Fitness of Spontaneous Rifampicin-Resistant Staphylococcus aureus Isolates in a Biofilm Environment

Biofilms of S. aureus accumulate cells resistant to the antibiotic rifampicin. We show here that the accumulation of rifampicin resistant mutants (RifR) in biofilms is not equable but rather is a local event, suggesting that the growth of a few locally emerged mutants is responsible for this. Competition assays demonstrated that, compared to wild-type bacteria, the isolated RifR mutants have a growth advantage in biofilms, but not in planktonic culture. To gain insight into the mechanism of the growth advantage, we tested the involvement of the two-component systems (TCS) that sense and respond to environmental changes. We found that a deletion of SrrAB or NreBC has a drastic effect on the growth advantage of RifR mutants, suggesting the importance of oxygen/respiration responses. All six of the RifR isolates tested showed increased resistance to at least one of the common stresses found in the biofilm environment (i.e., oxidative, nitric acid, and organic acid stress). The RifR mutants also had a growth advantage in a biofilm flow model, which highlights the physiological relevance of our findings

Fitness of Spontaneous Rifampicin-Resistant Staphylococcus aureus Isolates in a Biofilm Environment

Biofilms of S. aureus accumulate cells resistant to the antibiotic rifampicin. We showhere that the accumulation of rifampicin resistant mutants (RifR) in biofilms is notequable but rather is a local event, suggesting that the growth of a few locally emergedmutants is responsible for this. Competition assays demonstrated that, compared towild-type bacteria, the isolated RifR mutants have a growth advantage in biofilms, butnot in planktonic culture. To gain insight into the mechanism of the growth advantage,we tested the involvement of the two-component systems (TCS) that sense andrespond to environmental changes. We found that a deletion of SrrAB or NreBC hasa drastic effect on the growth advantage of RifR mutants, suggesting the importanceof oxygen/respiration responses. All six of the RifR isolates tested showed increasedresistance to at least one of the common stresses found in the biofilm environment (i.e.,oxidative, nitric acid, and organic acid stress). The RifR mutants also had a growthadvantage in a biofilm flow model, which highlights the physiological relevance ofour findings

Endotoxin, Capsule, and Bacterial Attachment Contribute to Neisseria meningitidis Resistance to the Human Antimicrobial Peptide LL-37▿ †

Pathogenic bacteria have evolved numerous mechanisms to evade the human immune system and have developed widespread resistance to traditional antibiotics. We studied the human pathogen Neisseria meningitidis and present evidence of novel mechanisms of resistance to the human antimicrobial peptide LL-37. We found that bacteria attached to host epithelial cells are resistant to 10 μM LL-37 whereas bacteria in solution or attached to plastic are killed, indicating that the cell microenvironment protects bacteria. The bacterial endotoxin lipooligosaccharide and the polysaccharide capsule contribute to LL-37 resistance, probably by preventing LL-37 from reaching the bacterial membrane, as more LL-37 reaches the bacterial membrane on both lipooligosaccharide-deficient and capsule-deficient mutants whereas both mutants are also more susceptible to LL-37 killing than the wild-type strain. N. meningitidis bacteria respond to sublethal doses of LL-37 and upregulate two of their capsule genes, siaC and siaD, which further results in upregulation of capsule biosynthesis

Lactobacilli Reduce Cell Cytotoxicity Caused by Streptococcus pyogenes by Producing Lactic Acid That Degrades the Toxic Component Lipoteichoic Acid▿ †

Lactobacilli are known to prevent colonization by many pathogens; nevertheless, the mechanisms of their protective effect are largely unknown. In this work, we investigated the role of lactobacilli during infection of epithelial cells with group A streptococci (GAS). GAS cause a variety of illnesses ranging from noninvasive disease to more severe invasive infections, such as necrotizing fasciitis and toxic shock-like syndrome. Invasion of deeper tissues is facilitated by GAS-induced apoptosis and cell death. We found that lactobacilli inhibit GAS-induced host cell cytotoxicity and shedding of the complement regulator CD46. Further, survival assays demonstrated that lactic acid secreted by lactobacilli is highly bactericidal toward GAS. In addition, lactic acid treatment of GAS, but not heat killing, prior to infection abolishes the cytotoxic effects against human cells. Since lipoteichoic acid (LTA) of GAS is heat resistant and cytotoxic, we explored the effects of lactic acid on LTA. By applying such an approach, we demonstrate that lactic acid reduces epithelial cell damage caused by GAS by degrading both secreted and cell-bound LTA. Taken together, our experiments reveal a mechanism by which lactobacilli prevent pathogen-induced host cell damage

Meningococcal Outer Membrane Protein NhhA Is Essential for Colonization and Disease by Preventing Phagocytosis and Complement Attack▿

Neisseria meningitidis is a leading cause of meningitis and septicemia worldwide, with a rapid onset of disease and a high morbidity and mortality. NhhA is a meningococcal outer membrane protein included in the family of trimeric autotransporter adhesins. The protein binds to the extracellular matrix proteins heparan sulfate and laminin and facilitates attachment to host epithelial cells. In this study, we show that NhhA is essential for bacterial colonization of the nasopharyngeal mucosa in a murine model of meningococcal disease. Successful colonization depends on bacterial attachment but also to the capacity to overcome innate host immune responses. We found that NhhA protected bacteria from phagocytosis, which is important for the mucosal survival of bacteria. In addition, NhhA mediated extensive serum resistance that increased bacterial survival in blood and promoted lethal sepsis. The presence of NhhA protected bacteria from complement-mediated killing by preventing the deposition of the membrane attack complex. Taken together, the results of this work reveal that NhhA inhibits phagocytosis and protects bacteria against complement-mediated killing, which enhances both nasal colonization and the development of sepsis in vivo

Beneficial Antimicrobial Effect of the Addition of an Aminoglycoside to a β-Lactam Antibiotic in an E. coli Porcine Intensive Care Severe Sepsis Model.

This study aimed to determine whether the addition of an aminoglycoside to a ß-lactam antibiotic increases the antimicrobial effect during the early phase of Gram-negative severe sepsis/septic shock. A porcine model was selected that considered each animal's individual blood bactericidal capacity. Escherichia coli, susceptible to both antibiotics, was given to healthy pigs intravenously during 3 h. At 2 h, the animals were randomized to a 20-min infusion with either cefuroxime alone (n = 9), a combination of cefuroxime+tobramycin (n = 9), or saline (control, n = 9). Blood samples were collected hourly for cultures and quantitative polymerase chain reaction (PCR). Bacterial growth in the organs after 6 h was chosen as the primary endpoint. A blood sample was obtained at baseline before start of bacterial infusion for ex vivo investigation of the blood bactericidal capacity. At 1 h after the administration of the antibiotics, a second blood sample was taken for ex vivo investigation of the antibiotic-induced blood killing activity. All animals developed severe sepsis/septic shock. Blood cultures and PCR rapidly became negative after completed bacterial infusion. Antibiotic-induced blood killing activity was significantly greater in the combination group than in the cefuroxime group (p<0.001). Growth of bacteria in the spleen was reduced in the two antibiotic groups compared with the controls (p<0.01); no difference was noted between the two antibiotic groups. Bacterial growth in the liver was significantly less in the combination group than in the cefuroxime group (p<0.05). High blood bactericidal capacity at baseline was associated with decreased growth in the blood and spleen (p<0.05). The addition of tobramycin to cefuroxime results in increased antibiotic-induced blood killing activity and less bacteria in the liver than cefuroxime alone. Individual blood bactericidal capacity may have a significant effect on antimicrobial outcome

Physiological and laboratory parameters reflecting circulation, hypoperfusion and organ dysfunction (n = 27).

<p>Values are mean±SD, except creatinine clearance, which is given as median (interquartile range).</p><p>MAP = mean arterial pressure, CI = cardiac index, MPAP = mean pulmonary arterial pressure, PaO₂ = arterial partial pressure of oxygen, FiO₂ = inspired fraction of oxygen, LVSWI = left ventricular stroke work index.</p

Bacterial DNA in the blood after <i>ex vivo</i> addition of live <i>E. coli</i>.

<p>Bacteria added to a concentration of 1×10⁵ CFU×mL⁻¹ to blood obtained from animals treated with cefuroxime alone or the combination of cefuroxime and tobramycin and from control animals at 3∶20 h, i.e. 1 h after completion of the administration of treatment. The time points for sampling are the same as those in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090441#pone-0090441-g003" target="_blank">Figure 3</a>, in which the antibiotic-induced killing activity significantly differed between the groups. The quantity of bacterial DNA is expressed as CFU equivalents. Values are mean±SE (n = 3 in each group).</p