Cell Wall Antibiotics Research Articles

Surviving Bacterial Sibling Rivalry: Inducible and Reversible Phenotypic Switching in Paenibacillus dendritiformis

ABSTRACTNatural habitats vary in available nutrients and room for bacteria to grow, but successful colonization can lead to overcrowding and stress. Here we show that competing sibling colonies of Paenibacillus dendritiformis bacteria survive overcrowding by switching between two distinct vegetative phenotypes, motile rods and immotile cocci. Growing colonies of the rod-shaped bacteria produce a toxic protein, Slf, which kills cells of encroaching sibling colonies. However, sublethal concentrations of Slf induce some of the rods to switch to Slf-resistant cocci, which have distinct metabolic and resistance profiles, including resistance to cell wall antibiotics. Unlike dormant spores of P. dendritiformis, the cocci replicate. If cocci encounter conditions that favor rods, they secrete a signaling molecule that induces a switch to rods. Thus, in contrast to persister cells, P. dendritiformis bacteria adapt to changing environmental conditions by inducible and reversible phenotypic switching.

Improved Oxacillin Treatment Outcomes in Experimental Skin and Lung Infection by a Methicillin-Resistant Staphylococcus aureus Isolate with a vraSR Operon Deletion

Methicillin-resistant Staphylococcus aureus (MRSA) strains are major pathogens causing infections of the skin and soft tissues and more serious, life-threatening diseases, including sepsis and necrotizing pneumonia. The vraSR operon encodes the key regulatory system that modulates the stress response of S. aureus elicited upon exposure to cell wall antibiotics. Mutation of vraS and vraR results in decreased oxacillin resistance in vitro. We investigated the effect of oxacillin treatment in experimental models employing a clinical USA300 MRSA strain (strain 923) and an isogenic vraSR deletion mutant (strain 923-M23). In a murine model of S. aureus necrotizing pneumonia, animals were treated with oxacillin, beginning 15 min after inoculation. Among mice infected with mutant strain 923-M23, oxacillin treatment significantly improved survival compared with saline treatment, whereas oxacillin treatment had no effect in mice infected with strain 923. Similarly, treatment with oxacillin decreased the bacterial burden among animals infected with strain 923-M23 but not among animals infected with strain 923. In a murine skin infection model, oxacillin eliminated the development of dermonecrosis among 923-M23-infected mice and decreased the bacterial burden in the lesions, but not among strain 923-infected mice. We conclude that deletion of the vraSR operon allowed an oxacillin regimen to be effective in murine models of MRSA pneumonia and skin infection. These findings provide proof-of-principle for development of a new antibiotic that could restore the usefulness of oxacillin against MRSA by inhibiting VraS or VraR.

In-Depth Profiling of the LiaR Response of Bacillus subtilis

The Lia system, a cell envelope stress response module of Bacillus subtilis, is comprised of the LiaRS two-component system and a membrane-anchored inhibitor protein, LiaF. It is highly conserved in the Firmicutes bacteria, and all orthologs investigated so far are activated by cell wall antibiotics. In response to envelope stress, the systems in Firmicutes cocci induce the expression of a number of genes that are involved in conferring resistance against its inducers. In contrast, a complete picture of the LiaR regulon of B. subtilis is still missing and no phenotypes could be associated with mutants lacking LiaRS. Here, we performed genome-wide transcriptomic, proteomic, and in-depth phenotypic profiling of constitutive "Lia ON" and "Lia OFF" mutants to obtain a comprehensive picture of the Lia response of Bacillus subtilis. In addition to the known targets liaIH and yhcYZ-yhdA, we identified ydhE as a novel gene affected by LiaR-dependent regulation. The results of detailed follow-up gene expression studies, together with proteomic analysis, demonstrate that the liaIH operon represents the only relevant LiaR target locus in vivo. It encodes a small membrane protein (LiaI) and a phage shock protein homolog (LiaH). LiaH forms large oligomeric rings reminiscent of those described for Escherichia coli PspA or Arabidopsis thaliana Vipp1. The results of comprehensive phenotype studies demonstrated that the gene products of the liaIH operon are involved in protecting the cell against oxidative stress and some cell wall antibiotics. Our data suggest that the LiaFSR system of B. subtilis and, presumably, other Firmicutes bacilli coordinates a phage shock protein-like response.

PepD Participates in the Mycobacterial Stress Response Mediated through MprAB and SigE

Currently, one-third of the world's population is believed to be latently infected with Mycobacterium tuberculosis. The mechanisms by which M. tuberculosis establishes latent infection remain largely undefined. mprAB encodes a two-component signal transduction system required by M. tuberculosis for aspects of persistent infection. MprAB regulates a large and diverse group of genetic determinants in response to membrane stress, including the extracytoplasmic function (ECF) sigma factor sigE and the HtrA-like serine protease pepD. Recent studies have demonstrated that PepD functions as both a protease and chaperone in vitro. In addition, inactivation of pepD alters the virulence of M. tuberculosis in a mouse model system of infection. Here, we demonstrate that PepD plays an important role in the stress response network of Mycobacterium mediated through MprAB and SigE. In particular, we demonstrate that the protease activity of PepD requires the PDZ domain, in addition to the catalytic serine at position 317. pepD expression initiates from at least three promoters in M. tuberculosis, including one that is regulated by SigE and is located upstream of the mprA coding sequence. Deletion of pepD or mprAB in Mycobacterium smegmatis and M. tuberculosis alters the stress response phenotypes of these strains, including increasing sensitivity to SDS and cell wall antibiotics and upregulating the expression of stress-responsive determinants, including sigE. Taking these data together, we hypothesize that PepD utilizes its PDZ domain to recognize and process misfolded proteins at the cell membrane, leading to activation of the MprAB and SigE signaling pathways and subsequent establishment of a positive feedback loop that facilitates bacterial adaptation.

Regulatory Overlap and Functional Redundancy amongBacillus subtilisExtracytoplasmic Function σ Factors

Bacillus subtilis encodes seven extracytoplasmic function (ECF) sigma factors that regulate partially overlapping regulons related to cell envelope homeostasis and antibiotic resistance. Here, we investigated their physiological role by constructing a mutant set of single, double, triple, and quadruple ECF sigma factor deletions in the undomesticated B. subtilis strain NCIB3610. This mutant set was subsequently screened for defects in motility, multicellular differentiation, and sensitivity to more than 200 chemicals by using Phenotype MicroArrays. A quadruple mutant strain, harboring deletions of the sigV, sigY, sigZ, and ylaC gene, behaved indistinguishably from the wild-type strain, indicative of either regulatory redundancy or very specific functions of these four ECF sigma factors. In contrast, a triple mutant, inactivated for the sigM, sigW, and sigX genes (but none of the corresponding double mutants), showed a biphasic growth behavior and a complete loss of multicellular differentiation, as judged by both colony formation and the inability to form a pellicle. This triple mutant also displayed a greatly increased sensitivity to detergents and several cell wall antibiotics including beta-lactams, polymyxin B, and d-cycloserine. In several cases, these antibiotic-sensitive phenotypes are significantly enhanced in the triple mutant strain relative to strains lacking only one or two sigma factors.

TLC Separation of Cephalosporins: Searching for Better Selectivity

Cephalosporins are broad‐spectrum bactericidal cell wall antibiotics which are very similar in structure and action to penicillins. They are used mainly for treating staphylococcal and streptococcal infections in patients who cannot use penicillins. Cephalosporins are also often used in veterinary medicine. They are commonly divided into four groups which differ slightly in spectrum and toxicity. Cephalosporins can be detected in pharmaceuticals, body fluids, and food by microbiological and immunological assays, capillary electrophoresis, chromatography, and other physicochemical methods. In this paper, the optimal conditions for thin‐layer chromatographic analysis of eight cephalosporins, i.e., cefaclor, cefoperazone, cefazolin, cefotaxime, cefoxitin, cefuroxime, cephalotin, and p‐chlorophenacyl cephalothin ester were established. Retention parameters for various chromatographic systems were compared. Good separations were achieved for several phases on plane silica gel and silica gel with boned diol‐, amino‐ and cyanopropyl chains.

Regulation of LiaRS-Dependent Gene Expression inBacillus subtilis: Identification of Inhibitor Proteins, Regulator Binding Sites, and Target Genes of a Conserved Cell Envelope Stress-Sensing Two-Component System

The regulatory network of the cell envelope stress response in Bacillus subtilis involves both extracytoplasmic function sigma-factors and two-component signal transducing systems. One such system, LiaRS, responds to cell wall antibiotics that interfere with the undecaprenol cycle and to perturbation of the cytoplasmic membrane. It is encoded by the last two genes of the liaIHGFSR locus. Here, we analyzed the expression of two LiaR-dependent operons, liaIHGFSR and yhcYZ-yhdA, and characterized a palindromic sequence required for LiaR-dependent activation. Since induction of the strong liaI promoter leads to both liaIH and liaIHGFRS transcripts, LiaR is positively autoregulated. Systematic deletion analysis of the liaI operon revealed that LiaF is a potent negative regulator of LiaR-dependent gene expression: a nonpolar liaF deletion led to constitutive activation of both characterized LiaR-dependent promoters. The liaF gene is conserved in both sequence and genomic context in the Firmicutes group of gram-positive bacteria, located directly upstream of liaSR orthologs. LiaH, a homolog of Escherichia coli phage shock protein A, also plays a more subtle role in negatively modulating the bacitracin-inducible expression from LiaR-dependent promoters. Our results support a model in which the LiaFRS module integrates both positive and negative feedback loops to transduce cell envelope stress signals.

The Cell Wall Stress Stimulon of Staphylococcus aureus and Other Gram- Positive Bacteria

By the early nineteen seventies the mechanism of inhibition of peptidoglycan biosynthesis by various cell wallactive antibiotics was well established, and the Gram-positive bacterium Staphylococcus aureus had often been used in the studies. From the early days of penicillin it was known that cell wall-active antibiotics are typically bactericidal causing cell death and lysis, a phenomenon that has recently been described as programmed cell death. Uncovering the details of the molecular and cellular events occurring subsequent to inhibition of peptidoglycan biosynthesis, a legitimate aspect of knowledge of the mode of action of these agents, has had to await the advent of the omics era-genomics, proteomics and transcriptomics-over the past several years. Genome-wide transcriptional profiling using DNA microarrays of the response of S. aureus to challenge by cell wall active antibiotics has revealed a cell wall stress stimulon of genes upregulated in their expression by these agents. Several of the genes encode proteins involved in cell wall metabolism and their induction can be regarded as a response of the organism to preserve and repair the compromised cell wall. Expression of a significant number of cell wall stress stimulon member genes is controlled by a two-component regulatory system. Cell wall stress stimulons regulated by two-component systems have also been described in Bacillus subtilis and Listeria monocytogenes, and are probably common to all Gram-positive bacteria. Unfortunately S. aureus has not remained susceptible to cell wall-active antibiotics and methicillin-resistant strains are common, and vancomycinintermediate and-resistant strains have arisen. Interestingly, some member genes of the cell wall stress stimulon have been previously encountered in the context of methicillin-resistance or vancomycin-intermediate resistance. There is a threat of pan-resistant S. aureus strains and new antimicrobial agents are needed. Cell wall biosynthesis remains a viable target for new drugs, and recognition of the transcriptomic signature of the cell wall stress stimulon can be used to indicate a cell wall mode of action. Individual cell wall stress member genes may form the basis of a screen for cell wall-active agents. Finally, agents targeting cell wall stress stimulon member gene expression or proteins might enhance the activity of cell wall-active agents that induce the cell wall stress stimulon. Keywords: cell wall stress stimulon, cell wall antibiotics, gram-positive bacteria, staphylococcus aureus

SigM, an extracytoplasmic function sigma factor of Bacillus subtilis, is activated in response to cell wall antibiotics, ethanol, heat, acid, and superoxide stress.

The extracytoplasmic function sigma M of Bacillus subtilis is required for normal cell growth under salt stress. It is expressed maximally during exponential growth and is further induced by the addition of 0.7 M NaCl. The promoter region of the sigM operon contains two promoters; one (P(A)) is sigma A dependent, and the other (P(M)) is sigma M dependent. These have been placed separately at the amy locus, directing expression of a lacZ reporter gene. Only the P(M) fusion responded to salt induction. This promoter, which was responsive to the level of active sigma M in the cell, was also induced by 5% ethanol, by vancomycin, bacitracin, or phosphomycin (inhibitors of cell wall biosynthesis; 2 micro g per ml), and by heat shock of 50 degrees C for 10 min. It was very strongly induced by acid (pH 4.3) and 80 micro M paraquat, but after a 15- to 30-min delay. There was no induction by alkali (pH 9), 5 mM H(2)O(2), the detergents 0.1% Triton X-100 and 0.1% Tween 20, or 50 micro M monensin. In addition to their reduced tolerance to salt, null mutants of sigM were unable to grow at pH 4.3 and lysed after exposure to 5% ethanol. Genes regulated by SigM were also tested for their response to pH 4.3, 5% ethanol, and 2 micro g of vancomycin per ml. Expression of the genes may have been activated by increased levels of sigma M, but at least some were also subject to additional controls, as they responded to one type of stress but not another. Expression of yrhJ, which encodes a cytochrome P450/NADPH reductase, was induced in response to acid and vancomycin. yraA expression was acid, ethanol, and vancomycin induced, whereas yjbD showed only ethanol induction. YraA protein was extremely important to acid survival-a mutation in yraA, like a sigM mutation, resulted in the failure of B. subtilis to grow at pH 4.3. Sigma M is therefore involved in maintaining membrane and cell wall integrity in response to several different stresses in exponential growth phase and is activated by such stresses.

Genetic evidence that the bacteriophage phi X174 lysis protein inhibits cell wall synthesis.

Protein E, a 91-residue membrane protein of phiX174, causes lysis of the host in a growth-dependent manner reminiscent of cell wall antibiotics, suggesting E acts by inhibiting peptidoglycan synthesis. In a search for the cellular target of E, we previously have isolated recessive mutations in the host gene slyD (sensitivity to lysis) that block the lytic effects of E. The role of slyD, which encodes a FK506 binding protein-type peptidyl-prolyl cis-trans isomerase, is not fully understood. However, E mutants referred to as Epos (plates on slyD) lack a slyD requirement, indicating that slyD is not crucial for lysis. To identify the gene encoding the cellular target, we selected for survivors of Epos. In this study, we describe the isolation of dominant mutations in the essential host gene mraY that result in a general lysis-defective phenotype. mraY encodes translocase I, which catalyzes the formation of the first lipid-linked intermediate in cell wall biosynthesis. The isolation of these lysis-defective mutants supports a model in which translocase I is the cellular target of E and that inhibition of cell wall synthesis is the mechanism of lysis.

Subinhibitory concentrations of gentamicin reduce production of listeriolysin, the main virulence factor of Listeria monocytogenes

The facultatively intracellular Gram-positive rod Listeria monocytogenes is the causative agent of severe infections in humans and animals, e.g. sepsis and meningoencephalitis. The treatment of choice is ampicillin in combination with gentamicin [1Jones EM MacGowan AP Antimicrobial chemotherapy of human infection due to Listeria monocytogenes.Eur J Clin Microbiol Infect Dis. 1995; 14: 165-175Crossref PubMed Scopus (99) Google Scholar]. Because of poor penetration through the blood-brain barrier and into the cytoplasm of cells where the listeriae multiply, these antibiotics are likely to reach concentrations below the levels obtained in the blood at the site of infection. Certain antibiotics are able to retard growth of bacteria at concentrations below the minimal inhibitory concentration assessed visually. In addition, some antibiotics are capable of selectively inhibiting the production of virulence factors by pathogenic bacteria [2Doss SA Tillotson GS Amyes SG Effect of sub-inhibitory concentrations of antibiotics on the virulence of Staphylococcus aureus.J Appl Bacteriol. 1993; 75: 123-128Crossref PubMed Scopus (31) Google Scholar]. Ampicillin has recently been shown to reduce the production of listeriolysin [3Nichterlein T Domann E Kretschmar M et al.Sub-inhibitory concentrations of β-lactams and other cell-wall antibiotics inhibit listeriolysin production by Listeria monocytogenes.Int J Antimicrob Agents. 1996; 7: 75-81Abstract Full Text PDF PubMed Scopus (17) Google Scholar]. Listeriolysin is an essential virulence factor of L. monocytogenes because it enables the bacterium to reach the cytoplasm of infected cells, and to start multiplication [4Portnoy DA Chakraborty T Goebel W Cossart P Molecular determinants of Listeria monocytogenes pathogenesis.Infect Immun. 1992; 60: 1263-1270Crossref PubMed Google Scholar]. Inhibition of the production of listeriolysin could also contribute to the therapeutic action of gentamicin on human listeriosis. Therefore, the effect of gentamicin in subinhibitory concentrations on the expression of this virulence factor was determined. This was done as previously described [3Nichterlein T Domann E Kretschmar M et al.Sub-inhibitory concentrations of β-lactams and other cell-wall antibiotics inhibit listeriolysin production by Listeria monocytogenes.Int J Antimicrob Agents. 1996; 7: 75-81Abstract Full Text PDF PubMed Scopus (17) Google Scholar], using an assay measuring the hemolytic activity in the culture supernatants of L. monocytogenes EGD with and without the plasmids containing the β-galactosidase fusions and measuring the activity of the listeriolysin promoter contained on a plasmid fused to the lacZ gene of Escherichia coli. This allowed the measurement of the activity of the listeriolysin promoter as β-galactosidase activity in the transformed strain. The expression of β-galactosidase from this promoter was compared with that of a plasmid contained in L. monocytogenes EGD harboring the promoter of a streptococcal protease fused at the same EcoRI site to the lacZ gene. Subinhibitory concentrations of gentamicin were able to suppress the expression of β-galactosidase from the listeriolysin promoter in concentrations that did not affect growth or the expression of β-galactosidase from the streptococcal promoter (Figure 1). Therefore, gentamicin is able to selectively suppress the production of this virulence factor. Because gentamicin inhibits bacterial protein synthesis by binding to the ribosome, this action might be due to a selective suppression of the translation of mRNAs deriving from the virulence gene itself or from the regulatory gene prfA, whose product regulates the production of listeriolysin and several other virulence factors of L. monocytogenes [4Portnoy DA Chakraborty T Goebel W Cossart P Molecular determinants of Listeria monocytogenes pathogenesis.Infect Immun. 1992; 60: 1263-1270Crossref PubMed Google Scholar]. In contrast to the β-galactosidase assay, the reduction of the hemolytic activity of culture supernatants of the wild-type strains and the transformed strains was not significant. This may be explained by the fact that in contrast to β-galactosidase, listeriolysin is rapidly inactivated at the incubation temperature of 37 °C ([5Geoffroy C Gaillard J-L Alouf JE Berche P Production of thiol-dependent haemolysins by Listeria monocytogenes and related species.J Gen Microbiol. 1989; 135: 481-487PubMed Google Scholar] and own unpublished data), which may have led to greater standard deviations in this assay that make it difficult to demonstrate minor effects on listeriolysin production. In conclusion, subinhibitory concentrations of gentamicin that do not affect growth are able to reduce the production of an essential virulence factor of L. monocytogenes. In combination with a similar effect of ampicillin [3Nichterlein T Domann E Kretschmar M et al.Sub-inhibitory concentrations of β-lactams and other cell-wall antibiotics inhibit listeriolysin production by Listeria monocytogenes.Int J Antimicrob Agents. 1996; 7: 75-81Abstract Full Text PDF PubMed Scopus (17) Google Scholar], this might contribute to the therapeutic action of the two antibiotics in human listeriosis.

Subinhibitory concentrations of β-lactams and other cell-wall antibiotics inhibit listeriolysin production by Listeria monocytogenes

In the present study we tested the ability of β-lactam antibiotics and other cell-wall antibiotics to inhibit the production of listeriolysin which is the main virulence factor of L. monocytogenes. The amount of listeriolysin produced was measured in the supernatants with a sensitive hemolysin assay and as β-galactosidase expression from the listeriolysin promoter. When tested in concentrations that did not affect growth of the bacteria, all β-lactam antibiotics and all other cell-wall antibiotics tested were able to reduce the listeriolysin production in at least one of the tests used. Yet, no change in β-galactosidase activity was detected in a strain harboring a control β-galactosidase fusion. We therefore demonstrate for the first time that subinhibitory concentrations of antibiotics specifically reduce the production of a virulence factor of L. monocytogenes.

Inorganic Pyrophosphatase-Based Detection Systems: II. Detection and Quantification of Cell Lysis and Cell-Lysing Activity

A novel technique, useful for detection of cell lysis and cell-lysing activity, has been developed. The method can be used for detection of cell lysis, both induced and natural, in all types of cells. The technique can be used for detection and quantification of all types of cell-lysing activities, e.g., cell wall hydrolases, toxicants, phospholipases, and antibiotics. The method relies on the detection, by a very sensitive enzymatic luminometric inorganic pyrophosphate detection assay (ELIDA; P. Nyrén and A. Lundin (1985) Anal. Biochem. 151, 504-509) of an enzyme, inorganic pyrophosphatase, which is constitutively expressed in all cells. The fraction of lysed cells in a sample can be assessed by determining the activity in the absence and in the presence of total lysing activity. The technique was used for determination of the effect of storage conditions on the intactness of two different cells, Micrococcus luteticus and Saccharomyces cerevisiae. In a model system, the approach was also used for detection of cell-lysing activity. The activity of a cell wall hydrolase (lysozyme) and a surfactant (Triton X-100) was quantified. M. luteticus cells were incubated with a specific buffer containing the lysing activity and inorganic pyrophosphate. The amount of unhydrolyzed PP i was determined by the ELIDA. The amount of PP i hydrolyzed was proportional to the amount of lysozyme present. The sensitivity of the assay was dependent on several factors, such as amount of cells used, incubation time, and incubation temperature. Lysozyme at concentrations below 5 ng/ml (<50 pg) could be detected. The possibility of using the approach for detection of other types of cell-lysing activities is discussed. Possible applications for the approach in a wide variety of areas such as clinical laboratories, food and dairy industries, and the pharmaceutical industry are also discussed.

Sphingosines: antimicrobial barriers of the skin.

Among the factors that control the survival of microorganisms on human stratum corneum are skin lipids, including sphingosines. Because the antibacterial spectrum of sphinganine resembles that of cell wall antibiotics, electron microscopy of sphinganine-treated and untreated S. aureus was performed; the lipid induced multiple lesions of cell wall, membrane evaginations and loss of ribosomes. However, comparisons of minimal inhibitory concentration of sphinganine for coccal forms and L-forms of S. aureus, which lack cell walls, and of the respective dose-related reductions in colony-forming units demonstrated both the susceptibility of L-forms and their superior resistance. Therefore, cell wall lesions are sequelae of a probable membrane reaction. Candida albicans was susceptible to sphinganine, sphingosine, dimethylsphingosine, and to a lesser degree, stearylamine. Liquid assays of these lipids against Trichophyton mentagrophytes, T. tonsurans and Epidermatophyton floccosum established their high susceptibility to sphingosine and stearylamine. Sphinganine was the least effective, perhaps due to the presence of L isomers; T. tonsurans was the most sensitive. These four lipids were found to be fungistatic, preventing germination and retarding thalli. Antifungal efficacy was confirmed in vitro on stratum corneum.

D-alanyl-lipoteichoic acid in Lactobacillus casei: secretion of vesicles in response to benzylpenicillin.

Vesicles containing lipoteichoic acid (LTA) have been isolated from Lactobacillus casei ATCC 7469 grown in the presence of either benzylpenicillin or D-cycloserine. These cell wall antibiotics enhanced the rate of LTA and lipid secretion 6.7 times, whereas chloramphenicol inhibited their release. The formation of these vesicles from peripheral and septal wall regions did not appear to be the result of bacteriolysis. The vesicle composition of LTA and lipid was similar to that of the cytoplasmic membrane whereas the protein composition was dissimilar. The size of these vesicles ranged from 20 to 40 nm and the length of LTA ranged from 5 to 50 glycerol phosphate residues. The isolation of these vesicles provides a potential in vitro acceptor system for studying the D-alanylation of lipoteichoic acid.

Influence of antibiotics on fibronectin binding and phagocytosis of staphylococci by human polymorphonuclear leukocytes.

The effect of subbactericidal concentrations of cell wall and protein synthesis-inhibiting antibiotics on fibronectin-staphylococcal interactions was examined. Exposure of staphylococci to protein synthesis inhibitors resulted in a dose-dependent decrease in fibronectin's ability to bind to and promote the phagocytosis of treated organisms by human polymorphonuclear leukocytes. Cell wall inhibitors, on the other hand, had little or no effect on fibronectin binding or phagocytosis. Serum remained an effective opsonin despite antibiotic modulation of fibronectin binding site expression.

Rifampin Resistance

The incidence of methicillin-resistant staphylococcal infections, for which vancomycin hydrochloride remains the only active cell-wall antibiotic therapy, is rising. Some physicians have been combining other antibiotics with vancomycin in hopes of obtaining a more effective regimen for the therapy of these infections. Rifampin has been advocated as a concurrent second antibiotic because of its extraordinary potent bactericidal activity for Staphylococcus aureus. When rifampin is used in combination with a cell-wall antibiotic, suppression of the development of rifampin resistance has been thought possible. We report a case of infection caused by a methicillin-resistant S aureus in which the rifampin resistance occurred during therapy with vancomycin and rifampin. The rifampin resistance was stable and was present after ten serial broth and agar passages. Physicians are cautioned against the indiscriminant or routine use of rifampin as a second antibiotic in combination with vancomycin for the therapy of infections caused by S aureus.

Effects of cell wall inhibitors on cell division in Streptococcus mutans.

The addition of inhibitors of cell wall biosynthesis to exponential-phase cultures of Streptococcus mutans may do one of three things, depending on the concentrations used: (i) prevent cell division at a time coincident with the onset of chromosome replication, (ii) prevent cell division later in the cell cycle coincident with or near completion of septation, or (iii) lead to limited cell lysis. The relative tolerance of S. mutans to inhibitors of cell wall biosynthesis may be due to the fact that S. mutans cultures treated with low levels of cell wall antibiotics seem to be blocked at a stage before initiation of autolytic activity, whereas cultures treated with high levels of these antibiotics seem to be blocked after termination of the autolytic phase. Thus, the cells escape the lytic death that is seen in other streptococci exposed to inhibitors of cell wall biosynthesis.

Extracellular proteases increase tolerance of Bacillus subtilis to nafcillin.

Mutants of Bacillus subtilis capable of secreting high amounts of protease were highly tolerant to the lethal and lytic effects of nafcillin. Protease-deficient mutants were more susceptible. However, when subtilisin was added to exogenously to a protease-deficient strain, the organism assumed the characteristics of nafcillin tolerance. Similarly, when phenylmethylsulfonyl fluoride, a serine protease inhibitor, was added to the tolerant strains, they became susceptible to nafcillin-induced lysis. The effects of nafcillin on B. subtilis were studied with both viability determinations and assay of cellular lysis. The minimum inhibitory concentrations of nafcillin tended to be higher for the protease hyperproducing strains, but these values could be reduced by the protease inhibitor. No loss of antibiotic activity was observed when nafcillin was incubated with either subtilisin or trypsin. Furthermore, protease and autolysin from B. subtilis were not modified by nafcillin. The results showed that extracellular proteases could render B. subtilis relatively tolerant to the killing and lytic effects of a cell wall antibiotic. The proteases were probably acting on the autolysins of the organism, thereby increasing tolerance to nafcillin.

Lipoteichoic acid inhibition of phagocytosis of Staphylococcus aureus by human polymorphonuclear leukocytes

Under the influence of cell wall antibiotics, tolerant strains of Staphylococcus aureus have been shown to excrete increased amounts of lipoteichoic acid (LTA) into the growth medium, relative to nontolerant strains. The effect of LTA on the phagocytic and bactericidal rate of isolated neutrophils for S. aureus was studied to determine if the enhanced production of LTA by tolerant strains might account in part for the increased severity of infections due to tolerant organisms. Phagocytosis was measured as leukocyte-associated radioactivity after incubation of neutrophils, human serum, and radiolabeled S. aureus in microtiter wells. The addition of LTA, at final concentrations ranging from 0–5 nmol/ml, to the incubation mixtures resulted in a dose-dependent inhibition of phagocytosis. The decreased uptake of S. aureus by the neutrophils was accompanied by an increase in the number of organisms surviving in the presence of phagocytes. Such an inhibition was not observed when preopsonized bacteria were employed. These results suggest that LTA is able to interfere with the normal rapid opsonization of S. aureus in in vitro phagocytic systems.