Neal R. Chamberlain

Insertional Inactivation of Branched-Chain α-Keto Acid Dehydrogenase in Staphylococcus aureus Leads to Decreased Branched-Chain Membrane Fatty Acid Content and Increased Susceptibility to Certain Stresses

ABSTRACT Staphylococcus aureus is a major community and nosocomial pathogen. Its ability to withstand multiple stress conditions and quickly develop resistance to antibiotics complicates the control of staphylococcal infections. Adaptation to lower temperatures is a key for the survival of bacterial species outside the host. Branched-chain α-keto acid dehydrogenase (BKD) is an enzyme complex that catalyzes the early stages of branched-chain fatty acid (BCFA) production. In this study, BKD was inactivated, resulting in reduced levels of BCFAs in the membrane of S. aureus. Growth of the BKD-inactivated mutant was progressively more impaired than that of wild-type S. aureus with decreasing temperature, to the point that the mutant could not grow at 12°C. The growth of the mutant was markedly stimulated by the inclusion of 2-methylbutyrate in the growth medium at all temperatures tested. 2-Methylbutyrate is a precursor of odd-numbered anteiso fatty acids and bypasses BKD. Interestingly, growth of wild-type S. aureus was also stimulated by including 2-methylbutyrate in the medium, especially at lower temperatures. The anteiso fatty acid content of the BKD-inactivated mutant was restored by the inclusion of 2-methylbutyrate in the medium. Fluorescence polarization measurements indicated that the membrane of the BKD-inactivated mutant was significantly less fluid than that of wild-type S. aureus. Consistent with this result, the mutant showed decreased toluene tolerance that could be increased by the inclusion of 2-methylbutyrate in the medium. The BKD-inactivated mutant was more susceptible to alkaline pH and oxidative stress conditions. Inactivation of the BKD enzyme complex in S. aureus also led to a reduction in adherence of the mutant to eukaryotic cells and its survival in a mouse host. In addition, the mutant offers a tool to study the role of membrane fluidity in the interaction of S. aureus with antimicrobial substances.

Evidence for persisters in Staphylococcus epidermidis RP62a planktonic cultures and biofilms

The pathogenesis of Staphylococcus epidermidis in foreign device-related infections is attributed primarily to its ability to form biofilms on a polymer surface. One mechanism proposed for the survival of organisms in a biofilm is the presence of persister cells. Persister cells survive antibiotic treatment without acquiring heritable antibiotic resistance. This study was conducted to determine if S. epidermidis RP62a growing in planktonic cultures and biofilms could survive as persister cells following treatment with levofloxacin and vancomycin. S. epidermidis RP62a produced a small percentage of persisters (levofloxacin, 3.09×10⁻⁷%; vancomycin, 8.21×10⁻⁵ %) when grown to exponential phase, whereas biofilms contained 28 and 94 % persisters, following exposure to levofloxacin and vancomycin, respectively. The highest percentages of persisters were obtained during stationary phase in planktonic cultures and the lowest percentages of persisters were obtained during mid-exponential phase. An increase in persister number was not due to activation of quorum-sensing regulons. Confocal laser scanning microscopy images of biofilms exposed to levofloxacin demonstrated that the antibiotic was able to kill bacteria throughout the biofilm. Our results suggest that antibiotic tolerance in biofilms and in planktonic cultures of S. epidermidis RP62a is due in part to the presence of persister cells.

Genetic regulation of fatty acid modifying enzyme from Staphylococcus aureus

Fatty acid modifying enzyme (FAME) is an extracellular enzyme that inactivates staphylocidal lipids by catalysing the esterification of these lipids to cholesterol. In-vitro expression of FAME began at the start of the stationary phase. This expression of FAME was very similar to other staphylococcal extracellular proteins controlled by the global regulators Agr and Sar. A staphylococcus aureus strain ISP546 (Agr-) produced c. 80% less FAME than an isogenic Agr+ strain ISP479C. Similar results were obtained with the isogenic Agr+/Agr- strain pair RN6390 and RN6911. A S. aureus strain R (Sar-) produced c. 86% less FAME than an isogenic Sar+ strain RN6390. However, lipase assays on the same culture filtrates from the Sar+/Sar- strains did not demonstrate any affect on lipase production by the sar mutation.

Utilization of case presentations in medical microbiology to enhance relevance of basic science for medical students

Abstract Background : Small-group case presentation exercises (CPs) were created to increase course relevance for medical students taking Medical Microbiology (MM) and Infectious Diseases (ID) Methods : Each student received a unique paper case and had 10 minutes to review patient history, physical exam data, and laboratory data. Students then had three minutes to orally present their case and defend why they ruled in or out each of the answer choices provided, followed by an additional three minutes to answer questions. Results : Exam scores differed significantly between students who received the traditional lecture-laboratory curriculum (Group I) and students who participated in the CPs (Group II). In MM, median unit exam and final exam scores for Group I students were 84.4% and 77.8%, compared to 86.0% and 82.2% for Group II students (P < 0.018; P < 0.001; Mann-Whitney Rank Sum Test). Median unit and final ID exam scores for Group I students were 84.0% and 80.0%, compared to 88.0% and 86.7% for Group II students (P < 0.001; P < 0.001). Conclusion : Students felt that the CPs improved their critical thinking and presentation skills and helped to prepare them as future physicians.

Growth cycle-induced changes in sensitivity of Staphylococcus aureus to bactericidal lipids from abscesses

Three Staphylococcus aureus strains (303, 18Z and TG), exhibiting various patterns of survival within abscesses, were significantly more sensitive to the bactericidal activity of oleic acid during the log phase of growth than at other stages of the growth cycle. Cells entering the stationary phase showed diminished sensitivity to the fatty acid. These changes were reflected by changes in the LD50 and also by differences in the rate of killing by oleic acid. Additional changes were noted: the rate of killing by oleic acid declined over a 4-day period; a progressively greater proportion of the staphylococcal population became resistant to even high concentrations of oleic acid; from the fourth day onwards c. 50-55% of the cocci were totally resistant to the fatty acid. Strains 303 and 18Z became more sensitive to mono-olein during the log phase of growth, but strain TG was very resistant to mono-olein throughout the growth cycle. Growth in the presence of glycine 6% to reduce cross-links in the peptidoglycan did not alter bacterial sensitivity to oleic acid. However, all three S. aureus strains exhibited significant increases in membrane fluidity during the log phase of growth, but upon entering the stationary phase membrane fluidity again decreased. Concomitant changes in carotenoid content occurred during the growth cycle, but these changes did not appear to be solely responsible for the changes in sensitivity to the lipids.

Effect of clpP and clpC deletion on persister cell number in Staphylococcus aureus.

Staphylococcus aureus is responsible for a wide variety of infections that include superficial skin and soft tissue infections, septicaemia, central nervous system infections, endocarditis, osteomyelitis and pneumonia. Others have demonstrated the importance of toxin-antitoxin (TA) modules in the formation of persisters and the role of the Clp proteolytic system in the regulation of these TA modules. This study was conducted to determine the effect of clpP and clpC deletion on S. aureus persister cell numbers following antibiotic treatment. Deletion of clpP resulted in a significant decrease in persister cells following treatment with oxacillin and erythromycin but not with levofloxacin and daptomycin. Deletion of clpC resulted in a decrease in persister cells following treatment with oxacillin. These differences were dependent on the antibiotic class and the CFU ml-1 in which the cells were treated. Persister revival assays for all the bacterial strains in these studies demonstrated a significant delay in resumption of growth characteristic of persister cells, indicating that the surviving organisms in this study were not likely due to spontaneous antibiotic resistance. Based on our results, ClpP and possibly ClpC play a role in persister cell formation or maintenance, and this effect is dependent on antibiotic class and the CFU ml-1 or the growth phase of the cells.

Identification and partial characterisation of an extracellular activator of fatty acid modifying enzyme (FAME) expression in Staphylococcus epidermidis.

Fatty acid modifying enzyme (FAME) is an extracellular enzyme that inactivates bactericidal fatty acids by esterifying them to cholesterol. Inactivation of these fatty acids may allow Staphylococcus epidermidis to live for long periods of time on the skin. This study describes the identification and partial characterisation of an extracellular activator of FAME production. Addition of FAME-free concentrated culture filtrate (activator) to S. epidermidis cultures (OD600 = 0.05) caused a 3-5-fold increase in FAME activity. Addition of the activator did not increase the amount of exopolysaccharide produced by S. epidermidis. The mol. wt of this activator was <3000 kDa and it was quite resistant to boiling. Treatment of the activator with proteinase K did not destroy its ability to induce FAME expression. Addition of S. aureus activator to S. epidermidis cultures also increased FAME expression. However, when S. epidermidis activator was added to S. aureus cultures no increase or inhibition in FAME production was observed.