Deoxyribonucleic acid homologies among staphylococci: Coagulase-positive reference strains

DNA hybridization results confirm the proposed separation of coagulase-positive staphylococci into two distinct species. Strains ofStaphylococcus aureus representing the various biotypes and different phage typing groups of the human biotype gave high values of reassociation with DNA fromS. aureus reference strain RN 450, at both optimal and restrictive reassociation temperatures. Similar results were obtained between strains ofS. intermedius and its reference strain K 3. Interspecific reassociation between the two coagulase-positive species was low, and each reference strain showed low DNA sequence homology with 10 coagulase-negative species.S. staphylolyticus, strain PS 73, and putative pleiotropic mutants ofS. aureus were shown to be unrelated toS. aureus.     

An assessment of the inhibitory activity of rat serum on staphylococci

A study has been made on the effect of rat serum on staphylococci. By following the respiration and growth of 5 strains ofStaphylococcus aureus and an equal number ofS. epidermidis in fresh, normal rat serum, we found thatS. aureus grew in, and oxidized rat serum better thanS. epidermidis. Difference in growth was not correlated with nutritional requirements. The antibacterial agent of fresh, normal, undiluted rat serum was stable to heating at 56 C for 1 hr, but its activity was completely destroyed after heating at 60 C for 2 hr. A 50 per cent dilution of rat serum with nutrient broth significantly reduced the antibacterial activity and treatment of rat serum with 0.4m solutions of sodium citrate reduced it drastically. Once the serum had been treated with sodium citrate, or oxalate, addition of equimolar solutions of calcium chloride or magnesium chloride failed to restore the antibacterial activity. Addition of ferric ions in high concentrations allowed the coagulase-negative strains of staphylococci to grow well in this serum. The antibacterial agent of rat serum was absorbed by heat-killed cells ofStaphylococcus aureus andS. epidermidis but not byStreptococcus pyogenes andEscherichia coli. Treatment of rat serum with bentonite at a concentration of 100 mg per ml decreased its antibacterial activity.     

A comparative immunological study of catalases from coagulase-positive staphylococci

Protein homology studies with catalase as a reference point were carried out with coagulasepositive staphylococci belonging to Staphylococcus aureus, S. intermedius and S. hyicus. Antisera against catalases of S. aureus ATCC 12600 and S. aureus ATCC 12601 reacted very weakly employing double immunodiffusion and quantitative microcomplementfixation assay with cell-free extracts or catalase enriched preparations of S. intermedius or S. hyicus. The differences between coagulase-positive staphylococci could be confirmed by using the antiserum against S. intermedius H 11 catalase. Within the strains of the species S. intermedius immunological distances ranging up to 25 indicate a heterogeneity which justify the separation of the biotypes E and F on a subspecies level. Coagulase-positive strains of S. hyicus revealed neither a close relationship to S. aureus nor to S. intermedius.     

Construction ofClostridium butyricum hybrid plasmids and transfer toBacillus subtilis

Summary Small plasmids were isolated from type strains ofClostridium butyricum. Strain NCIB 7423 carries one plasmid (pCBU1) of 6.4 kb, whereas strain NCTC 7423 carries two unrelated plasmids of 6.3 kb (pCBU2) and 8.4 kb (pCBU3). Cleavage sites for 18 restriction endonucleases have been mapped on these plasmids and detailed physical maps are presented. For the purpose of developing vector plasmids for gene cloning in saccharolytic clostridia these crypticC. butyricum plasmids were joined to a selectable marker that will likely be expressed in clostridia. This was achieved by cloning the clostridial plasmids into theE. coli vector pBR322 carrying the chloramphenicol acetyltransferase (CAT) gene from theStaphylococcus aureus plasmid pC194. The recombinant plasmids were tested for their ability to confer chloramphenicol resistance toBacillus subtilis. Hybrid plasmids (pHL105, pHL1051) derived from pCBU2 were identified, which are capable of replication and expression of theS. aureus drug resistance marker in bothE. coli andB. subtilis. No structural instability was detected upon retransformation of pHL105 fromB. subtilis intoE. coli. The recombinant plasmids might thus be useful as shuttle vectors for the gene transfer betweenE. coli and a wide range of bacilli and clostridia.     

Mobilization functions of the bacteriocinogenic plasmid pRJ6 of <Emphasis Type="Italic">Staphylococcus aureus</Emphasis>

Plasmid pRJ6 is the first known bacteriocinogenic mobilizable (Mob) plasmid of Staphylococcus aureus. Its Mob region is composed of four mob genes (mobCDAB) arranged as an operon, a genetic organization uncommon among S. aureus Mob plasmids. oriT _pRJ6 was detected in a region of 431 bp, positioned immediately upstream of mobC. This region, when cloned into pCN37, was able to confer mobilization to the re-combinant plasmid only in the presence of pRJ6. The entire Mob region, including oriT _pRJ6, is much more similar to Mob regions from several coagulase-negative staphylococci plasmids, although some remarkable similarities with S. aureus Mob plasmids can also be noted. These similarities include the presence within oriT _pRJ6 of the three mcb (MobC binding sites), firstly described in pC221 and pC223, an identical nick site also found in these same plasmids, and a nearly identical sra _pC223 site (sequence recognized by MobA). pRJ6 was successfully transferred to S. epidermidis by conjugation in the presence of the conjugative plasmid pGOl. Altogether these findings suggest that pRJ6 might have been originally a coagulase-negative staphylococci plasmid that had been transferred successfully to S. aureus.     

Selective antimicrobial action of chitosan against spoilage yeasts in mixed culture fermentations

The effect of chitosan on Saccharomyces cerevisiae (the yeast that carries out alcohol fermentation), Brettanomyces bruxellensis and Brettanomyces intermedius (contaminants of alcohol fermentations), was investigated. The effect of chitosan was tested on each yeast, as well as on mixed cultivations of S. cerevisiae + B. bruxellensis and S. cerevisiae + B. intermedius. Chitosan enhanced the lag period of both strains of Brettanomyces (80 h for B. bruxellensis and 170 h for B. intermedius with 6 and 2 g/l chitosan, respectively). The growth rate of S. cerevisiae was inversely proportional to the chitosan concentration; the former was 50% when 6 g/l polysaccharide was used. Moreover, in mixed cultivations of S. cerevisiae and Brettanomyces strains, it was found that both B. bruxellensis and B. intermedius failed to grow while growth of S. cerevisiae was not affected (using 3 and 6 g/l chitosan, respectively). An interesting collateral result was that the presence of chitosan accelerated the consumption of glucose in the mixed cultivations (60 h instead of 120 h).     

Identification andin silico characterisation of putative conjugative transfer genes onGeobacillus stearothermophilus plasmids

We repor the first data demonstrating the presence of putative conjugative transfer genes on plasmids of the speciesGeobacillus stearothermophilus. Partial sequence analysis of the plasmid pGS18 fromG. stearothermophilus 18 was determined. It contained eleven complete open reading frames. Five of them encoded proteins which are homologous toBacillus megaterium pBM300 Mob/TraA,Lactococcus lactis pMRC01 TrsD and TrsE,Staphylococcus aureus pGO1 TrsG andS. aureus subsp.aureus pUSA03 TraL, the proteins that are associated with conjugative plasmid transfer. Southern hybridizations were performed on two other plasmids isolated fromG. stearothermophilus 3 andG. stearothermophilus 19 strains using the most homologous parts of those five genes as probes. Data from different hybridization patterns show a close homology of putative conjugative transfer genes between pGS18 and pGS3 hypothesizing a similar molecular organization of putative conjugative plasmid transfer region of both plasmids.     

Deoxyribonucleic acid reassociation in the classification of coagulase-positive staphylococci

DNA-DNA reassociation studies were performed with coagulase-positive staphylococci belonging to the biotypes A, B, C, D, E and F. These studies present genetic evidence for the existence of at least two distinct species within this group of organisms. The common Staphylococcus aureus strains were represented by organisms from biotypes A to D, and their DNA revealed over 80% nucleotide sequence homology under restrictive conditions. Less than 15% DNA homology was detected between strains from biotypes A to D (S. aureus) and those from biotypes E and F. The DNA of organisms from either the biotypes E or F displayed over 70% homology. Together, both biotypes are considered to represent the species S. intermedius. However, DNA homology values dropped to 50–65% between strains from different biotypes. This may justify the separation of S. intermedius biotypes E and F on a subspecies level.Abbreviations O.D. optical density - SSC standard saline citrate buffer (0.15 M NaCl, 0.015 M sodium citrate, pH 7.0) This work was supported by Deutsche Forschungsgemeinschaft     

Donor/recipient interactions affecting plasmid transfer amongStreptomyces species: A conjugative plasmid will mobilize nontransferable plasmids in soil

Streptomyces parvulus was used as the recipient for plasmid pIJ303 and pIJ211, two conjugative plasmids derived from the self-transmissible plasmid pIJ101. One of the resulting transconjugantS. parvulus strains containing plasmid pIJ303 was used withS. lividans to evaluate the effects of the host strain on the frequency of pIJ303 transfer betweenStreptomyces species. Only 30% ofS. parvulus cells acquired plasmid pIJ303 in crosses in whichS. lividans was the donor, whereas 100% ofS. lividans cells acquired the plasmid whenS. parvulus was the donor. This indicates that the frequency of transfer of the conjugative plasmid was determined by the recipient. The other resulting transconjugantS. parvulus strain containing plasmid pIJ211 was evaluated for its ability to mobilize the nonconjugative plasmid pIJ702 fromS. lividans, on agar and in sterile soil. AfterS. lividans containing pIJ702 was crossed on agar and in sterile soil withS. parvulus containing pIJ211, recombinantS. parvulus colonies carrying pIJ702 and expressing pigments characteristic of both species were recovered, from both agar and soil. Although a large percentage ofS. parvulus transconjugants lost pIJ211 during incubation in soil, the mobilization of pIJ702 fromS. lividans intoS. parvulus still occurred. Plasmid integration into the chromosome of the donor and the transconjugant was evaluated by Southern blot hybridization. Hybridization of plasmid pIJ303, with chromosomal DNA fromS. lividans andS. parvulus transconjugants, using biotinylated DNA, indicated that no integration had occurred. Genetic exchange betweenStreptomyces species also occurred in a liquid medium. The finding of plasmid mobilization in soil is significant. It demonstrates that genetic exchange in the environment can occur between released genetically engineeredStreptomyces species and nativeStreptomyces species that contain conjugative plasmids.Paper of the Idaho Agricultural Experiment Station.     

Expression of the gene encoding protein A in Staphylococcus aureus and coagulase-negative staphylococci

Two shuttle vectors containing the gene for protein A (spa) from Staphylococcus aureus have been constructed to study expression of the gene in various strains of S. aureus and in the coagulase-negative species Staphylococcus epidermidis, Staphylococcus capitis, and Staphylococcus xylosus. One plasmid, pSPA15, contains the complete structural gene for protein A, which binds to the cell wall in various Staphylococcus species. The other plasmid, pSPA16, codes for a truncated protein A lacking the C-terminal part called region X. The latter is exclusively extracellular in all Staphylococcus species tested, which confirms the importance of region X for cell wall binding. The expression of the plasmid-coded protein A in various strains of S. aureus is strongly correlated to the expression of the chromosomal spa gene. The coagulase-negative species expressing plasmid-encoded protein A produce 12 to 30% of the amount coded by the chromosomal spa gene in S. aureus strains Cowan I and A676.     

Characterization of a complex restriction-modification system detected in<Emphasis Type="Italic">Staphylococcus aureus</Emphasis> and<Emphasis Type="Italic">Streptococcus agalactiae</Emphasis> strains isolated from infections of domestic animals

Characterization of classic type II restriction-modification systems (RMS) (restriction endonucleases and modification methyltransferases) was carried out in isolates ofStaphylococcus aureus andStreptococcus agalactiae obtained from clinical material. Among the 100 isolates ofS. aureus two different RMS type II were detected. The first was expressed in isolates 32 and 33 (Sau32 I andSau33 I); the targeting sequence was determined as 5′-GGN CC-3′ (Sau96 I isoschizomer). The second was found in isolates no. 90, 93, 96*, and 98 (Sau90 I,Sau93 I,Sau96* I,Sau98 I) and enzymes recognized sequence 5′-CTY RAG-3′ (SmlI isoschizomer). Analysis of 40 isolates ofS. agalactiae revealed only one RMS; it was detected in two isolates (no. 16 and 23;Sag16 I andSag23 I). Restriction endonuclease expressed by these isolates cleaved DNA in sequence 5′-CTG CA/G-3′ (PstI isoschizomer). In RMS-positiveS. aureus andS. agalactiae isolates plasmid DNA capable of replication inEscherichia coli andBacillus subtilis was also detected and isolated. This research was supported by VEGA grant of theSlovak Academy of Sciences no. 2/2059/22 and grant no. 2003 SP27/0208E 02/028/0E02 of theMinistry of Agriculture of the Slovak Republic.     

Distribution and analysis of plasmids inStreptococcus thermophilus

Summary In a survey of 35 strains ofStreptococcus thermophilus, 13 strains were found to harbor plasmid DNA. Most of these strains contained plasmid species varying in size from 2.2 to 7.15 kilobases. Only three strains had more than one plasmid species. Each of the nine distinct types of plasmid DNAs identified had two or more unique recognition sites for restriction endonucleases. The characteristics of the indigenous cryptic plasmids ofS. thermophilus may allow their development as cloning vectors useful in the genetic engineering of this species and other streptococci that are important in food production     

Plasmid content and homology of 16 strains of filamentous,nonheterocystous cyanobacteria

We have developed several strain-specific, rapid, small-scale plasmid isolation procedures in order to characterize the plasmid profiles of 16 filamentous, nonheterocstous cyanobacteria. At least one distinct plasmid was found in eight strains, with seven of these containing two or more different plasmids. Eight strains were found to be without plasmid DNA. Both the large, 12.9 kb, and the small, 1.6 kb, plasmids fromPlectonema boryanum 581 were isolated, purified, and cloned. Southern blots of plasmid DNAs from the eight strains were probed with these cloned DNAs and also with ultra-pure plasmid DNA fromPhormidium liridum 426. Four strains ofP. boryanum (485, 581, 594, 1542) andP. luridum 426 have identical plasmid profiles, and plasmid homology is extensive.     

The enumeration ofStaphylococcus aureus in foods

Summary Staphylococcus aureus can be enumerated in foods by the use ofChapman’s special medium (3), under application of the drop plate technique, recommended byMiles andMisra (6). Colonies ofS.aureus so obtained can easily be macroscopically differentiated from aerobic sporeformers which grow also on this medium. The colonies can be used immediately for testing their coagulase reaction.     

Detection of the intercellular adhesion loci (ica) in clinicalStaphylococcus aureus strains responsible for hospital acquired auricular infection

In clinicalStaphylococcus aureus strains, the presence of theica genes, biofilm formation and susceptibility to antibiotics are considered important factors of virulence. In this study, 35 strains ofS. aureus, isolated from auricular infection, were investigated for slime production using Congo red agar (CRA) method, antibiotic susceptibility, presence ofmecA gene, and presence oficaA andicaD gene. The results show that 60% of strains weremecA positive when tested by PCR although 25.7% of strains were oxacillin resistant when tested with ATB STAPH. Qualitative slime production ofS. aureus using CRA revealed that 74.3% ofS. aureus were slime producers. All the strains carried theica gene.     

Resistance to cadmium salts and metal absorption by different microbial species

The present study evaluates the inhibitory activity and the absorption of cadmium (Cd) salts by different microbial species, including Gram-positive (Staphylococcus aureus andS. epidermidis), Gram-negative (Pseudomonas aeruginosa, Escherichia coli, Salmonella typhimurium, andProteus mirabilis) bacteria and one yeast (Candida albicans). The metal absorption by growing cells was considered both in liquid and in solid medium. For one strain ofP. aeruginosa the presence of Cd deposits inside the cell was investigated by transmission electron microscopy. Generally, the Gram-negative species tested proved to be highly resistant to Cd ions and accumulated great amounts of Cd during growth. Two strains ofP. aeruginosa showed a high degree of resistance to Cd and were particularly efficient in removing the metal from solutions. The Gram-positive bacteria showed a heterogeneous behavior: anS. aureus strain susceptible to Cd absorbed, at low metal concentrations, higher amounts of metal than a Cd-resistant one. The metal absorption for Gram-negative species was dose dependent, while for the Cd-resistant staphylococci it reached a plateau. Our results suggest that microorganisms can represent a good model to study the interactions between heavy metals and living organisms.     

Bacteroides gingivalis,Bacteroides asaccharolyticus,andBacteroides melaninogenicus subspecies: Cell surface morphology and adherence to erythrocytes and human buccal epithelial cells

All study strains ofBacteroides gingivalis, B. asaccharolyticus, andB. melaninogenicus subspecies possessed numerous pilus-like fibers and capsule-like outer surface structures. The capsular morphology varied between the different species and subspecies.B. gingivalis strongly agglutinated 16 erythrocyte species studied.B. asaccharolyticus showed variable and weak agglutination of only a few erythrocyte species.B. melaninogenicus subsp.intermedius strains strongly agglutinated rabbit erythrocytes and exhibited variable, often weak agglutination of 8 other erythrocyte species. Preparations of capsular polysaccharide or lipopolysaccharide fromB. gingivalis failed to agglutinate human erythrocytes, while pili preparations from the same organisms possessed marked hemagglutinating activity.B. gingivalis cells adhered in high numbers to human buccal epithelial cells, whereas strains ofB. asaccharolyticus failed to show measurable adherence. Oral strains ofB. melaninogenicus subsp.intermedius feebly adhered to the buccal epithelial cells. Pretreatment ofB. gingivalis cells with serum or saliva prevented the adherence to epithelial cells. Our findings suggest that cell surfaces with distinct properties exist on the various black-pigmentedBacteroides species and subspecies and this may accout for markedly differing ability of these organisms to attach to mammalian cells.     

Phage Type 187 as a Separate Subunit MboI Restriction Site Within the Staphylococcus aureus Species

The aim of this study was to use MboI restriction of pta gene fragment to compare the strains of Staphylococcus aureus phage type 187 and other phage types of S. aureus isolated from humans and dogs, as well as canine S. intermedius group strains. The study included 395 human and canine staphylococcal strains representing S. aureus, S. intermedius, and S. pseudintermedius species. The strains were identified with classic phenotypic methods and by the presence of species-specific thermostable nuclease (nuc SA) gene. All the strains were subjected to the analysis of MboI restriction site of pta gene fragment with PCR–RFLP method. Nearly, all human and animal strains of S. aureus possessed 156- and 164-bp restriction fragments. One of the human strains lacked the 320-bp amplification product. In the case of all S. aureus phage type 187, the amplification product of pta gene was insusceptible to cutting with MboI restrictase. None of S. intermedius strains possessed restriction sites present in the product of amplification of pta gene, while all the strains of S. pseudintermedius had 213- and 107-bp restriction fragments. In conclusion, our findings regarding S. aureus phage type 187 reveal that within the population of strains of S. aureus species, these bacteria represent a group with distinct properties.     

Multiplex PCR for simultaneous detection of enterococcal genes vanA and vanB and staphylococcal genes mecA, ileS-2 and femB

The experimental transfer of the vanA gene cluster from Enterococcus faecalis to Staphylococcus aureus has raised fears about the occurrence of such genetic transfer in clinical isolates of methicillin-resistant staphylococci. Recently, infections by a S. aureus strain carrying the enterococcal vancomycin resistance vanA gene cluster were reported. The possible emergence and dissemination of these strains is a serious health threat and makes optimization of prevention strategies and fast detection methods absolutely necessary. In the present study, we developed a PCR protocol for simultaneous detection of enterococcal vanA and vanB genes , the staphylococcal methicillin and mupirocin resistance markers mecA and ileS-2, and identification of S. aureus. As no vancomycin-resistant S. aureus isolates were available for our study, we used mixtures of enterococcal and staphylococcal colonies that harbored the different resistance markers to show that these genes could be detected simultaneously. This protocol could be used to facilitate the detection and identification of predictable S. aureus or methicillin-resistant strains carrying vanA or vanB.     

Absence of a role for lytic microorganisms in the decline of bacteria andSaccharomyces introduced into soil

The populations ofKlebsieila pneumoniae, Escherichia coli, Enterobacter aerogenes, andPseudomonas sp. fell following their addition to soil, but species lysing these gram-negative bacteria were not detected. The numbers ofStaphylococcus aureus andMicrococcus flavus fell by more than four orders of magnitude and ofSaccharomyces cerevisiae by more than two orders after their addition to soil. Organisms lysing these gram-positive bacteria were present in soil, but their numbers did not increase as a result of the additions. Lytic activity againstS. aureus was detected in soil filtrates, but this activity was not enhanced by inoculation of soil with the bacterium. Addition of cycloheximide to soil suspensions delayed the fall in abundance ofM. flavus but did not suppress the lytic populations. We conclude that lysis is not responsible for the decline of bacteria orS. cerevisiae added to soil.