NOVEL DISINFECTANT/ANTISEPTIC-SELECTED ANTIMICROBIAL-REDUCED SUSCEPTIBILITY MECHANISM IN STAPHYLOCOCCUS AUREUS.

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: TERMINATED
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 1006570
• Project No.: OKL03002
• Project Start Date: Oct 1, 2015
• Project End Date: Sep 30, 2020

Project Director
Gustafson, JO.

Recipient Organization
OKLAHOMA STATE UNIVERSITY
(N/A)
STILLWATER,OK 74078

Performing Department
Biochemistry & Molecular Biology

Non Technical Summary
The research proposed will characterize a novel multiple antimicrobial resistance mechanism discovered in a common pathogen of livestock and humans. The methods utilized in this study are designed to characterize the complex bacterial phenotype expressed by these laboratory-selected mutants. Common methods employed will include general bacteriology aseptic techniques, antimicrobial (e.g. ethanol) reduced susceptibility selection, measurement of pigment production, antibiotic susceptibility testing and electron microscopy to investigate cell wall alterations and cell size in the mutants. We will also employ specific techniques required to measure the modification of an essential enzyme required for bacterial fatty acid synthesis. In addition, gene specific inactivation and replacement techniques will also be used to created specific mutants of a bacterial pathogen to investigate mutations proposed to affect fatty acid synthesis. Understanding this multiple antimicrobial resistance mechanism at the molecular level will help identify targets in bacterial pathogens for the development of future antibiotics. We will also acquire a better understanding of essential pathways required by bacterial pathogens to cause disease.

Animal Health Component

0%

Research Effort Categories

Basic

100%

Applied

(N/A)

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
712	4010	1100	100%

Knowledge Area
712 - Protect Food from Contamination by Pathogenic Microorganisms, Parasites, and Naturally Occurring Toxins;

Subject Of Investigation
4010 - Bacteria;

Field Of Science
1100 - Bacteriology;

Keywords

staphylococcus aureus

antimicrobial resistance

fatty acid biosynthesis

ethanol

small colony variant

Goals / Objectives
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.

Project Methods
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.

Progress 10/01/15 to 09/30/20

Outputs
Target Audience:Researchers interested in antibiotic and antiseptic/disinfectant reduced susceptibility in bacterial pathogens. Changes/Problems:None since last report - once again, it is hoped that the experiments conducted in 2020 will provide enough data to successfully acquire external competitive research funding. What opportunities for training and professional development has the project provided?Thus far, 8 undergraduates and 1 Ph.D. student have benefitted from this project. A Masters's student from Microbiology and Molecular Genetics also benefited from this project. Dr. Nathanial Torres graduated and has taken up a postdoctoral position at the University of Southern Florida. This work is also helping to train an OSU postdoctoral student. How have the results been disseminated to communities of interest?We have presented some of our work at the Oklahoma Academy of Sciences meetings and the American Society for Biochemistry and Molecular Biology annual meeting. We have published results of our studies in the Canadian Journal of Microbiology and the journal Antibiotics. What do you plan to do during the next reporting period to accomplish the goals?Publish on the ethanol-selected mutants of S. aureus. We have completed the proteomics and mutational analysis of SCV-ERS mutants which are presently being analyzed and the results are being prepared for publication with the help of 2 undergraduate students. This publication will require additional work by a postdoctoral student who is determining if the SCV-ERS mutants have an iron deficiency. We will also determine the importance of the identified mutations in both SCV-ERS SH1000 mutants to determine which mutation(s) are important for the expression of the SCV-ERS phenotype.

Impacts
What was accomplished under these goals? Staphylococcus aureus is a target for alcohol-based hand rub (ABHR) antisepsis procedures that are used in part to prevent infections caused by this organism. Utilizing an in vitro approach, we show that selection with growth-inhibitory concentrations of ethanol produced stable S. aureus mutants that demonstrated a small colony variant (SCV) phenotype, ethanol reduced susceptibility (ERS), and slowed growth. SCV-ERS mutants were selected from a clinical vancomycin-intermediate S. aureus (VISA) strain MM66, a vancomycin-susceptible methicillin-resistant strain LP9, and the standard laboratory antibiotic susceptible strain SH1000. In a strain-dependent manner, the SCV-ERS mutants also demonstrated altered susceptibility to the mechanistically unrelated antimicrobials, isopropanol, tea tree oil, triclosan, oxacillin, and vancomycin. All SCV-ERS mutants derived from SH1000 and LP9 demonstrated reduced susceptibility to the primary antistaphylococcal drug vancomycin. On the other hand, all SCV-ERS mutants derived from the VISA MM66 parent demonstrated increased susceptibility to vancomycin. This study demonstrates that ethanol can select for S. aureus SCV-ERS mutants which express altered susceptibility to multiple antimicrobials and should provide an incentive for a widespread investigation on alcohol susceptibility in clinical S. aureus isolates. We have completed the proteomic and mutational analysis of select MM66 and SH1000 SCV-ERS mutants. In the proteomics study, we have identified all proteins that were increased or decreased 2-fold in production in the SCV-ERS mutants compared to their parent strains. From this study, we have identified a number of cell wall metabolism proteins including cell wall degradative proteins (e.g. LytM, Alt) that were upregulated in the SCV-ERS mutants in a strain-dependent manner. Whole-cell autolysis assays indicated that the autolysis of MM66 SCV-ERS mutants compared to MM66 was slower, while the autolysis of SH1000 SCV-ERS mutants was comparable to parent strain SH1000. We also noticed the upregulation of a number of proteins (e.g. SbnB, SbnC, SbnE, and SbnF) involved with the production of staphyloferrin B which is intimately involved with iron sequestration in SH1000 SCV-ERS mutants compared to parent strain SH1000. Experiments are underway to determine if the SH100 SCV-ERS mutants accumulate less iron than parent strain SH1000. Overall, we are now trying to determine if alterations in cell wall metabolism or iron acquisition are linked to the SCV-ERS phenotype. The mutational analysis of both MM66 SCV-ERS mutants revealed the presence of non-synonomous mutations in two genes (mutS and mutL) encoding proteins important in DNA mismatch repair. In one MM66 SCV-ERS mutant (MM66-113), there were 2,299 non-synonomous mutations in protein-encoding open reading frames and 3,626 mutations in non-coding regions. In another MM66 SCV-ERS mutant (MM66-115), there were 5,209 non-synonomous mutations in protein-encoding open reading frames and 3,584 mutations in non-coding regions. We suspect that the reason for this incredibly large number of mutations is directly due to the mutations in mutS and mutL, which would lead to an increased mutation rate in the MM66 SCV-ERS mutants. Such a number of mutations due to selection with an antimicrobial has not yet been reported on in S. aureus to our knowledge. It is unlikely that all of these mutations are required for the ERS-SCV phenotype in MM66 SCV-ERS mutants. In contrast, we identified 3 mutations in fnbB which encodes fibronectin-binding protein B, and 5 additional intergenic mutations in both SCV-ERS SH1000 mutants compared to parent strains. We are now going to investigate each one of these mutations to determine if one or more contribute to the SCV-ERS phenotype expressed in the ethanol-selected SH1000 mutants.

Publications

• Type: Journal Articles Status: Published Year Published: 2019 Citation: Torres N. J., S. D. Hartson, J. Rogers, and J. E. Gustafson. 2019. Proteomic and metabolomic analysis of a tea tree oil-selected Staphylococcus aureus small colony variant. Antibiotics 8, 248; doi:10.3390/antibiotics8040248.
• Type: Theses/Dissertations Status: Other Year Published: 2019 Citation: Nathanial Torres: Investigation of a Fusidic Acid-Induced Operon and Characterization of Tea Tree Oil and Ethanol Selected Small Colony Variants of Staphylococcus aureus
• Type: Other Status: Other Year Published: 2019 Citation: Invited lectures: Mechanism of reduced susceptibility to a multitargeting antimicrobial: a foundation in growth inhibition. Department of Biological Sciences, Mississippi State University, 10/18/19.

Progress 10/01/18 to 09/30/19

Outputs
Target Audience:Researchers interested in antibiotic and antiseptic/disinfectant reduced susceptibility in bacterial pathogens. Changes/Problems:None since the last report - It is hoped that the experiments conducted this year will provide enough data to successfully acquire external competitive research funding. What opportunities for training and professional development has the project provided?Thus far, 6 undergraduates and 1 Ph.D. student have benefitted from this project. A Masters's student from Microbiology and Molecular Genetics also benefited from this project. Dr. Nathanial Torres graduated and has taken up a postdoctoral position at the University of Southern Florida. How have the results been disseminated to communities of interest?We have presented some of our work at the Oklahoma Academy of Sciences meetings and the American Society for Biochemistry and Molecular Biology annual meeting. We have also published some of our results in the Canadian Journal of Microbiology. We are in the process of preparing another manuscript for publication. What do you plan to do during the next reporting period to accomplish the goals?Publish on the TTO-selected mutants of S. aureus. We have completed the proteomics analysis of select ethanol reduced susceptibility strains which are presently being analyzed and the results are being prepared for publication with the help of an undergraduate honors student. This publication will require that we complete metabolomics and genomic analysis of these strains in the coming year with the help of a Postdoctoral student.

Impacts
What was accomplished under these goals? Staphylococcus aureus is a target for alcohol-based hand rub (ABHR) antisepsis procedures that are used in part to prevent infections caused by this organism. Utilizing an in vitro approach, we show that selection with growth-inhibitory concentrations of ethanol produced stable S. aureus mutants that demonstrated a small colony variant (SCV) phenotype, ethanol reduced susceptibility (ERS) and slowed growth. In a strain-dependent manner, the SCV-ERS mutants also demonstrated altered susceptibility to the mechanistically unrelated antimicrobials, isopropanol, tea tree oil, triclosan, oxacillin, and vancomycin. All SCV-ERS mutants derived from two vancomycin-susceptible parent strains demonstrated reduced susceptibility to the primary antistaphylococcal drug vancomycin. On the other hand, all SCV-ERS mutants derived from a vancomycin-intermediate S. aureus parent demonstrated increased susceptibility to vancomycin. This study demonstrates that ethanol can select for S. aureus SCV-ERS mutants which express altered susceptibility to multiple antimicrobials, and should provide an incentive for a widespread investigation on alcohol susceptibility in clinical S. aureus isolates.

Publications

• Type: Other Status: Other Year Published: 2018 Citation: Invited lectures: From tea tree oil and alcohol abuse, rises Staphyloccus! National Institute for microbial forensics and food and agricultural biosecurity 3/16/18.

Progress 10/01/17 to 09/30/18

Outputs
Target Audience:Researchers interested in antibiotic and antiseptic/disinfectant reduced susceptibility in bacterial pathogens. Changes/Problems:As with the previous report, we have abandoned cloning and replacement of the acpP mutated gene observed in the TTO-selected mutant. What opportunities for training and professional development has the project provided?Thus far, 6 undergraduates and 1 Ph.D. student have benefitted from this project. A Masters student from Microbiology and Molecular Genetics also benefited from this project. Dr. Nathanial Torres graduated and has taken up a Postdoctoral position at the University of Southern Florida. How have the results been disseminated to communities of interest?We have presented some of our work at the Oklahoma Academy of Sciences meeting and the American Society for Biochemistry and Molecular Biology annual meeting. We have also published some of our results in the Canadian Journal of Microbiology. We are in the process of preparing another 1, maybe 2, manuscripts for publication. What do you plan to do during the next reporting period to accomplish the goals?Present or publish on the ethanol selected mutants of S. aureus. The perform proteomics analysis of select ethanol reduced susceptibility strains has been completed and is presently being analyzed.

Impacts
What was accomplished under these goals? Tea tree oil (TTO) kills bacteria by denaturing membrane and protein structures. A Staphylococcus aureus small colony variant (SCV) selected with TTO (SH1000-TTORS-1) demonstrated: slowed growth; reduced susceptibility to TTO; a diminutive cell size; and a thinned cell wall. Utilizing a proteomics and metabolomics approach, we revealed that the TTO-selected SCV mutant demonstrated: a reduction of proteins critical for active growth and translation; defective fatty acid synthesis; an alteration in the expression of genes and metabolites associated with central metabolism; and the induction of a general stress response. SH1000-TTORS-1 also demonstrated an increase in amino acid accumulation and a decrease in sugar content. The reduction in glycolytic pathway proteins and sugar levels indicated that carbon flow through glycolysis and gluconeogenesis is reduced in SH1000-TTORS-1. The increase in amino acid accumulation coincides with the reduced production of translation specific proteins and the induction of proteins associated with the stringent response. The decrease in sugar content likely deactivates catabolite repression and the increased amino acid pool observed in SH1000-TTORS-1 represents a potential energy and carbon source which could maintain carbon flow through the TCA cycle. It is noteworthy that processes that contribute to the production of the multiple TTO targets (proteins and membrane) are reduced in SH1000-TTORS-1. This is one of a few studies describing a mechanism that bacteria utilize to withstand the action of an antiseptic which is thought to inactivate multiple cellular targets.

Publications

• Type: Theses/Dissertations Status: Accepted Year Published: 2017 Citation: Dr. Nathanial Torres: INVESTIGATION OF A FUSIDIC ACID-INDUCED OPERON AND CHARACTERIZATION OF TEA TREE OIL AND ETHANOL SELECTED SMALL COLONY VARIANTS IN STAPHYLOCOCCUS AUREUS

Progress 10/01/16 to 09/30/17

Outputs
Target Audience:Researchers interested in antibiotic and antiseptic/disinfectant reduced susceptibility in bacterial pathogens. Changes/Problems:Try as we might we still cannot clone the acp or mutated forms of acp from the S. aureus. This is hampering our work. We are alos still working on developing the anti-sense acpP system should allow us to regulate acpP expression and determine the effects of acpP downregulation on antimicrobial resistance. A student picked to work on this project was moved to a non-thesis option, so this portion of the work is on hold. What opportunities for training and professional development has the project provided?Thus far, 4 undergraduates and a Ph.D. student has benefitted from this project. A masters student from Microbiology and Molecular Genetics also benefited from this project. How have the results been disseminated to communities of interest?Yes. We have presented some of our work at the Oklahoma Academy of Sciences meeting and the American Society for Biocemistry and Molecular Biology annual meeting. We have also published some of our results in the Canadian Journal of Microbiology. What do you plan to do during the next reporting period to accomplish the goals?Present or publish on the ethanol selected mutants of S. aureus and the proteomics of the tea tree oil selected mutants. We also intend to perform proteomics and/or metabolomics analysis of select ethanol reduced susceptibility strains.

Impacts
What was accomplished under these goals? A new procedure for the isolation of ethanol reduced susceptibility mutants of S. aureus was devised using increasing ethanol concentrations in liquid media. As of the writing of this report we have selected mutants from strains SH1000 with 9 % v/v ethanol, MM66 and LP9 with 10 % v/v ethanol. All ten SH1000 and LP9 mutants demonstrated reduced susceptibility to ethanol. Only one of the five MM66 mutants investigated demonstrated elevated ethanol resistance. Most of these mutants also demonstrated altered resistance to isopropanol and tea tree oil. With MM66, which is a heterogeneous vancomycin intermediate S. aureus (VISSA) strain, all isolates demonstrated elevated resistance to the non-selecting drug vancomycin which was unexpected. This demonstrates that ethanol selection can increase vancomycin resistance in VISA strains. We completed a publication on the ethanol-induced stress response of Staphylcoccus aureus. Transcriptional profiles of 2 unrelated clinical methicillin-resistant S. aureus (MRSA) isolates were analyzed following 10% (v/v) ethanol challenge (15 min), which arrested growth but did not reduce viability. Ethanol-induced stress (EIS) resulted in differential gene expression of 1091 genes, 600 common to both strains, of which 291 were upregulated. With the exception of the downregulation of genes involved with osmotic stress functions, EIS resulted in the upregulation of genes that contribute to stress response networks, notably those altered by oxidative stress, protein quality control in general, and heat shock in particular. In addition, genes involved with transcription, translation, and nucleotide biosynthesis were downregulated. relP, which encodes a small alarmone synthetase (RelP), was highly upregulated in both MRSA strains following ethanol challenge, and relP inactivation experiments indicated that this gene contributed to EIS growth arrest. A number of persistence associated genes were also upregulated during EIS, including those that encode toxin-antitoxin systems. Overall, transcriptional profiling indicated that the MRSA investigated responded to EIS by entering a state of dormancy and by altering the expression of elements from cross protective stress response systems in an effort to protect preexisting proteins.

Publications

• Type: Conference Papers and Presentations Status: Other Year Published: 2017 Citation: Torres, N. J., S. D. Hartson, J. Rogers, K. A. Abdulhafid and J. E. Gustafson. 2017. Proteomics Analysis of Tea Tree Oil-Selected Staphylococcus aureus Small Colony Variant. American Society for Biochemistry and Molecular Biology Annual Meeting Chicago 3/22-3/26.
• Type: Journal Articles Status: Published Year Published: 2017 Citation: Pando, J. M., R. F. Pfeltz, J. A. Cuaron, V. Nagarajan, S. Yang, A. K. Singh, M. N. Mishra, N. J. Torres, M. O. Elasri, B. J. Wilkinson, and J. E. Gustafson. 2017. Ethanol-induced stress response of Staphylococcus aureus. Can. J. Microbiol. 63:1-3.

Progress 10/01/15 to 09/30/16

Outputs
Target Audience:Researchers working on antibiotic and antiseptic resistance. Changes/Problems:We are having trouble cloning the wild-type and mutated forms of the Staphylococcus aureus acpP genes, so we are trying to develop an anti-sense acpP system. It seems that the Escherichia coli cloning system and S. aureus acpP amplicons we are producing are incompatible at this time, but we are still working on this issue. The anti-sense acpP system should allow us to regulate acpP expression and determine the effects of acpP downregulation on antimicrobial resistance. The anti-sense work will be taken up by a new Masters student in the laboratory. Critical analysis of our suspected Staphylococcus aureus SH1000 small colony variants colonies that survived 12% (v/v) ethanol challenge (8-8-ETHSCV and 8-4-ETHSCV) by MALDI-TOF, revealed these two strains to be S. epidermidis contaminants, which is a set-back. This work was conducted by an undergraduate student and represents the first time in 20 years our laboratory has run into a contamination issue like this. We are making additional attempts to isolate S. aureus mutants with altered susceptibility to ethanol, and we have one mutant (4-2 described above) confirmed by MALDI-TOF without an acpP mutation which demonstrates increased tolerance to ethanol. What opportunities for training and professional development has the project provided?Thus far, 3 undergraduates and a Ph.D. student has benefitted from this project. A new Masters student from Microbiology and Molecular Genetics will embark on research related to this grant proposal this year. How have the results been disseminated to communities of interest?We have presented some of our work at the Oklahoma Academy of Sciences meeting and a regional American Society for Microbiology meeting. What do you plan to do during the next reporting period to accomplish the goals?We have submitted an abstract to the American Society for Biochemistry and Molecular Biology that we hope is accepted. We also intend to publish our manuscript entitled, "Ethanol-induced bacteriostasis of Staphylococcus aureus" which while unrelated to the work proposed adds significantly to our understanding of how this pathogen reacts to alcohols.

Impacts
What was accomplished under these goals? As expected, the addition of 12 % ethanol reduced Staphylococcus aureus strain SH1000 CFUs/ml from 2 X 109 at time zero, to 4 CFUs/ml following 8 hr of ethanol selection. Colonies that survived 4 hr (strain 4-2), 6 hr (strain 6-0A), and 8 hr (strains 8-0A, 8-0B) of ethanol challenge were randomly picked and further characterized. All of these strains were confirmed by MALDI-TOF to be S. aureus. Strains SH1000, 4-2, 6-0A, 8-0A and 8-0B all produced the same size colonies on MHA, produced the same amount of staphyloxanthin, and grew at the same rate in MHB. None of the strains investigated demonstrated the presence of an acpP mutation. In essence, these mutants are unlike the TTO reduced-susceptibility S. aureus mutants, which we did not expect. All ethanol-selected strains demonstrated altered susceptibility to two or more of the antimicrobials tested. Only strain 4-2 demonstrated a greater distance grown on both the ethanol and isopropanol gradients. Strain 6-0A also grew to a greater distance on the isopropanol gradient. Strain 4-2 also demonstrated increased distances grown on the triclosan and vancomycin gradients. Both 8-0A and 8-0B grew to shorter distances on the tea tree oil gradient, and greater distances on the triclosan gradient, compared to SH1000. 8-0B also grew to the top of the vancomycin and oxacillin gradients, while 8-0A grew to shorter distances on these gradients compared to SH1000. These results demonstrate that selection with ethanol can alter resistances to other antimicrobials. Due to issues we are having with our original ethanol-selected mutants (see CHNAGES PROBLEMS SECTION below), we began a new project that compares and contrasts the proteome of a TTO reduced-susceptibility (TTORS) S. aureus mutant (TTORS-1) harboring a mutation within acpP which encodes the acyl carrier protein (ACP) essential for fatty acid biosynthesis. Comparative proteomics revealed that TTORS-1 demonstrated increases in 39 proteins and decreases in 74 proteins compared to parent strain SH1000. In TTORS-1, the fatty acid biosynthesis proteins FabF (3-oxoacyl-synthase) and FabD (malonyl CoA-acyl carrier protein transacylase) and the bifunctional phosphopantothenoylcysteine decarboxylase/phosphopantothenate-cysteine ligase were found in greater abundance. This latter enzyme is required for the synthesis of 4'-phosphopantetheine, which when linked to ACP acts as the anchor on which fatty acid biosynthesis takes place. Furthermore, RT-PCR analysis revealed that the 5 gene fapR operon gene member plsX (encodes glycerol-3-phosphate acyltransferase), was up-regulated in TTORS-1. These findings indicate an alteration in fatty acid biosynthesis in TTORS-1. S. aureus SCVs can result from the deletion of menB or cold shock protein cspB, and menB SCVs demonstrate a decrease in citric acid cycle activity. TTORS-1 harbored less menaquinone biosynthetic protein MenB (1,4-dihydroxy-2-naphthoyl-CoA synthase), cold shock proteins CspB and CspC, ATP synthase subunit gamma, and proteins involved with the citric acid cycle. Collectively this data indicates that fatty acid biosynthesis is altered in TTORS-1, as would be expected in an acpP mutant. We also demonstrate the reduced synthesis of certain proteins in TTORS-1 mirroring what has been observed in previously described SCV phenotypes.

Publications

• Type: Conference Papers and Presentations Status: Other Year Published: 2015 Citation: Simenson, A. J., N. J. Torres, J. E. Gustafson. 2015. Isolation of ethanol reduced susceptibility Staphylococcus aureus. Joint Meeting of the Missouri Valley and Missouri Valley Branches of the American Society for Microbiology - March 26-27, 2015. First Prize for Undergraduate Research Poster Presentation.
• Type: Conference Papers and Presentations Status: Other Year Published: 2016 Citation: Torres, N., Hartson S., Rogers, J. and J. E. Gustafson. 2016. Proteomics analysis of a Staphylococcus aureus tea tree oil-selected small colony variant. Oklahoma Academy of Sciences Technical Meeting, Tulsa 11/4/2016.
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2016 Citation: Torres, N. J., S. D. Hartson, J. Rogers, K. A. Abdulhafid and J. E. Gustafson. SUBMITTED 2016. Proteomics Analysis of Tea Tree Oil-Selected Staphylococcus aureus Small Colony Variant. American Society for Biochemistry and Molecular Biology Annual Meeting Chicago.