RESUMO
OBJECTIVES: The present study reports the draft genome sequence of Staphylococcus aureus 4181, a strain involved in bovine mastitis that produces aureocin 4181, a broad-spectrum antimicrobial peptide (AMP). Inhibition of multidrug-resistant (MDR) staphylococci involved in human infections by S. aureus 4181 was also investigated. METHODS: A sequencing library was constructed using a Nextera XT DNA Library Preparation Kit (Illumina). Whole-genome shotgun sequencing was performed using an Illumina MiSeq System. The A5-miseq pipeline was employed for de novo genome assembly. Genome annotation was performed by the RAST server. The online automated tools BAGEL4 and antiSMASH v.5.0 were used for mining gene clusters encoding AMP production. The virulence potential of the strain was investigated employing online tools. Its inhibitory activity toward MDR staphylococcal isolates associated with human infections was tested by the deferred antagonism assay on brain-heart infusion agar medium. RESULTS: The total scaffold size was determined to be 2 719 949 bp, with a G + C content of 32.7%. Genome analyses revealed 2504 protein-coding sequences and 74 RNA-coding sequences as well as several genes encoding drug resistance and a single AMP gene cluster coding for aureocin 4181. Staphylococcus aureus 4181 exhibited a pathogenic potential and inhibited all MDR staphylococcal isolates tested as a target. CONCLUSIONS: This study describes the main features of the draft genome of S. aureus 4181, a strain that produces the third four-component bacteriocin described in the literature, namely aureocin 4181. This bacteriocin is a potential alternative drug to control MDR staphylococcal isolates involved in human infections.
Assuntos
Bacteriocinas , Infecções Estafilocócicas , Animais , Bovinos , Feminino , Humanos , Proteínas Citotóxicas Formadoras de Poros , Staphylococcus/genética , Staphylococcus aureus/genéticaRESUMO
Staphylococcins are antimicrobial peptides or proteins produced by staphylococci. They can be separated into different classes, depending on their amino acid composition, structural complexity, and steps involved in their production. In this review, an overview of the current knowledge on staphylococcins will be presented with emphasis on the information collected in the last decade, including a brief description of new peptides. Most staphylococcins characterized to date are either lantibiotics or linear class II bacteriocins. Recently, gene clusters coding for production of circular bacteriocins, sactipeptides, and thiopeptides have been mined from the genome of staphylococcal isolates. In contrast to class II bacteriocins, lantibiotics, sactipeptides, and thiopeptides undergo post-translational modifications that can be quite extensive, depending on the peptide. Few staphylococcins inhibit only some staphylococcal species, but most of them have proven to target pathogens belonging to different genera and involved in a variety of infectious diseases of clinical or agronomic importance. Therefore, these peptides exhibit potential application as anti-infective drugs in different areas. This review will also cover this diverse and remarkable potential. To be commercialized, however, staphylococcin production should be cost-effective and result in high bacteriocin yields, which are not generally achieved from the culture supernatant of their native producers. Such low yields make their production quite costly and not suitable at large industrial scale. Efforts already made to overcome this limitation, minimizing costs and time of production of some staphylococcins and employing either chemical synthesis or in vivo biosynthesis, will be addressed in this review as well. KEY POINTS: ⢠Staphylococci produce a variety of antimicrobial peptides known as staphylococcins. ⢠Most staphylococcins are post-translationally modified peptides. ⢠Staphylococcins exhibit potential biotechnological applications. Graphical abstract.
Assuntos
Anti-Infecciosos , Bacteriocinas , Antibacterianos , Proteínas Citotóxicas Formadoras de Poros , StaphylococcusRESUMO
Bacteriocins are ribosomally synthesized antimicrobial peptides produced by prokaryotes. Here, the molecular characterization of aureocin 4181, a bacteriocin produced by Staphylococcus aureus 4181, a strain involved in bovine mastitis, is presented. Aureocin 4181 gene cluster (aurRID1CBAT) was mined from scaffold 15 of the draft genome of its producer strain. Three (AurABC) out of the four structural peptides of aureocin 4181 are identical to those of aureocin A70, except for AurD1 of aureocin 4181, which showed a conservative substitution of Leu29 to Phe29 when compared to AurD of aureocin A70. According to molecular mass determination and peptide sequencing, combined with genome sequencing data, aureocin 4181 is an N-formylated variant of aureocin A70. The analysis of its antimicrobial spectrum was extended to include strains of the two major contagious pathogens involved in bovine mastitis, S. aureus and Streptococcus agalactiae. Aureocin 4181 exhibited a striking activity against S. aureus, inhibiting most strains tested. Besides having a broader spectrum of activity, aureocin 4181 exhibited a stronger bacteriolytic action against the target strains and proved to be from two- to fourfold more active than aureocin A70 against S. aureus. Aureocin 4181 has potential to become an alternative drug for prevention and control of mastitic staphylococci, a pathogen that imposes a huge economic burden to dairy industry worldwide. It also represents the third four-component bacteriocin described in the literature, the second in staphylococci.
Assuntos
Antibacterianos/farmacologia , Bacteriocinas/farmacologia , Mastite Bovina/microbiologia , Infecções Estafilocócicas , Staphylococcus aureus , Animais , Brasil , Bovinos , Feminino , Infecções Estafilocócicas/microbiologia , Infecções Estafilocócicas/veterinária , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/metabolismoRESUMO
Reducing salt content in foods such as cheeses, while limiting the growth of spoilage microorganisms and foodborne pathogens, is a difficult challenge. One method that may prove useful is use of staphylococcins, which are bacteriocins produced by staphylococci. Therefore, staphylococcin antimicrobial activity against six strains of S. aureus isolated from cheese was tested aiming at their industrial application in biopreservation of Minas fresh (Frescal) cheese with reduced sodium content. Three staphylococcins were selected for these tests: Pep 5, aureocin A53 and lysostaphin. All three staphylococcins proved to be bacteriolytic against all six strains of S. aureus. The antimicrobial activity of the partially purified staphylococcins was subsequently investigated against strains S. aureus Q1 and QJ3 in cheese matrices (6.0 log CFU/g) with different NaCl contents (control, a 25% reduction, and a 50% reduction), kept under refrigeration at 4⯰C, for 21â¯days. Both strains were shown to be of concern for food industry as they carry the SEA, SEB and SEH enterotoxin genes, and are resistant to ß-lactam drugs and moderate biofilm formers when grown in TSB. When used singly, Pep5, aureocin A53 and lysostaphin reduced approximately 95%, 99% and 99.99% of the viable cell counts, respectively, irrespective of the sodium content of the cheese matrix. The combined action of aureocin A53 and Pep5 resulted in an additional and significant reduction (pâ¯<â¯0.05) of ~1.0 log CFU/g when compared with the reduction caused by the use of either one singly. The combined action of lysostaphin and aureocin A53 or lysostaphin and Pep5 resulted in a reduction similar to or slightly smaller (pâ¯>â¯0.05) than that observed when lysostaphin was employed singly. Lysostaphin also proved to reduce the number of the staphylococcal viable cells to a level (~ 2.0 log CFU/g) at which enterotoxin production should not reach a sufficient quantity to cause food poisoning. Therefore, lysostaphin may have a practical application in the food industry to control staphylococcal contamination of Minas fresh cheese with a sodium content reduced up to 50%, providing consumers with more safe options to reduce their intake of sodium.
Assuntos
Antibacterianos/farmacologia , Bacteriocinas/farmacologia , Queijo/microbiologia , Doenças Transmitidas por Alimentos/prevenção & controle , Lisostafina/farmacologia , Peptídeos/farmacologia , Staphylococcus aureus/efeitos dos fármacos , Peptídeos Catiônicos Antimicrobianos , Enterotoxinas/análise , Doenças Transmitidas por Alimentos/microbiologia , Cloreto de Sódio/análise , Staphylococcus aureus/isolamento & purificação , Staphylococcus aureus/metabolismoRESUMO
OBJECTIVES: The aim of this study was to report the draft genome sequence of the bacteriocinogenic strain Enterococcus faecium E86. Bacteriocins are prokaryotic peptides or proteins with antimicrobial activity. The genome information may contribute to the identification of enterocins produced by this strain that exhibit inhibitory activity against the foodborne pathogen Listeria monocytogenes and vancomycin-resistant enterococci (VRE) involved in human infections, among other bacterial genera and species. METHODS: An Illumina MiSeq platform was used for genome sequencing. De novo assembly of 5 735 838 paired-end reads was done using the A5-miseq pipeline, yielding >300-fold average genome coverage. Genome annotation was performed by the RAST server, and mining of the bacteriocinogenic gene clusters was done using the BAGEL3 and antiSMASH v.4 platforms. RESULTS: The total scaffold size was determined to be 2 689 107 bp, approximately 2.7 Mbp, featuring a G + C content of 38.1%. The genome contains 2858 coding sequences and 74 RNA genes. Genome analyses revealed the presence of: 30 genes involved in drug resistance; 2 bacteriocinogenic gene clusters (for enterocin P and enterocin TW21); EntiTW21, a novel bacteriocin immunity protein and a novel multilocus sequence type (ST1500). CONCLUSION: This work highlights the potential biotechnological application of this strain for the production of enterocin P, a bacteriocin that can be employed in the food industry as a biopreservative against L. monocytogenes and as an alternative to classical antibiotics against VRE.
Assuntos
Peptídeos Catiônicos Antimicrobianos/biossíntese , Bacteriocinas/biossíntese , Enterococcus faecium/genética , Genoma Bacteriano , Antibacterianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/genética , Peptídeos Catiônicos Antimicrobianos/farmacologia , Bacteriocinas/genética , Bacteriocinas/farmacologia , Farmacorresistência Bacteriana/genética , Humanos , Listeria monocytogenes/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Família Multigênica , Análise de Sequência de DNA , Enterococos Resistentes à Vancomicina/efeitos dos fármacos , Sequenciamento Completo do GenomaRESUMO
The phylum Actinobacteria, which comprises a great variety of Gram-positive bacteria with a high G+C content in their genomes, is known for its large production of bioactive compounds, including those with antimicrobial activity. Among the antimicrobials, bacteriocins, ribosomally synthesized peptides, represent an important arsenal of potential new drugs to face the increasing prevalence of resistance to antibiotics among microbial pathogens. The actinobacterial bacteriocins form a heterogeneous group of substances that is difficult to adapt to most proposed classification schemes. However, recent updates have accommodated efficiently the diversity of bacteriocins produced by this phylum. Among the bacteriocins, the lantibiotics represent a source of new antimicrobials to control infections caused mainly by Gram-positive bacteria and with a low propensity for resistance development. Moreover, some of these compounds have additional biological properties, exhibiting activity against viruses and tumour cells and having also potential to be used in blood pressure or inflammation control and in pain relief. Thus, lantibiotics already described in Actinobacteria exhibit potential practical applications in medical settings, food industry and agriculture, with examples at different stages of pre-clinical and clinical trials.
Assuntos
Actinobacteria/efeitos dos fármacos , Actinobacteria/metabolismo , Antibacterianos/farmacologia , Infecções Bacterianas/tratamento farmacológico , Bacteriocinas/biossíntese , Antibacterianos/biossíntese , Bacteriocinas/farmacologia , Testes de Sensibilidade MicrobianaRESUMO
Hyicin 3682, the first bacteriocin reported for Staphylococcus hyicus, is a BsaCOL variant produced by S. hyicus 3682, a strain isolated from bovine milk. Hyicin 3682 is found in the culture supernatant, is bactericidal and its producing strain exhibits a much broader spectrum of antimicrobial activity than the producing strain of BsaCOL against several Gram-positive bacteria, which include foodborne pathogens, food-spoilage microorganisms and bacterial species of medical and veterinary importance. Sequencing of the genome of S. hyicus 3682 provided the nucleotide sequence of the entire gene cluster involved in hyicin 3682 production, which seems to be located on pRJ109, the single plasmid carried by this strain. This gene cluster is expressed and consists of 8525bp and of eight genes (hyiA, hyiB, hyiC, hyiD, hyiP, hyiF, hyiE and hyiG) encoded on the same DNA strand. The mature lantibiotic exhibits 91% identity to BsaCOL and its molecular mass was found to be â¼26Da higher due to two amino acid substitutions. S. hyicus 3682 proved to be only partially immune to its cognate bacteriocin up to 1024 AU/ml. Therefore, hyicin 3682, the first Bsa variant reported in coagulase-negative staphylococci, does exhibit antimicrobial and siblicidal activities.
Assuntos
Antibacterianos/metabolismo , Bacteriocinas/genética , Bacteriocinas/metabolismo , Staphylococcus hyicus/genética , Staphylococcus hyicus/metabolismo , Sequência de Aminoácidos , Animais , Antibacterianos/química , Bacteriocinas/química , Vias Biossintéticas/genética , Bovinos , Ordem dos Genes , Genes Bacterianos , Genoma Bacteriano , Bactérias Gram-Positivas/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Leite/microbiologia , Dados de Sequência Molecular , Peso Molecular , Família Multigênica , Análise de Sequência de DNA , Staphylococcus hyicus/isolamento & purificaçãoRESUMO
Aureocin A70 is the only four-component bacteriocin described to date. As it inhibits the growth of a wide range of Gram-positive bacteria, including Listeria monocytogenes strains isolated from food, its potential for improving food safety was investigated in this study. Aureocin A70 (10,240AU/mL) proved to be bactericidal, but not extensively lytic, against listerial strains. The antibacterial activity of aureocin A70 (16AU/mL) was then tested in UHT-treated skimmed milk inoculated with the food-associated L. monocytogenes L12 strain (4-log CFU/mL) during storage at 4°C for one week. Aureocin A70 caused a time-dependent reduction in the listerial viable cell counts (5.51-log units) up to 7days of incubation. Aureocin A70 was neither toxic to the Vero and the L-929 cell lines nor exhibited a hemolytic activity against sheep red blood cells. Aureocin A70 proved to be completely stable for one month at 25°C, 16weeks at 4°C and 20weeks at -20°C. Aureocin A70 exhibited a time-dependent susceptibility to simulated gastric juice and bile salts mimicking gastrointestinal conditions. The entrapment of aureocin A70 in an alginate/gelatin matrix revealed that this bacteriocin can be released from this matrix. Moreover, it remained adsorbed to and active on a low-density polyethylene plastic surface suggesting that aureocin A70 may be employed in bioactive packaging to control the growth of undesirable bacteria. Taken together these results suggest that aureocin A70 is a promising alternative to be used in food applications.
Assuntos
Bacteriocinas/farmacologia , Conservantes de Alimentos/farmacologia , Animais , Microbiologia de Alimentos , Conservação de Alimentos/instrumentação , Listeria monocytogenes/efeitos dos fármacos , Listeria monocytogenes/crescimento & desenvolvimento , Leite/microbiologia , Ovinos , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/crescimento & desenvolvimentoRESUMO
The coagulase-negative staphylococci are known for their ability to acquire resistance genes, which limits the choice of therapeutic options for the treatment of infections caused by these microorganisms. In this study, the diversity of high-level mupirocin resistance plasmids (Mup(R) ) was investigated in four strains of Staphylococcus haemolyticus belonging to different pulsed-field gel electrophoresis (PFGE) types or subtypes, isolated in a Brazilian hospital. These strains harbor the mupA gene in large plasmids. In addition, the presence of IS257 sequences flanking the mupA gene was also shown. Two isolates belonging to two different PFGE types exhibited a similar polymorphism for a fragment of the mupA gene and the closest proximal flanking copies of the IS257, suggesting horizontal transmission of S. haemolyticus mupirocin resistance plasmids in the environment and a role of this species as a reservoir of the mupA gene.
Assuntos
Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Farmacorresistência Bacteriana , Transferência Genética Horizontal , Mupirocina/farmacologia , Proteínas Nucleares/genética , Plasmídeos , Staphylococcus haemolyticus/efeitos dos fármacos , Técnicas de Tipagem Bacteriana , Brasil , Eletroforese em Gel de Campo Pulsado , Genótipo , Hospitais , Humanos , Tipagem Molecular , Polimorfismo Genético , Infecções Estafilocócicas/microbiologia , Staphylococcus haemolyticus/classificação , Staphylococcus haemolyticus/genética , Staphylococcus haemolyticus/isolamento & purificaçãoRESUMO
Fifty strains of Staphylococcus spp. isolated from bovine mastitis cases in several herds from different Argentinian provinces were screened for antimicrobial substances. Twelve strains exhibited a high antagonistic activity against the indicator strain (Corynebacterium fimi) and were chosen for further characterisation. The antimicrobial substances were sensitive to proteolytic enzymes suggesting that they might be bacteriocins (Bac). These strains were identified as S. aureus by the amplification of the femA gene. Plasmid profile analysis of these strains revealed the presence of at least one plasmid. Eleven strains carried a plasmid with a size similar to that of pRJ6 (8.0kb), which encodes aureocin A70, a bacteriocin produced by the Brazilian S. aureus strain A70 isolated from commercial milk. The other strain harboured a much larger plasmid. PCR experiments, using specific primers for amplification of the bacteriocin operon found in pRJ6, showed that all strains had the expected 525bp amplicon, suggesting that the bacteriocin produced may be related to aureocin A70. The genomic DNA of all Bac(+) strains was then analysed by pulsed-field gel electrophoresis (PFGE) in order to investigate clonal relationships amongst strains. Based on the results of PFGE experiments, 10 out of the 12 Bac(+) strains belonged to the same clone. The remaining two strains are possibly related to the prevalent clone. The aureocin A70 producer-strain belonged to a distinct clone.