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<p>Methicillin-resistant S. aureus (MRSA) are considered multiply antibiotic-resistant and therapeutic options for infections caused by MRSA are seriously limited. MRSA cause greater human mortality than HIV/AIDS in the US. Given the real threat posed by multiply antibiotic-resistant S. aureus, aspects of this organisms biology including mechanisms of antimicrobial resistance and physiology are currently being investigated, in part to support the search for new antistaphylococcal agents. S. aureus is also a common livestock pathogen and cause of food-poisoning. MRSA transmission has been reported to occur between animals, and from animals to humans and visa-versa. MRSA carriage by livestock also correlates with the MRSA colonization of farm families and veterinarian. To date, S. aureus has been isolated from horses, sheep, pigs, dairy cows, veal calves and fowl. S. aureus is also a major cause of bovine mastitis responsible for substantial dairy industry economic loss and one of the most common agents implicated in food poisoning worldwide.Hand disinfection with alcohol-based hand rubs (ABHR) is associated with a reduction in S. aureus infections. The bactericidal affect of alcohol is attributed to its ability to denature proteins and membrane structures. Tea tree oil (TTO) is a popular plant-derived disinfectant/antiseptic that exhibits a mechanism of bactericidal action similar to alcohol. Certain strains of S. aureus exhibit a small colony variant (SCV) phenotype which are of particular concern since they are linked to recurrent, chronic and antibiotic-resistant infections. We have isolated and partially characterized TTO-reduced susceptibility SCV mutants of S. aureus (TTOSCV) that demonstrate impaired growth, reduced cell diameter, a thinned peptidoglycan layer and reduced susceptibility to alcohols, TTO, TTO terpenes, and the cell wall active antibiotics oxacillin, and vancomycin. The TTOSCV mutants analyzed all have a mutation in acpP which encodes the acyl carrier protein (ACP) which is an essential component of fatty acid synthase II and required for fatty acid biosynthesis in bacteria. We have also isolated two SCV-acpP mutants with ethanol (ETHSCV), which leads us to hypothesis that ethanol can select for SCVs that exhibit altered susceptibility to multiple antimicrobials and harbor acpP mutations.The goals of this proposal are: 1. to characterize S. aureus SCVs selected with ethanol; 2. compare the relative levels of apo-ACP and holo-ACP in TTORS and ETHRS mutants and the parent strain and; 3. determine if acpP mutations are responsible for the TTOSCV and ETHSCV phenotypes.Because of the importance of ABHR in diminishing the spread of infectious disease, investigating mechanisms that bacteria utilize to withstand the cidal action of alcohol or related biocides is worthy of investigation. The long term goal of this research is to investigate mechanisms that S. aureus employs to survive in the presence of alcohol and our findings and strains produced may be utilized to improve ABHR formulation and effectiveness. This work will also begin to analyze the role that acpP has on clinically-relevant characteristics of a major livestock and human pathogen. </p>
<p>a. Ethanol-reduced susceptibility (ERS) mutants of S. aureus. Gradient plates expose S. aureus populations to an antimicrobial gradient, so this provides a greater possibility of producing a "goldilocks" ERS mutant selection concentration.Gradient plates will be prepared as previously described utilizing MHA and square petri plates. After drying the first tilted MHA layer, the second MHA layer infused with 20 % filtered (0. 2 µm Nylon, Fisher Scientific, Pittsburgh, PA) ethanol will be poured onto the first layer and allowed to dry (4 hr). One hundred ?l of SH1000 or JE2 overnight cultures will then be spread onto the plates which will be incubated (48 hr). Ten colonies that appear at the highest ethanol concentration will then be selected using toothpicks and spread onto drug-free MHA plates which will be incubated (24 hr). Following overnight growth we will screen for SCV variants and all putative ERS mutants (stored at -80°C) will be characterized.b. TTOSCV, RV and ETHSCV mutants/revertants and ERS mutant analysis. Ethanol, isopropanol and TTO used for MIC and MBC determination will filter sterilized. MICs and MBCs for all liquid antimicrobials will be determined in screw top test tubes to prevent evaporation. Growth in all tubes will be initiated by combining 1 ml of diluted overnight MHB cultures (final OD625 = 0.01) with 1 ml of MHB containing the antimicrobial analyzed. MICs will be visually determined after 24 h static incubation at 37ºC. MBCs will be determined by spotting 5 ?l aliquots from the MIC and all tubes above the MIC (previously vortexed for 5 sec to resuspend cells) onto MHA and scoring for visible growth following 24 h incubation. MICs/MBCs for will be determined utilizing 0.025% to 0.25% v/v TTO, in 0.025% increments and 3% to 17%, v/v ethanol and isopropanol in 1% increments. Vancomycin, oxacillin and triclosan MICs and MBCs will be performed according to CLSI guidelines as previously described. All confirmed ERS mutants will then be further characterized.Vancomycin resistance population analyses will be performed as previously described. All isolates will initially be grown overnight in 2 ml MHB, and then serially diluted in sterile MHB. Ten ?l aliquots of all dilutions will then be pipetted at separate positions on the surface of MHA containing various concentrations of vancomycin and allowed to absorb into the agar. Plates will then be incubated (48 h) and CFUs within the dilution positions will be scored.Growth curves (200 rpms, 37°C) will be performed in 20 ml MHB cultures inoculated with overnight cultures to reach an initial OD580 of 0.01, and the OD580 will be monitored for 48 h.acpP amplicon sequencing will be performed as previously described with forward (GGA GGT GAA TCG ACG TGG) and reverse primers (GTC GAC AAT ACT GAC GAC CCA G) [37]. All PCR mixtures will then be loaded into a 1% agarose gels and the acpP amplicons will be gel purified using a Wizard® SV Gel and PCR Clean-Up System kit (Promega, Madison, WI). The PCR isolates will then be sequenced utilizing an ABI/Hitachi 3730 DNA Analyzer using POP-7 polymer and BigDye (R) v3.1 reagents (Life Technologies, Foster City, CA).Staphyloxanthin production will be assessed as previously described. Initially cells will be harvested from MHB cultures (OD580 = 1.0) and washed in cold (4°C) distilled water. The cell pellets will then be extracted with 55°C methanol (3 min) and the OD465 will be measured to estimate the carotenoid content, and related to bacterial dry weight from culture OD580 measurements.Preparation and fixing of S. aureus samples for scanning- and transmission-electron microscopy (SEM and TEM) will be carried out within the OSU Microscopy Laboratory. SEM will be used to determine the cell diameter and TEM will be utilized to determine the cell wall width, of 100 individual cells of each strain investigated.c. ACP immunoblotting. Initially 20 ml MHB cultures of SH1000, the three SH1000-TTOSCV and the two ETHSCV mutants will be grown to an A6oo = 0.7, and the cells will be harvested by centrifugation (4,000 X g, 5 min) and the cell pellets will be washed twice with MHB medium containing 10 mg/ml bovine serum albumin. The cell pellets will then be reharvested and the cells will be resuspended in 110 μl of phosphate-buffered saline containing 1 mg/ml lysostaphin, 0.1 mg/ml DNase I and a protease inhibitor cocktail before incubation on ice for 3 hr. The extracts will then be centrifuged at 20,000 X g for 15 min and the supernatant will be removed and the protein content will be quantified using the Bradford assay. Portions of the cell lysates (10 μg) will then be electrophoresized in a 2.5 M urea, 15% acrylamide conformationally sensitive gel and the separated proteins will then be transferred to a polyvinylidene difluoride membrane by electroblotting. apo-ACP and holo-ACP standards will also be included on this gel. The primary anti-ACP antibody will then be used at 1:500 dilution and secondary anti-rabbit IgG conjugated with alkaline phosphatase at a 1:5000 dilution. The blot will then be developed using the ECF substrate developer (GE Healthcare) and the fluorescent signals will be recorded using a Typhoon PhosphoImager 9500 and compared.d. Wild type acpP replacement with acpP-A34D and acpP-A34N and complementation of acpP mutation in TTOSCV and ETHSCV mutants. The replacement plasmid pKOR1 employs antisense secY RNA expression for counter-selection and a lambda recombination cassette (Gateway Technology, Invitrogen) that permits rapid cloning of genes without the use of restriction enzymes and ligases.Initially, the acpP-A34D and acpP-A34N mutant gene and ~ 100 bps up and downstream of these genes will be PCR amplified from SH1000-TTOSCV-1 and 8-4-ETHSCV chromosomal DNA with the appropriate attB1 and attB2 sequences and BamHI linker sequences. These amplions will then be digested with BamHI and ligated with T4 DNA ligase and inserted into pKOR1 utilizing the BP clonase enzyme mix (Invitrogen) and transformed into Escherichia coli DH5α. Plasmid DNA containing the acpP-A34D and acpP-A34N mutated genes will then be isolated and electroporated [102]first into S. aureus strain RN4220 to modify the DNA and then subsequently into SH1000 and JE2. Plasmid integration into the chromosome of SH1000 and JE2 will then be induced by growth at the non-permissive temperature of 43°C for pKOR1. Anhydrotetracycline induction of the pKOR1 encoded secY antisense transcript via the Pxyl/tetO promoter will then cause the excision of the integrated plasmid. The replacement of the wildtype acpP in SH1000 and JE2 with the integrated acpP-A34D and acpP-A34N versions will then be confirmed by acpP amplication and sequencing.acpP will be PCR-amplified from SH1000 and cloned into the EcoRI site upstream of the anhydrotetracycline-inducible promoter within the E. coli-S. aureus shuttle vector expression plasmid pALC2073. The correct acpP orientation in reference to the pALC2073 anhydrotetracycline promoter and acpP sequence, will be confirmed by PCR amplification and sequencing. pALC2073 and pALC2073::acpP will then be electroporated first into S. aureus strain RN4220 to modify the DNA and both plasmids will then be isolated from RN4220. SH1000-TTOSCV-1 and ETHSCV will then be transformed with the modified pALC2073 and pALC2073::acpP to produce shuttle vector control strains and complemented strains SH1000-TTOSCV-1-pALC2073::acpP and ETHSCV-pALC2073::acpP. Both pALC2073 and pALC2073::acpP constructs will be isolated from the control and complemented strains to confirm their sequences. </p>