Lipidomics of homeoviscous adaptation to low temperatures in Staphylococcus aureus utilizing exogenous straight-chain unsaturated fatty acids.

It is well established that Staphylococcus aureus can incorporate exogenous straight-chain unsaturated fatty acids (SCUFAs) into membrane phospho- and glyco-lipids from various sources in supplemented culture media and when growing in vivo during infection. Given the enhancement of membrane fluidity when oleic acid (C18:1Δ9) is incorporated into lipids, we were prompted to examine the effect of medium supplementation with C18:1Δ9 on growth at low temperatures. C18:1Δ9 supported the growth of a cold-sensitive, branched-chain fatty acid (BCFA)-deficient mutant at 12°C. Interestingly, we found similar results in the BCFA-sufficient parental strain, supported by the fact that the incorporation of C18:1Δ9 into the membrane increased membrane fluidity in both strains. We show that the incorporation of C18:1Δ9 and its elongation product C20:1Δ11 into membrane lipids was required for growth stimulation and relied on a functional FakAB incorporation system. Lipidomics analysis of the phosphatidylglycerol and diglycosyldiacylglycerol lipid classes revealed major impacts of C18:1Δ9 and temperature on lipid species. Growth at 12°C in the presence of C18:1Δ9 also led to increased production of the carotenoid pigment staphyloxanthin. The enhancement of growth by C18:1Δ9 is an example of homeoviscous adaptation to low temperatures utilizing an exogenous fatty acid. This may be significant in the growth of S. aureus at low temperatures in foods that commonly contain C18:1Δ9 and other SCUFAs in various forms. IMPORTANCE: We show that Staphylococcus aureus can use its known ability to incorporate exogenous fatty acids to enhance its growth at low temperatures. Individual species of phosphatidylglycerols and diglycosyldiacylglycerols bearing one or two degrees of unsaturation derived from the incorporation of C18:1Δ9 at 12°C are described for the first time. In addition, enhanced production of the carotenoid staphyloxanthin occurs at low temperatures. The studies describe a biochemical reality underlying membrane biophysics. This is an example of homeoviscous adaptation to low temperatures utilizing exogenous fatty acids over the regulation of the biosynthesis of endogenous fatty acids. The studies have likely relevance to food safety in that unsaturated fatty acids may enhance the growth of S. aureus in the food environment.

Lipidomics of homeoviscous adaptation to low temperatures in Staphylococcus aureus utilizing exogenous straight-chain unsaturated fatty acids over biosynthesized endogenous branched-chain fatty acids.

It is well established that Staphylococcus aureus can incorporate exogenous straight-chain unsaturated fatty acids (SCUFAs) into membrane phospho- and glyco-lipids from various sources in supplemented culture media, and when growing in vivo in an infection. Given the enhancement of membrane fluidity when oleic acid (C18:1Δ9) is incorporated into lipids, we were prompted to examine the effect of medium supplementation with C18:1Δ9 on growth at low temperatures. C18:1Δ9 supported the growth of a cold-sensitive, branched-chain fatty acid (BCFA)-deficient mutant at 12°C. Interestingly, we found similar results in the BCFA-sufficient parental strain. We show that incorporation of C18:1Δ9 and its elongation product C20:1Δ9 into membrane lipids was required for growth stimulation and relied on a functional FakAB incorporation system. Lipidomics analysis of the phosphatidylglycerol (PG) and diglycosyldiacylglycerol (DGDG) lipid classes revealed major impacts of C18:1Δ9 and temperature on lipid species. Growth at 12°C in the presence of C18:1Δ9 also led to increased production of the carotenoid pigment staphyloxanthin; however, this was not an obligatory requirement for cold adaptation. Enhancement of growth by C18:1Δ9 is an example of homeoviscous adaptation to low temperatures utilizing an exogenous fatty acid. This may be significant in the growth of S. aureus at low temperatures in foods that commonly contain C18:1Δ9 and other SCUFAs in various forms.

Effect of continuous in-feed administration of tylosin to feedlot cattle on macrolide and tetracycline resistant enterococci in a randomized field trial.

Liver abscess causes substantial economic loss to the beef cattle industry through liver condemnation, reduced animal performance, and carcass yield. Continuous in-feed use of tylosin is the most effective and a commonly used practice in beef cattle production to prevent liver abscess. However, such mass medication can increase the level of antimicrobial resistant bacteria. We investigated the effect of continuous in-feed use of tylosin in feedlot cattle on (i) concentrations and prevalence of erythromycin-resistant (ERYr) and tetracycline-resistant (TETr) enterococci; (ii) associated antimicrobial resistance genes (ARGs) for resistance; (iii) species distribution; iv) macrolide and tetracycline resistance gene concentrations; and (v) tylosin concentration. A cohort of weaned calves were randomized to receive tylosin-medicated feed (Tylosin; n = 10) or nonmedicated feed (Control; n = 10) for a full feedlot cycle. Feces, feed and pen-surface samples were collected and processed by culture, droplet digital PCR, and liquid chromatography/mass spectroscopy for bacterial enumeration, detection and characterization, ARG quantification, and tylosin concentration, respectively. Data were analyzed by mixed effects linear- or binary-regression models depending on the outcomes. Tylosin administration significantly increased fecal concentration (P < 0.001) and prevalence (P = 0.021) of ERYr enterococci and erm(B) gene concentration (P < 0.001), compared to the control group. Interestingly, tylosin administration significantly reduced (P = 0.037) fecal TETr enterococci concentration compared to the control group, with no significant effect (P = 0.758) on fecal tet(M) concentration. In both treatment groups, enterococci concentrations increased over time, peaking on 174 days in feed before returning to the baseline. ERYr enterococci concentration was significantly (P = 0.012) higher in tylosin medicated feeds, with no significant effect (P = 0.321) on TETr enterococci concentration. Pen-surface concentration of ermB was significantly (P = 0.024) higher in the tylosin group, with no significant effect (P > 0.05) on bacterial concentrations. Increased diversity and a shift in the composition of enterococcal species and ARGs were observed over time, although tylosin use did not significantly affect (P > 0.05) their prevalence. Tylosin concentration was significantly higher in the feces of tylosin administered cattle (P < 0.001) and medicated feed (P = 0.027), with numerically higher pen-surface concentration (P = 0.065) in the tylosin group. In conclusion, continuous in-feed use of tylosin in feedlot cattle increases macrolide resistant enterococci and its fecal excretion, while decreasing tetracycline resistance. Two medically important species, E. faecium and E. faecalis, were predominant regardless of resistance status or sample source. Risk-based approaches including label changes to limit tylosin use such as withdrawal period, and development of effective manure treatments are potential areas of research to reduce environmental and public health impacts.

Lagoon, Anaerobic Digestion, and Composting of Animal Manure Treatments Impact on Tetracycline Resistance Genes.

Increased demand for animal protein is met by increased food animal production resulting in large quantities of manure. Animal producers, therefore, need sustainable agricultural practices to protect environmental health. Large quantities of antimicrobials are used in commercial food animal production. Consequently, antimicrobial-resistant bacteria and the resistance genes emerge and are excreted through feces. Manure management is essential for the safe disposal of animal waste. Lagoons, with or without covers, and anaerobic digesters, with the primary purpose of methane production, and composting, with the primary purpose of producing organic fertilizer, are widely used methods of manure treatment. We reviewed manure management practices and their impact on tetracycline resistance genes. Lagoons are maintained at ambient temperatures; especially uncovered lagoons are the least effective in removing tetracycline resistance genes. However, some modifications can improve the performance of lagoons: sequential use of uncovered lagoons and the use of covered lagoons resulted in a one-log reduction, while post-treatments such as biofiltration following covered lagoon treatment resulted in 3.4 log reduction. Mesophilic digestion of animal manure did not have any significant effect; only a 0.7 log reduction in tet(A) was observed in one study. While thermophilic anaerobic digesters are effective, if properly operated, they are expensive for animal producers. Aerobic thermophilic composting is a promising technology if optimized with its economic benefits. Composting of raw animal manure can result in up to a 2.5 log reduction, and postdigestion composting can reduce tetracycline resistance gene concentration by >80%. In general, manure management was not designed to mitigate antimicrobial resistance; future research is needed to optimize the economic benefits of biogas or organic fertilizer on the one hand and for the mitigation of foodborne pathogens and antimicrobial resistance on the other.

Revealing Fatty Acid Heterogeneity in Staphylococcal Lipids with Isotope Labeling and RPLC-IM-MS.

Up to 80% of the fatty acids in Staphylococcus aureus membrane lipids are branched, rather than straight-chain, fatty acids. The branched fatty acids (BCFAs) may have either an even or odd number of carbons, and the branch position may be at the penultimate carbon (iso) or the antepenultimate (anteiso) carbon of the tail. This results in two sets of isomeric fatty acid species with the same number of carbons that cannot be resolved by mass spectrometry. The isomer/isobar challenge is further complicated when the mixture of BCFAs and straight-chain fatty acids (SCFAs) are esterified into diacylated lipids such as the phosphatidylglycerol (PG) species of the S. aureus membrane. No conventional chromatographic method has been able to resolve diacylated lipids containing mixtures of SCFAs, anteiso-odd, iso-odd, and iso-even BCFAs. A major hurdle to method development in this area is the lack of relevant analytical standards for lipids containing BCFA isomers. The diversity of the S. aureus lipidome and its naturally high levels of BCFAs present an opportunity to explore the potential of resolving diacylated lipids containing BCFAs and SFCAs. Using our knowledge of lipid and fatty acid biosynthesis in S. aureus, we have used a stable-isotope-labeling strategy to develop and validate a 30 min C18 reversed-phase liquid chromatography method combined with traveling-wave ion mobility-mass spectrometry to provide resolution of diacylated lipids based on the number of BCFAs that they contain.

Recent applications of mass spectrometry in bacterial lipidomics.

The popularity of mass spectrometry-based lipidomics has soared in the past decade. While the majority of the lipidomics work is being performed in mammalian and other eukaryotic systems, there is also a growing rise in the exploration of bacterial lipidomics. The lipids found in bacteria can be substantially different from those in eukaryotic systems, but they are equally important for maintaining the structure of the bacteria and providing protection from the surrounding environment. In this article, recent applications of lipidomics in combination with molecular biology and applications in microbial strain identification and antibiotic susceptibility are highlighted. The authors' perspectives on current challenges facing the field and future directions are also provided.

Anaerobic digestion of livestock and poultry manures spiked with tetracycline antibiotics.

We investigated the anaerobic degradation of tetracycline antibiotics (tetracycline [TC], oxytetracycline [OTC] and chlortetracycline [CTC]) in swine, cattle, and poultry manures. The manures were anaerobically digested inside polyvinyl chloride batch reactors for 64 days at room temperature. The degradation rate constants and half-lives of the parent tetracyclines were determined following first-order kinetics. For CTC the fastest degradation rate was observed in swine manure (k = 0.016 ± 0.001 d-1; half-life = 42.8 days), while the slowest degradation rate was observed in poultry litter (k = 0.0043 ± 0.001 d-1; half-life = 161 days). The half-lives of OTC ranged between 88.9 (cattle manure) and 99.0 days (poultry litter), while TC persisted the longest of the tetracycline antibiotics studied with half-lives ranging from 92.4 days (cattle manure) to 330 days (swine manure). In general, the tetracyclines were found to degrade faster in cattle manure, which had the lowest concentrations of organic matter and metals as compared to swine and poultry manures. Our results demonstrate that tetracycline antibiotics persist in the animal manure after anaerobic digestion, which can potentially lead to emergence and persistence of antibiotic resistant bacteria in the environment when anaerobic digestion byproducts are land applied for crop production.