Involvement of multiple genetic determinants in high-level methicillin resistance in Staphylococcus aureus.

A methicillin-susceptible, novobiocin-resistant strain of Staphylococcus aureus (RN2677; methicillin MIC, 0.8 micrograms/ml) was transformed with DNA prepared from highly and homogeneously methicillin-resistant S. aureus strains (methicillin MIC, greater than or equal to 400 micrograms/ml) or from heterogeneous strains in which the majority of cells had a low level of resistance (methicillin MIC, 6.3 micrograms/ml). All methicillin-resistant transformants showed low and heterogeneous resistance (methicillin MIC, 3.1 micrograms/ml) irrespective of the resistance level of DNA donors. All transformants examined produced normal amounts of the low-affinity penicillin-binding protein (PBP) 2a, and methicillin resistance and the capacity to produce PBP 2a showed the same degree of genetic linkage to the novobiocin resistance marker with both homogeneous and heterogeneous DNA donors. Next, we isolated a methicillin-susceptible mutant from a highly and homogeneously resistant strain which had a Tn551 insertion near or within the PBP 2a gene and thus did not produce PBP 2a. With this mutant used as the recipient, genetic transformation of the methicillin resistance gene was repeated with DNA isolated either from highly and homogeneously resistant strains or from heterogeneous (low-resistance) strains. All transformants obtained expressed high and homogeneous resistance and produced PBP 2a irrespective of the resistance level of the DNA donors. Our findings suggest that (i) the methicillin resistance locus is identical to the structural gene for PBP 2a, (ii) although the ability to produce PBP 2a is essential for resistance, the MICs for the majority of cells are not related to the cellular concentration of PBP 2a, and (iii) high MICs and homogeneous expression of resistance require the products of other distinct genetic elements as well.     

Nucleotide sequence and characterization of the Staphylococcus aureus norA gene, which confers resistance to quinolones.

The norA gene cloned from chromosomal DNA of quinolone-resistant Staphylococcus aureus TK2566 conferred relatively high resistance to hydrophilic quinolones such as norfloxacin, enoxacin, ofloxacin, and ciprofloxacin, but only low or no resistance at all to hydrophobic ones such as nalidixic acid, oxolinic acid, and sparfloxacin in S. aureus and Escherichia coli. The 2.7-kb DNA fragment containing the norA gene had a long open reading frame coding for 388 amino acid residues with a molecular weight of 42,265, which was consistent with the experimental value of about 49,000 obtained on DNA-directed translation. The deduced NorA polypeptide has 12 hydrophobic membrane-spanning regions and is partly homologous to tetracycline resistance protein and sugar transport proteins. The uptake of a hydrophilic quinolone, enoxacin, by S. aureus harboring a plasmid carrying the norA gene was about 50% that by the parent strain lacking the plasmid, but it increased to almost the same level as that by the latter strain with carbonyl cyanide m-chlorophenyl hydrazone. On the other hand, the uptake of a hydrophobic quinolone, sparfloxacin, was similar in the two strains. These results suggest that the NorA polypeptide may constitute a membrane-associated active efflux pump of hydrophilic quinolones.     

Cold-sensitive variants of Staphylococcus aureus and their stimulation by penicillins and cephalosporins.

Cold-sensitive (Cs) variants were obtained from ageing broth cultures of the Oxford strain of Staphylococcus aureus (NCTC 6571) and other penicillin-sensitive strains of this species. Growth of all strains was severely retarded on certain media at 30 degrees C but was stimulated by the addition of penicillins and cephalosporins. Revertants were derived which behaved as the parent cells did with respect to growth temperature requirements and response to these antibiotics.     

Studies of intracellular thymidine nucleotides. Relationship between the synthesis of deoxyribonucleic acid and the thymidine triphosphate pool in Escherichia coli K12.

The two types of mutant strains which show resistance to T-even phage infection have been isolated and been shown to have either a higher or lower ratio of dTDP-sugar to dTTP than that of the parent strains. The one with a higher ratio of dTDP-sugar to dTTP than the parents has a large dTDP-sugar pool and small dTTP pool, and a high level of dTDPG pyrophosphorylase activity. The other one, with a lower ratio of dTDP-sugar to dTTP than the parents, has a small dTDP-sugar pool and large dTTP pool, and a low or deficient level of this enzyme activity. They form an entirely mucoid colony in the synthetic agar plate. Mutant cells (Ter-6 and Ter-21) which have deficient dTDPG pyrophosphorylase activity show 2 -- 3 times higher activity of UDPG pyrophosphoyrlase than that of parent cells. The dTDPG pyrophosphorylase-deficient mutants (Ter-15 and Ter-21) have a 3 -- 4 times higher concentration of dTTP and a faster rate of DNA synthesis and cell division than those of parent strains in growth with external thymine. The dTDPG pyrophosphorylase constitutive mutant (Ter-4) has a 0.5 -- 0.33 smaller dTTP pool and a slower rate of DNA synthesis and cell division than those of parent cells grown in the same medium. In the Ter-15 and Ter-21 mutants, the intracellular dTTP-dependent DNA synthesis rapidly disappeared in thymine suboptimal concentration, but the Ter-4 mutant maintained its dTTP-dependent DNA synthesis over a 20 muM concentration of external thymine. In high concentration (100 muM) of external thymidine, the thymidine effects on the intracellular dTTP concentration do not significantly appear in these enzyme-deficient mutants (Ter-15 and Ter-21). Also, the concentration of intracellular dTTP in the cell growth with external thymidine is 2.5 times greater than that with external thymine in these enzyme-deficient mutants (Ter-15 and Ter-21).     

Significance of chromogenic variants in studies of virulence factors of Staphylococcus aureus

Parisi, Joseph T. (Duquesne University, Pittsburgh, Pa.). Significance of chromogenic variants in studies of virulence factors of Staphylococcus aureus. J. Bacteriol. 92:589-591. 1966.-Large numbers of chromogenic variants were isolated from cultures of a parent strain of Staphylococcus aureus growing in Brain Heart Infusion (Difco). The parent strain and four selected chromogenic variants were tested for either quantitative or qualitative differences in the production of extracellular substances associated with virulence. Quantitative differences were found in the ability of these strains to produce coagulase and hyaluronidase, whereas qualitative differences were found in the production of plate hemolysins, bound coagulase, opacity in an egg yolk medium, and a proteinase. In view of the rate and extent of the occurrence of these chromogenic variants, their presence in an inoculum could lead to inaccurate results in in vitro studies of staphylococcal virulence.     

Characteristics of Staphylococcus aureus, isolated from airways of cystic fibrosis patients, and their small colony variants

The colonization of respiratory tract by Staphylococcus aureus is a frequent feature of cystic fibrosis (CF), especially in pediatric patients. The formation of small colony variants (SCVs), which produce reduced amounts of alpha-toxin, is one of the proposed ways of staphylococcal accommodation in an intracellular niche. The aim of the present study was to compare some properties of S. aureus SCVs and their parent strains. A site-directed S. aureus hemB mutant and parent strain 8325-4 were included in the study (control pair). Normal and SCV strain pairs from CF patients as well as control strains were tested for the susceptibility to defensins, killing activity of professional phagocytes and adhesion to A549 cell line. Because S. aureus are exposed to many cationic proteins in the host, we challenged a clinical isolate with minimal subinhibitory concentration (subMIC) of protamine and found that hemin and menadione auxotrophic SCVs emerged. SCVs were more resistant than normal strains to protamine but not to dermaseptin. The susceptibility to the bactericidal activity of magainin was the same for normal and SCV strains. The protamine resistance of normal as well as SCVs was strongly enhanced by high salt concentration. The adhesion of some SCVs to A549 cells was higher than adhesion of parental strains. However, the number of adherent bacteria (SCVs) was diminished in the presence of hemin for hemin auxotrophs. The uptake of SCVs by granulocytes was lower than ingestion of normal strains, but SCVs were killed with equal or greater potency. SCVs are adapted to intracellular survival and persistence in the host under certain circumstances. The ability to form a variant subpopulation affords S. aureus additional survival options.     

Molecular analyses of conjugative, gentamicin-resistance plasmids from staphylococcal clinical isolates

Gentamicin-resistant Staphylococcus aureus and Staphylococcus epidermidis strains which were isolated from infants with staphylococcal bacteremia were analyzed for the presence of self-transmissible gentamicin-resistance (Gmr) plasmids. Conjugative GMr plasmids of approximately 43.8-63 kilobases (kb) were found in all S. aureus strains. Inter- and intra-species transfer of Gmr plasmids by conjugation was observed from S. aureus to S. aureus and to S. epidermidis recipient strains. However, neither inter- nor intra-species transfer of gentamicin resistance by conjugation was observed with nine out of nine S. epidermidis donor strains which were mated with either S. epidermidis or S. aureus recipient strains. These conjugative Gmr plasmids were unable to comobilize a smaller (15-kb) plasmid present in all but two S. aureus clinical isolates. Many of the conjugative Gmr plasmids also carried genetic determinants for kanamycin, tobramycin, neomycin, and ethidium bromide resistance, and for beta-lactamase synthesis. EcoRI restriction endonuclease digests of the S. aureus Gmr conjugative plasmids revealed three different digestion patterns. Four EcoRI restriction endonuclease digestion fragments of 15, 11.4, 6.3, and 4.6 kb in size were common to all plasmids. These plasmids and conjugative Gmr staphylococcal plasmids from other geographical regions shared restriction digestion fragments of similar molecular weights. DNA hybridization with biotinylated S. aureus plasmid pIZ7814 DNA revealed a high degree of homology among these plasmids. A 50.9-kb plasmid from one of the nonconjugative S. epidermidis clinical isolates showed homology with the probe DNA but lacked a portion of a 6.3-kb fragment which was present in all conjugative plasmids and believed to carry much genetic information for conjugation.     

Gamma-ray- and UV-sensitive strains of a radioresistant cell line: isolation and cross-sensitivity to other agents

Two gamma-ray-sensitive and two ultraviolet (UV)-sensitive variants were isolated from the gamma-ray- and UV-resistant TN-368 lepidopteran insect cell line. The isolation was performed by inducing mutations in the TN-368 cells using ethyl methanesulfonate, growing them for an expression period, irradiating with 137Cs gamma rays or 254-nm UV radiation, allowing cells to incorporate 5-bromodeoxyuridine (BrdU) in the presence of hydroxyurea (DNA repair synthesis), and finally irradiating with 365-nm UV radiation to cause DNA strand breakage at sites of BrdU incorporation with the intent of killing those cells that have undergone DNA repair synthesis and sparing those cells which, for a variety of reasons, did not. The survival of the Cs2 and Cs7 variants exposed to X rays is significantly different from the parent TN-368 line at the P less than 0.0001 level. The survival of the UV10 and UV19 variants exposed to UV radiation is different from the parent at the P less than 0.0001 and P less than 0.003 levels, respectively. In cross-sensitivity testing of the gamma-ray-sensitive variants, only Cs2 is more sensitive to 254-nm UV and only Cs7 is more sensitive to 44 degrees C heating; both are sensitive to PUVA. The UV-sensitive mutants are both sensitive to X irradiation, PUVA, and mitomycin C. However, UV10 is not sensitive to 44 degrees C heating while UV19 is, making UV19 the only variant strain sensitive to all agents examined. Despite the isolation procedure which was intended to select for DNA repair-deficient cells, the results suggest that a more general mechanism is responsible for the sensitivity of the variant cells to the agents tested.     

Rec-Mediated Recombinational Hot Spot Activity in Bacteriophage Lambda. I. Hot Spot Activity Associated with Spi- Deletions and bio Substitutions

In order to survey the distribution along the bacteriophage lambda chromosome of Rec-mediated recombination events, crosses are performed using conditions which block essentially all DNA synthesis. One parent is density-labeled and carries a genetic marker in the left terminal lambda gene (A), while the other parent is unlabeled and carries a genetic marker in the right terminal lambda gene (R). Both parents are deleted for the lambda recombination genes int and red, together with other recombination-associated genes, by virtue of either (1) a pure deletion or (2) a bio insertion-deletion. The distribution in a cesium density gradient of the resulting A+R+ recombinant phage reflects the chromosomal distribution of the recombination events which gave rise to those phage.Crosses employing either of two different pure deletion phage strains exhibit recombinational hot spot activity located near the right end of the lambda chromosome, between the cI and R genes. This hot spot activity persists when unlimited DNA synthesis is allowed. Crosses employing bio1-substituted phage strains exhibit recombinational hot spot activity located to the right of the middle of the chromosome and to the left of the cI gene. Crosses employing either bio1 or bio69-substituted phage strains indicate that the bio-associated hot spot activity occurs in the presence of DNA synthesis, but is dependent on a functional host recB gene.     

ADSRRS-fingerprinting and PCR MP techniques for studies of intraspecies genetic relatedness in Staphylococcus aureus

The present study was designed to evaluate the usefulness of two novel molecular typing methods, amplification of DNA fragments surrounding rare restriction sites (ADSRRS-fingerprinting) and the PCR melting profile (PCR MP), for Staphylococcus aureus strain differentiation. Thirty-seven S. aureus strains isolated from patients with a history of furunculosis were studied. The strains were identified by determining several phenotypic properties and were genotyped using three differentiation methods: macrorestriction analysis of the chromosomal DNA by pulsed-field gel electrophoresis (REA-PFGE), ADSRRS-fingerprinting, and PCR MP technique. In some cases the results obtained showed that the S. aureus isolated from the nose was identical to the one from the furuncle of the same patient. The same genotype was also identified for S. aureus strains isolated from two different members of a family with a history of recurrent furunculosis, although the active lesions were present in only one of them when the investigation was done. Results from strain genotyping illustrated that the recently developed ADSRRS-fingerprinting and PCR MP techniques are useful for studies of intraspecies genetic relatedness of S. aureus strains. They are as effective in discriminating closely related strains as the PFGE method, which is currently considered to be "the gold standard" for epidemiological studies.     

The innate immune modulators staphylococcal complement inhibitor and chemotaxis inhibitory protein of Staphylococcus aureus are located on beta-hemolysin-converting bacteriophages

Two newly discovered immune modulators, chemotaxis inhibitory protein of Staphylococcus aureus (CHIPS) and staphylococcal complement inhibitor (SCIN), cluster on the conserved 3' end of beta-hemolysin (hlb)-converting bacteriophages (betaC-phis). Since these betaC-phis also carry the genes for the immune evasion molecules staphylokinase (sak) and enterotoxin A (sea), this 8-kb region at the 3' end of betaC-phi represents an innate immune evasion cluster (IEC). By PCR and Southern analyses of 85 clinical Staphylococcus aureus strains and 5 classical laboratory strains, we show that 90% of S. aureus strains carry a betaC-phi with an IEC. Seven IEC variants were discovered, carrying different combinations of chp, sak, or sea (or sep), always in the same 5'-to-3' orientation and on the 3' end of a betaC-phi. From most IEC variants we could isolate active bacteriophages by mitomycin C treatment, of which lysogens were generated in S. aureus R5 (broad phage host). All IEC-carrying bacteriophages integrated into hlb, as was measured by Southern blotting of R5 lysogens. Large quantities of the different bacteriophages were obtained by mitomycin C treatment of the lysogens, and bacteriophages were collected and used to reinfect all lysogenic R5 strains. In total, five lytic families were found. Furthermore, phage DNA was isolated and digested with EcoR1, revealing that one IEC variant can be found on different betaI-phis. In conclusion, the four human-specific innate immune modulators SCIN, CHIPS, SAK, and SEA form an IEC that is easily transferred among S. aureus strains by a diverse group of beta-hemolysin-converting bacteriophages.     

A study of the polymorphism of mec dna in methycillin-resistant strains of Staphylococcus aureus isolated at permanent stations in different regions of Russia

Polymorphism of the chromosome staphylococcus cassette mec (SCCmec), a mobile and heterological genetic element providing resistance to beta-lactam antibiotics was studied in methycillin-resistant strains of Staphylococcus aureus (MRSA) isolated at permanent stations situated in different regions of Russia. Type SCCmec was identified using the PCR method by determining allotypes of 3 different structural genetic complexes incorporated in the cassettes mec. It was found that the isolates studied in this work contained 3 different types of SCCmec: I, III, and IVb. Both isolates containing 2 different copies of SCCmec and isolates containing defective copies of SCCmec were identified. It was demonstrated that determination of the SCC-mec type provided an opportunity to differentiate the isolates studied in this work from one another. The isolates attributed to the same genotype variant (identified by polymorphism of coagulase gene) but isolated at different hospitals located in different regions of Russia were found to contain the same type of the chromosome staphylococcus cassette mec, whereas the isolates of different coagulase groups (i.e., different genotype variants) contained different types of SCCmec. It was found that at least 2 epidemic strains circulated in the permanent hospitals of Russia. The strains differ from one another by the polymorphism of the coagulase gene and the mec DNA polymorphism. According to results of studies of several molecular markers (including mec DNA), these strains proved to be identical to the international strains EMRSA-1 and EMRSA-2. Possible mechanisms of MRSA formation and circulation in Russia and CIS countries are discussed.     

利用RAPD技术检测香菇双—单杂交后代

利用ＲＡＰＤ技术对香菇６个双单杂交菌株及其两个双核体基因组ＤＮＡ进行了检测,结果显示：１－３号杂交菌株之间的相似系数为０．８９３－０．９６２,它们与其亲本Ｓ之间的相似系数为０．８４２－０．８５９;４－６号杂交菌株之间的相似系数０．８５７－０．９２５,与其亲本ＳＳＯ１之间相似系数为０．７０８－０．９０２;表明杂交菌株与其双核体亲本基因组具有较大差异,杂交菌株之间存在着不同程度差异,６个杂交菌株是真正     

Jumping the barrier to beta-lactam resistance in Staphylococcus aureus

Although the staphylococcal methicillin resistance determinant, mecA, resides on a mobile genetic element, staphylococcus cassette chromosome mec (SCCmec), its distribution in nature is limited to as few as five clusters of related methicillin-resistant Staphylococcus aureus (MRSA) clones. To investigate the potential role of the host chromosome in clonal restriction of the methicillin resistance determinant, we constructed plasmid pYK20, carrying intact mecA, and introduced it into several methicillin-susceptible Staphylococcus aureus strains, five of which were naive hosts (i.e., mecA not previously resident on the host chromosome) and five of which were experienced hosts (i.e., methicillin-susceptible variants of MRSA strains from which SCCmec was excised). We next assessed the effect of the recipient background on the methicillin resistance phenotype by population analysis, by assaying the mecA expression of PBP2a by Western blot analysis, and by screening for mutations affecting mecA. Each experienced host transformed with pYK20 had a resistance phenotype and expressed PBP2a similar to that of the parent with chromosomal SCCmec, but naive hosts transformed with pYK20 selected against its expression, indicative of a host barrier. Either inducible beta-lactamase regulatory genes blaR1-blaI or homologous regulatory genes mecR1-mecI, which control mecA expression, acted as compensatory elements, permitting the maintenance and expression of plasmid-carried mecA.     

Rat hepatoma cell variants resistant to insulin-diphtheria toxin A fragment conjugates. Genetic evidence for the separate pathways for insulin receptor-mediated mitogenic and hormonal stimulation

The rat hepatoma H35 cells in serum-free medium produce tyrosine aminotransferase (TAT) and initiate DNA synthesis and cell division upon exposure to 10(-9)-10(-10) M insulin. This insulin-dependent hormonal and mitogenic stimulation is through the insulin receptors and not through the receptors for insulin-like growth factor type I. We have isolated genetic variants of H35 cells which are resistant to a cytotoxic insulin-diphtheria toxin A fragment conjugate. These variants showed different degrees of insulin binding capacity and exhibited different sensitivities to insulin-stimulated TAT and DNA synthesis. Variant DTaI-b had a slight decrease in number of insulin receptors but completely lost sensitivity to both TAT and DNA stimulation. Variant 11-1 had a reduced number of insulin receptors but retained both TAT and DNA inducibilities. Variant 14-1, which had a high number of insulin receptors, was not responsive to DNA synthesis but was responsive to TAT induction. The beta-subunit of insulin receptors in these cell variants had different sensitivities to their insulin-dependent autophosphorylation. The rat hepatoma HTC cells used as a control showed very low insulin binding, no stimulation of TAT and DNA synthesis by insulin, and no detectable insulin-enhancement of beta-subunit phosphorylation. These characteristics provide genetic evidence for the divergence of the insulin receptor-mediated mitogenic and hormonal signals during the post-receptor pathways which is apparently regulated by the insulin-dependent phosphorylation.     

An evaluation of the Drosophila zeste somatic eye mutation test for the detection of genotoxic azo dyes and an aromatic amine

The rate of DNA synthesis was studied in normal cell strain and in strains from patients suffering from inherited disorder ataxia telangiectasia (AT). After exposure to reactively low doses of oxic X-rays (0–4 krad) DNA synthesis was depressed in AT cell strains to a significantly lesser extent than in normal cells. This response was observed in both an “excision-deficient” and an “excision-proficient” strain. In contrast, there was no difference in DNA-synthesis inhibition between AT and normal cells after UV exposure. After X-irradiation of cells from patients with xeroderma pigmentosum, both complementation group A and XP variants, the observed rate of DNA synthesis was equal to that in normal cells. An exception was the strain XP3BR which has been shown to be X-ray sensitive. This strain exhibited diminished DNA synthesis inhibition after X-ray doses below 1 krad.These data suggest a relationship between hypersensitivity to X-rays and diminished depression of DNA synthesis.     

A genetic and monoclonal analysis of high-yielding reassortants of influenza A virus used for human vaccines

This paper describes two methods of analysis using monoclonal antibodies and RNA hybridization to characterize variation in the haemagglutinins of seven high-yielding influenza virus reassortants used for inactivated vaccine production. The results show that variants' were selected in producing these genetic reassortants. The haemagglutinins of two reassortants showed both antigenic and structural differences from their wild-type (wt) parents as detected by the two methods of analysis. These variants were more closely related to other subtype strains which had previously been differentiated from the wt parent by post-infection ferret sera and which also had amino acid sequence differences in antigenically significant sites on the HA 1 polypeptide chain of the haemagglutinin molecule. The haemagglutinins of four of the seven reassortants showed antigenic differences but no apparent structural differences from their wt parents. The haemagglutinin of only on reassortant was antigenically and structurally identical to its wt parent. The variants could not be reliably distinguished with hyperimmune rabbit serum or immune ferret serum to the wt parent virus. It is therefore important to use more discriminatory tests to identify influenza strains correctly. It is also essential for vaccine strains to be as completely characterized as possible. It is considered desirable that both methods of analysis be used to characterize influenza virus reassortant strains.