Genetic Pathway in Acquisition and Loss of Vancomycin Resistance in a Methicillin Resistant Staphylococcus aureus (MRSA) Strain of Clonal Type USA300

An isolate of the methicillin-resistant Staphylococcus aureus (MRSA) clone USA300 with reduced susceptibility to vancomycin (SG-R) (i.e, vancomycin-intermediate S. aureus, VISA) and its susceptible “parental” strain (SG-S) were recovered from a patient at the end and at the beginning of an unsuccessful vancomycin therapy. The VISA phenotype was unstable in vitro generating a susceptible revertant strain (SG-rev). The availability of these 3 isogenic strains allowed us to explore genetic correlates of antibiotic resistance as it emerged in vivo. Compared to the susceptible isolate, both the VISA and revertant strains carried the same point mutations in yycH, vraG, yvqF and lspA genes and a substantial deletion within an intergenic region. The revertant strain carried a single additional frameshift mutation in vraS which is part of two component regulatory system VraSR. VISA isolate SG-R showed complex alterations in phenotype: decreased susceptibility to other antibiotics, slow autolysis, abnormal cell division and increased thickness of cell wall. There was also altered expression of 239 genes including down-regulation of major virulence determinants. All phenotypic properties and gene expression profile returned to parental levels in the revertant strain. Introduction of wild type yvqF on a multicopy plasmid into the VISA strain caused loss of resistance along with loss of all the associated phenotypic changes. Introduction of the wild type vraSR into the revertant strain caused recovery of VISA type resistance. The yvqF/vraSR operon seems to function as an on/off switch: mutation in yvqF in strain SG-R turns on the vraSR system, which leads to increase in vancomycin resistance and down-regulation of virulence determinants. Mutation in vraS in the revertant strain turns off this regulatory system accompanied by loss of resistance and normal expression of virulence genes. Down-regulation of virulence genes may provide VISA strains with a “stealth” strategy to evade detection by the host immune system.     

Morphofunctional alterations in testicular cells of deslorelin-treated boars: an immunohistochemical study

In this study we thoroughly scrutinized testes morphology and investigated whether treatment of recipient boars with gonadotropin-releasing hormone (GnRH)-agonist deslorelin could alter the expression of 3beta-hydroxysteroid dehydrogenase (3beta-HSD), luteinizing hormone receptors (LHRs), and androgen receptors (ARs) in testicular cells. An implant containing 4.7 mg of the GnRH-agonist deslorelin was subcutaneously inserted into crossbred male pigs at 91 and 147 days of age. Testicular traits, morphology of the testes, the proteins' expression, and testosterone concentration in blood plasma were analyzed in all boars after slaughter at 175 days of age. Histological analysis revealed significant alterations in both the interstitial tissue and seminiferous tubules of experimental animals after 28 and 84 days of deslorelin treatment. The intensity of the AR immunostaining within the testis appeared as a function of the severity of testicular dysgenesis. Time-dependent action of deslorelin on the expression of LHR and 3beta-HSD in Leydig cells was also detected. Staining for LHR and 3beta-HSD was very weak or the Leydig cells were immunonegative. Concomitantly, a significant decrease in plasma testosterone level was found in both groups of deslorelin-treated boars when compared with the control group. This is the first report showing the cellular distribution of AR, LHR, and 3beta-HSD in testicular cells of deslorelin-treated boars. It is concluded that morphological and immunohistochemical studies are important for the evaluation of testicular histoarchitecture and steroidogenic function. Subsequently, the endocrine control of reproduction in the GnRH-agonist deslorelin-treated males will be better understood.     

Interaction of the antitumor antibiotic mitomycin C with Z-DNA

Mitomycin C (MC), an antitumor antibiotic, alkylated Z-DNAs such as poly(dG-dC)/Co(NH3)3+(6), poly(dG-m5dC)/Mg2+ and brominated poly(dG-dC) upon reductive activation. Computer-generated energy-minimized molecular models indicated that monofunctional alkylation of Z-DNA at the N2-position of guanine by MC did not distort Z-DNA geometry, but bifunctional alkylation, leading to interstrand crosslinks between two N2-positions of guanine was sterically unfavorable. The above three Z-DNA's were exposed both to monofunctionally and bifunctionally activated MC in separate experiments and the resulting covalent MC-polynucleotide complexes were examined for conformation and for covalent MC-adducts, by circular dichroism (CD) spectroscopy and HPLC analysis of nuclease digests, respectively. Monofunctionally activated MC alkylated all three polynucleotides in their Z-forms, resulting in the same monofunctional N2-guanine adduct as that known to be formed with B-DNA. Upon bifunctional activation of MC, poly(dG-dC/Co(NH3)3+(6) reverted to the B-form and bifunctional (cross-link) adducts were detected, identical again with those formed with B-DNA. Poly(dG-m5dC), however, remained in the Z-form after the alkylation and only a monofunctional adduct could be detected. It was concluded that Z-DNA is subject to monofunctional alkylation by MC but cannot be cross-linked. The latter process occurs only when the Z-DNA is labile enough [as is in the case of poly(dG-dC)] to have some B-form in equilibrium at the site of the first formed monolinked adduct; the cross-linking then occurs at such local B-sites, pulling the overall B in equilibrium Z equilibrium irreversibly to the left. These results are in accord with the predictions from the above modeling. The irreversible "lock" by the MC cross-link on B-DNA may be exploited for probing Z-DNA intermediacy in various DNA functions.     

Separation of abnormal cell wall composition from penicillin resistance through genetic transformation of Streptococcus pneumoniae.

Compared with most penicillin-susceptible isolates of Streptococcus pneumoniae, penicillin-resistant clinical isolate Hun 663 contains mosaic penicillin-binding protein (PBP) genes encoding PBPs with reduced penicillin affinities, anomalous molecular sizes, and also cell walls of unusual chemical composition. Chromosomal DNA prepared from Hun 663 was used to transform susceptible recipient cells to donor level penicillin resistance, and a resistant transformant was used next as the source of DNA in the construction of a second round of penicillin-resistant transformants. The greatly reduced penicillin affinity of the high-molecular-weight PBPs was retained in all transformants through both genetic crosses. On the other hand, PBP pattern and abnormal cell wall composition, both of which are stable, clone-specific properties of strain Hun 663, were changed: individual transformants showed a variety of new, abnormal PBP patterns. Furthermore, while the composition of cell walls resembled that of the DNA donor in the first-round transformants, it became virtually identical to that of susceptible pneumococci in the second-round transformants. The findings indicate that genetic elements encoding the low affinity of PBPs and the penicillin resistance of the bacteria are separable from determinants that are responsible for the abnormal cell wall composition that often accompanies penicillin resistance in clinical strains of pneumococci.     

Sigma-B, a putative operon encoding alternate sigma factor of Staphylococcus aureus RNA polymerase: molecular cloning and DNA sequencing.

We have identified a gene cluster located on the chromosomal SmaI I fragment of a highly methicillin resistant strain of Staphylococcus aureus, consisting of four open reading frames (ORFs), named after the number of deduced amino acid residues, in the sequential order orf333-orf108-orf159-orf256. The gene cluster showed close similarities to the Bacillus subtilis sigB operon both in overall organization and in primary sequences of the gene products. The complete gene cluster (provisionally named sigma-B or sigB) was preceded by an sigmaA-like promoter (PA) and had an internal sigmaB-like promoter sequence (PB) between orf333 and orf108, suggesting a complex regulatory mechanism. The polypeptides encoded by orf333, -108, -159, and -256 showed 62, 67, 71, and 77% homologies, respectively, with the RsbU, RsbV, RsbW, and SigB polypeptides encoded by the B. subtilis sigB operon. A Tn551 insertional mutant, RUSA168 (insert in orf256 of the staphylococcal sigma-B operon), showed drastic reduction in methicillin resistance (decrease in MIC from 1,600 microg ml-1 to 12 to 25 microg ml-1off     

The Comparison of the Effects of Three Physiotherapy Techniques on Hamstring Flexibility in Children: A Prospective,Randomized, Single-Blind Study

The aim of the study was to evaluate changes in hamstring flexibility in 120 asymptomatic children who participated in a 6-week program consisting of one physiotherapy session per week and daily home exercises. The recruitment criteria included age (10–13 years), no pain, injury or musculoskeletal disorder throughout the previous year, physical activity limited to school sport. Subjects were randomly assigned to one of the three groups: (1) post-isometric relaxation – PIR (n = 40), (2) static stretch combined with stabilizing exercises – SS (n = 40) and (3) stabilizing exercises – SE (n = 40). Hamstring flexibility was assessed with straight leg raise (SLR), popliteal angle (PA) and finger-to-floor (FTF) tests. The examinations were conducted by blinded observers twice, prior to the program and a week after the last session with the physiotherapist. Twenty-six children who did not participate in all six exercise sessions with physiotherapists were excluded from the analysis. The results obtained by 94 children were analyzed (PIR, n = 32; SS, n = 31; SE, n = 31). In the PIR and SS groups, a significant (P<0.01) increase in SLR, PA, FTF results was observed. In the SE group, a significant (P<0.001) increase was observed in the SLR but not in the PA and FTF (P>0.05). SLR result in the PIR and SS groups was significantly (P<0.001) higher than in the SE group. As far as PA results are concerned, a significant difference was observed only between the SS and SE groups (P = 0.014). There were no significant (P = 0.15) differences regarding FTF results between the three groups. Post-isometric muscle relaxation and static stretch with stabilizing exercises led to a similar increase in hamstring flexibility and trunk forward bend in healthy 10–13-year-old children. The exercises limited to straightening gluteus maximus improved the SLR result, but did not change the PA and FTF results.     

Large BRCA1 and BRCA2 genomic rearrangements in Polish high-risk breast and ovarian cancer families

BRCA1 and BRCA2 are two major genes associated with familial breast and ovarian cancer susceptibility. In Poland standard BRCA gene test is usually limited to Polish founder BRCA1 mutations: 5382insC, C61G and 4153delA. To date, just a few single large genomic rearrangements (LGRs) of BRCA1 gene have been reported in Poland. Here we report the first comprehensive analysis of large mutations in BRCA1 and BRCA2 genes in this country. We screened LGRs in BRCA1 and BRCA2 genes by multiplex ligation-dependent probe amplification in 200 unrelated patients with strong family history of breast/ovarian cancers and negative for BRCA1 Polish founder mutations. We identified three different LGRs in BRCA1 gene: exons 13-19 deletion, exon 17 deletion and exon 22 deletion. No LGR was detected in BRCA2 genes. Overall, large rearrangements accounted for 3.7 % of all BRCA1 mutation positive families in our population and 1.5 % in high-risk families negative for Polish founder mutation.     

Low sequence similarity and gene content of symbiotic clusters of Bradyrhizobium sp. WM9 (Lupinus) indicate early divergence of “lupin” lineage in the genus Bradyrhizobium

Two sequenced nodulation regions of lupin Bradyrhizobium sp. WM9 carried the majority of genes involved in the Nod factor production. The nod region I harbored: nolA, nodD, nodA, nodB, nodC, nodS, nodI, nodJ, nolO, nodZ, fixR, nifA, fixA, nodM, nolK and noeL. This gene arrangement resembled that found in the nodulation region of Bradyrhizobium japonicum USDA110, however strain WM9 harbored only one nodD gene copy, while the nodM, nolK and noeL genes had no counterparts in the 410 kb symbiotic region of strain USDA110. Region II harbored nolL and nodW, but lacked an nodV gene. Both regions carried ORFs that lacked similarity to the published USDA110 sequences, though they had homologues in symbiotic regions of Rhizobium etli, Sinorhizobium sp. NGR234 and Mesorhizobium loti. These differences in gene content, as well as a low average sequence identity (70%) of symbiotic genes with respect to B. japonicum USDA110 were in contrast with the phylogenetic relationship of USDA110 and WM9 revealed by the analysis of 16S rDNA and dnaK sequences. This most likely reflected an early divergence of symbiotic loci, and possible co-speciation with distinct legumes. During this process the loss of a noeI gene and the acquisition of a nolL gene could be regarded as an adaptation towards these legumes that responded to Nod factors carrying 4-O-acetylfucose rather than 2-O-methylfucose. This explained various responses of lupins and serradella plants to infection by mutants in nodZ and nolL genes, knowing that serradella is a stringent legume while lupins are more promiscuous legumes. This revised version was published online in August 2006 with corrections to the Cover Date.