolates. All of the strains tested were resistant to a broad range of aminocyclitols and contained large plasmids which encoded a variety of aminocyclitol-modifying enzymes in addition to other antibiotic resistances. All strains expressed multiple aminocyclitol-modifying enzymes. The plasmids present in these strains appear to be related by virtue of their similar restriction endonuclease digestion patterns. The plasmids are related and differ by the gain or loss of small DNA segments, one of which encodes erythromycin and kanamycin resistance.     

Highly efficient Staphylococcus carnosus mutant selection system based on suicidal bacteriocin activation

Strains from various staphylococcal species produce bacteriocin peptides, which are thought to play important roles in bacterial competition and offer interesting biotechnological avenues. Many bacteriocins are secreted as inactive prepeptides with subsequent activation by specific proteolytic cleavage. By deletion of the protease gene gdmP in Staphylococcus gallinarum Tü3928, which produces the highly active lanthionine-containing bacteriocin gallidermin (lantibiotic), a strain was created producing inactive pregallidermin. On this basis, a new suicidal mutant selection system in the food-grade bacterium Staphylococcus carnosus was developed. Whereas pregallidermin was inactive against S. carnosus, it exerted potent bactericidal activity toward GdmP-secreting S. carnosus strains. To take advantage of this effect, gdmP was cloned in plasmid vectors used for random transposon mutagenesis or targeted allelic replacement of chromosomal genes. Both mutagenesis strategies rely on rare recombination events, and it has remained difficult and laborious to identify mutants among a vast majority of bacterial clones that still contain the delivery vectors. The gdmP-expressing plasmids pGS1 and pGS2 enabled very fast, easy, and reliable identification of transposon and gene replacement mutants, respectively. Mutant selection in the presence of pregallidermin caused suicidal inactivation of all clones that had retained the plasmids and allowed growth of only plasmid-cured mutants. Efficiency of mutant identification was several magnitudes higher than standard screening for the absence of plasmid-encoded antibiotic resistance markers and reached 100% specificity. Thus, the new pregallidermin-based mutant selection system represents a substantial improvement of staphylococcal mutagenesis methodology.     

Characterization of plasmids in aminocyclitol-resistant Staphylococcus aureus: electron microscopic and restriction endonuclease analysis

Plasmid species isolated from aminocyclitol-resistant Staphylococcus aureus have been analyzed by restriction endonuclease digestion and electron microscopy. These plasmids can be divided into two interrelated groups; intergroup variability is due to the gain or loss of defined DNA sequences. Plasmids pSJ1 and pSJ24 are related to staphylococcal penicillinase plasmid pI524 which was first described over 20 years ago. Both pSJ1 and pSJ24 differ from pI524 by the acquisition of 8 and 4 kbp, respectively, and encode additional resistance to the antibiotics erythromycin and kanamycin. The gain of these resistance determinants suggests that the evolution of staphylococcal resistance plasmids parallels that observed for plasmids of gram-negative bacteria and has serious implications for the spread of antibiotic resistance among the staphylococci.     

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.     

Issues in determining factors influencing bacterial attachment: a review using the attachment of Escherichia coli to abiotic surfaces as an example

The emergence of an increasing number of antibiotic resistant human clinical bacteria has been a great cause of concern for the last decades. As an example, Staphylococcus aureus isolates in the hospital environment are becoming more and more resistant to antibiotics including vancomycin which is considered as a last line of defence in treatment of Staphylococcus aureus -resistant methicillin. On the other hand, food safety is threatened by development of pathogenic bacteria including Listeria monocytogenes, Campylobacter jejuni, Salmonella enteritidis, Escherichia coli O157:H7 and Staphylococcus aureus. The use of antimicrobial peptides such as glycopeptides, semi-synthetic peptides, bacteriocins including lantibiotics offers a hope to face these clinical and food microbiology concerns. Clinical approval of new chemotherapeutic agents requires a long period of time. Research on bacteriocins has demonstrated potential use to fight against undesired foodborne pathogens but the use industrial use of bacteriocins is limited. To date only lantibiotic nisin and in class IIa bacteriocin Pediocin PA-1 are legally used as food preservative in many countries. The present minireview is focused on divercin V41 (DvnV41), a class IIa bacteriocin naturally produced by Carnobacterium divergens V41. The last decade has been the witness of intensive investigations carried out on this cationic peptide tempting to answer multiple questions covering basic and applied aspects. DvnV41 has shown a wide spectrum of activity either alone or in combination with nisin and/or polymixins (synergistic effect). This outcome indicates that Cb. divergens V41 could potentially be used for safe and efficient prevention of L. monocytogenes growth in cold smoked salmon.     

Plasmid-associated bacteriocin production by a strain of Carnobacterium piscicola from meat

Carnobacterium piscicola LV17 isolated from vacuum-packed meat produces bacteriocin(s) that is active against closely related lactic acid bacteria, Enterococcus spp., and a strain of Listeria monocytogenes but not against gram-negative bacteria. The bacteriocin has a bactericidal mode of action, is heat resistant, and is stable over a wide range of pH but is inactivated by proteolytic enzymes. Sensitive and resistant cells were shown to adsorb the bacteriocin, but cell death depended on contact of the bacteriocin with the cell membrane. Bacteriocin production is detected early in the growth cycle of the organism in APT broth, but it is not produced in APT broth adjusted to pH 5.5. Bacteriocin production and resistance to the bacteriocin produced are associated with two plasmids of 40 and 49 megadaltons. The possibility that two bacteriocins are produced is indicated because the inhibitory substances of the mutant strains containing either the 40- or 49-megadalton plasmids have different antimicrobial spectra.     

Plasmid-associated bacteriocin production by a strain of Carnobacterium piscicola from meat.

Carnobacterium piscicola LV17 isolated from vacuum-packed meat produces bacteriocin(s) that is active against closely related lactic acid bacteria, Enterococcus spp., and a strain of Listeria monocytogenes but not against gram-negative bacteria. The bacteriocin has a bactericidal mode of action, is heat resistant, and is stable over a wide range of pH but is inactivated by proteolytic enzymes. Sensitive and resistant cells were shown to adsorb the bacteriocin, but cell death depended on contact of the bacteriocin with the cell membrane. Bacteriocin production is detected early in the growth cycle of the organism in APT broth, but it is not produced in APT broth adjusted to pH 5.5. Bacteriocin production and resistance to the bacteriocin produced are associated with two plasmids of 40 and 49 megadaltons. The possibility that two bacteriocins are produced is indicated because the inhibitory substances of the mutant strains containing either the 40- or 49-megadalton plasmids have different antimicrobial spectra.