Staphylococcus aureus can utilize exogenous fatty acids for phospholipid synthesis. The fatty acid kinase FakA is essential for this utilization by phosphorylating exogenous fatty acids for incorporation into lipids. How FakA impacts the lipid membrane composition is unknown. In this study, we used mass spectrometry to determine the membrane lipid composition and properties of S. aureus in the absence of fakA. We found the fakA mutant to have increased abundance of lipids containing longer acyl chains. Since S. aureus does not synthesize unsaturated fatty acids, we utilized oleic acid (18:1) to track exogenous fatty acid incorporation into lipids. We observed a concentration-dependent incorporation of exogenous fatty acids into the membrane that required FakA. We also tested how FakA and exogenous fatty acids impact membrane-related physiology and identified changes in membrane potential, cellular respiration, and membrane fluidity. To mimic the host environment, we characterized the lipid composition of wild-type and fakA mutant bacteria grown in mouse skin homogenate. We show that wild-type S. aureus can incorporate exogenous unsaturated fatty acids from host tissue, highlighting the importance of FakA in the presence of host skin tissue. In conclusion, FakA is important for maintaining the composition and properties of the phospholipid membrane in the presence of exogenous fatty acids, impacting overall cell physiology. IMPORTANCE Environmental fatty acids can be harvested to supplement endogenous fatty acid synthesis to produce membranes and circumvent fatty acid biosynthesis inhibitors. However, how the inability to use these fatty acids impacts lipids is unclear. Our results reveal lipid composition changes in response to fatty acid addition and when S. aureus is unable to activate fatty acids through FakA. We identify concentration-dependent utilization of oleic acid that, when combined with previous work, provides evidence that fatty acids can serve as a signal to S. aureus. Furthermore, using mouse skin homogenates as a surrogate for in vivo conditions, we showed that S. aureus can incorporate host fatty acids. This study highlights how exogenous fatty acids impact bacterial membrane composition and function.

is capable of phosphorylating exogenous fatty acids via the fatty acid kinase FakA for incorporation into the bacterium's membrane. Additionally, FakA plays a significant role in virulence factor regulation and skin infections. We previously showed that a mutant displays altered growth kinetics observed during late-exponential phase of growth. Here, we demonstrate that the absence of FakA leads to key metabolic changes. First, the mutant has an altered acetate metabolism with acetate being consumed at an increased rate than the wild-type strain. Moreover, the growth benefit was diminished with inactivation of the acetate-generating enzyme AckA. Using a mass spectrometry-based approach, we identified altered concentrations of TCA cycle intermediates and both intracellular and extracellular amino acids. Together, these data demonstrate a change in carbohydrate carbon utilization and altered amino acid metabolism in the mutant. Energy status analysis revealed the mutant had a similar ADP/ATP ratio, but reduced adenylate energy charge. The inactivation of changed the NAD+/NADH and NADP+/NADPH ratio, indicating a more oxidized cellular environment. Evidence points towards the global metabolic regulatory proteins CcpA and CodY being important contributors for the altered growth in a mutant. Indeed, it was found that directing amino acids from the urea cycle into the TCA cycle via glutamate dehydrogenase was an essential component of growth after glucose depletion. Together, this data identifies a previously unidentified role for FakA in the global physiology of that links external fatty acid utilization and central metabolism. The fatty acid kinase, FakA, of plays several important roles in the cell. FakA is important for the activation of the SaeRS two-component system and secreted virulence factors like α-hemolysin. However, the contribution of FakA to cellular metabolism has not been explored. Here, we highlight the metabolic consequence of removal of FakA from the cell. The absence of FakA leads to altered acetate metabolism, altered redox balance, as well as a change in intracellular amino acids. Additionally, the use of environmental amino acids sources is affected by FakA. Together, these results demonstrate for the first time that FakA provides a link between the pathways for exogenous fatty acid use, virulence factor regulation, and other metabolic processes.

Antimicrobial treatments result in the host’s enteric bacteria being exposed to the antimicrobials. Pharmacodynamic models can describe how this exposure affects the enteric bacteria and their antimicrobial resistance. The models utilize measurements of bacterial antimicrobial susceptibility traditionally obtained in vitro in aerobic conditions. However, in vivo enteric bacteria are exposed to antimicrobials in anaerobic conditions of the lower intestine. Some of enteric bacteria of food animals are potential foodborne pathogens, e.g., Gram-negative bacilli Escherichia coli and Salmonella enterica. These are facultative anaerobes; their physiology and growth rates change in anaerobic conditions. We hypothesized that their antimicrobial susceptibility also changes, and evaluated differences in the susceptibility in aerobic vs. anaerobic conditions of generic E. coli and Salmonella enterica of diverse serovars isolated from cattle feces. Susceptibility of an isolate was evaluated as its minimum inhibitory concentration (MIC) measured by E-Test® following 24 hours of adaptation to the conditions on Mueller-Hinton agar, and on a more complex tryptic soy agar with 5% sheep blood (BAP) media. We considered all major antimicrobial drug classes used in the U.S. to treat cattle: β-lactams (specifically, ampicillin and ceftriaxone E-Test®), aminoglycosides (gentamicin and kanamycin), fluoroquinolones (enrofloxacin), classical macrolides (erythromycin), azalides (azithromycin), sulfanomides (sulfamethoxazole/trimethoprim), and tetracyclines (tetracycline). Statistical analyses were conducted for the isolates (n≥30) interpreted as susceptible to the antimicrobials based on the clinical breakpoint interpretation for human infection. Bacterial susceptibility to every antimicrobial tested was statistically significantly different in anaerobic vs. aerobic conditions on both media, except for no difference in susceptibility to ceftriaxone on BAP agar. A satellite experiment suggested that during first days in anaerobic conditions the susceptibility changes with time. The results demonstrate that assessing effects of antimicrobial treatments on resistance in the host’s enteric bacteria that are Gram negative facultative Anaerobe Bacilli requires data on the bacterial antimicrobial susceptibility in the conditions resembling those in the intestine.

According to the Centers of Disease Control and Prevention (CDC), approximately 2.8 million people in the U.S. are infected by antibiotic-resistant bacteria each year. It is estimated that antibioticresistant Staphylococcus aureus accounted for over 300,000 hospitalizations and over 10,000 deaths in 2017, indicating that S. aureus is a key contributor to the burden of antibiotic-resistant infections (CDC). S. aureus is a Gram-positive bacterium that asymptomatically colonizes 20%-30% of the population, predominately in the anterior nares and the skin (Lowy, 1998). Despite being asymptomatic, S. aureus can opportunistically establish infections in nearly every anatomical site but most commonly causes infections in the skin (Pallin et al., 2008;Suaya et al., 2014). This bacterium is responsible for most of the skin and soft-tissue infections observed in the clinic (Moran et al., 2006) and was estimated to cost the U.S. healthcare system $1.7 billion in 2017 (CDC). With the large burden that S. aureus places on the healthcare system, an emphasis on understanding what makes S. aureus such a successful pathogen and designing novel targeted therapeutics is needed. S. aureus must be able to adapt to individual niches on and withinthe host to ensure well-timed regulation of its virulence arsenal to be a successful pathogen. Environmental cues include changes in osmolarity, temperature, presence, or absence of oxygen, pH, and host-associated factors. Some of these signals are sensed by two-component systems (TCS) which allow bacteria to respond to stimuli and adjust target gene expression accordingly (Tiwari et al., 2017;Wang, 2012). S. aureus possesses 16 TCS that respond to

We review historical availability and regulation, and recent indications of antimicrobial drugs for food animals in the USA. We summarize the timeline of introduction of individual antimicrobial drug classes from the 1930s to present, history of regulation of antimicrobial drugs from the 1930s to present and indications of antimicrobial drugs in 1996-2014 for food animals in the USA. The history of antimicrobial drug regulation demonstrates a historical precedent for harmonized regulations of antimicrobials 'for human and other animals' in the USA.