Isolation and characterization of Staphylococcus aureus strains from a Paso del Norte dairy.

The primary purpose of this study was to determine if methicillin-resistant Staphylococcus aureus (MRSA) strains could be identified in the milk of dairy cattle in a Paso del Norte region dairy of the United States. Using physiological and PCR-based identification schemes, a total of 40 Staph. aureus strains were isolated from 29 raw milk samples of 133 total samples analyzed. Pulsed-field gel electrophoresis after digestion with the SmaI enzyme revealed that the 40 confirmed strains were represented by 5 pulsed-field types, which each contained 3 or more strains. Of 7 hospital strains isolated from cows undergoing antibiotic therapy, 3 demonstrated resistance to 3 or more antimicrobial classes and displayed similar pulsed-field gel electrophoresis patterns. A secondary purpose of this study was to elucidate the evolutionary relationships of strains isolated in this study to genomically characterized Staph. aureus strains. Therefore, Roche 454 GS (Roche Diagnostics Corp., Dallas, TX) pyrosequencing was used to produce draft genome sequences of an MRSA raw milk isolate (H29) and a methicillin-susceptible Staph. aureus (PB32). Analysis using the BLASTn database (http://blast.ncbi.nlm.nih.gov/) demonstrated that the H29 draft genome was highly homologous to the human MRSA strain JH1, yet the ß-lactamase plasmid carried by H29 was different from that carried by JH1. Genomic analysis of H29 also clearly explained the multidrug resistance phenotype of this raw milk isolate. Analysis of the PB32 draft genome (using BLASTn) demonstrated that this raw milk isolate was most related to human MRSA strain 04-02981. Although PB32 is not a MRSA, the PB32 draft genome did reveal the presence of a unique staphylococcal cassette mec (SCCmec) remnant. In addition, the PB32 draft genome revealed the presence of a novel bovine staphylococcal pathogenicity island, SaPIbovPB32. This study demonstrates the presence of clones closely related to human and (or) bovine Staph. aureus strains circulating in a dairy herd.

Genetic changes that correlate with the pine-oil disinfectant-reduced susceptibility mechanism of Staphylococcus aureus.

AIMS: To identify factors associated with the Staphylococcus aureus pine-oil disinfectant-reduced-susceptibility (PD(RS)) mechanism and to describe one possible PD(RS) model. METHODS AND RESULTS: Comparative genomic sequencing (CGS) and microarray analysis were utilized to detect mutations and transcriptome alterations that occur in a S. aureus PD(RS) mutant. Mutant analysis, antimicrobial gradient plates, growth studies and 3-hydroxy-3-methylglutaryl coenzyme A synthase assays were then performed to confirm the biological consequences of the 'omics' alterations detected in a PD(RS) mutant. CGS uncovered three mutations in a PD(RS) mutant in a(n): alcohol dehydrogenase (adh), catabolite control protein A (ccpA) and an NADPH-flavin oxidoreductase (frp). These mutations lead to increased growth rates; increased transcription of an NAD-dependent D-lactate dehydrogenase gene (ddh); and increased flux through the mevalonate pathway. PD(RS) mutants demonstrated reduced susceptibility to bacitracin and farnesol, and one PD(RS) mutant displayed upregulation of bacA, a bacitracin-resistance gene. Collectively, this evidence demonstrates altered undecaprenol metabolism in PD(RS) mutants. CONCLUSIONS: The PD(RS) mechanism proposed results from increased catabolic capabilities and increased flux through the mevalonate pathway as well as altered bactoprenol physiology. SIGNIFICANCE AND IMPACT OF THE STUDY: A novel mechanism that bacteria utilize to overcome the killing effects of PD formulations is proposed that is unique from the PD(RS) mechanism of the enterobacteraciae.

Characterization of Staphylococcus aureus mutants expressing reduced susceptibility to common house-cleaners.

AIMS: To characterize mutants of Staphylococcus aureus expressing reduced susceptibility to house cleaners (HC), assess the impact of the alternative sigma factor SigB on HC susceptibility, and determine the MIC of clinical methicillin-resistant S. aureus (MRSA) to a HC. METHODS AND RESULTS: Susceptibility to HC, HC components, H2O2, vancomycin and oxacillin and physiological parameters were determined for HC-reduced susceptibility (HCRS) mutants, parent strain COL and COLsigB::kan. HCRS mutants selected with three HC expressed reduced susceptibility to multiple HC, HC components, H2O2 and vancomycin. Two unique HCRS mutants also lost the methicillin resistance determinant. In addition, all HCRS mutants exhibited better growth at two temperatures, and one HCRS mutant expressed reduced carotenoid production. COLsigB::kan demonstrated increased susceptibility to all HC and many HC components. sigB operon mutations were not detected in one HCRS mutant background. Of 76 clinical MRSA, 20 exhibited reduced susceptibility to a HC. CONCLUSIONS: HCRS mutants demonstrate altered susceptibility to multiple antimicrobials. While sigB is required for full HC resistance, one HCRS mechanism does not involve sigB operon mutations. Clinical MRSA expressing reduced susceptibility to a common HC were detected. SIGNIFICANCE AND IMPACT OF THE STUDY: This study suggests that HCRS mutants are not protected against, nor selected by, practical HC concentrations.

The multidrug efflux pump NorA is not required for salicylate-induced reduction in drug accumulation by Staphylococcus aureus.

Growth of Staphylococcus aureus in the presence of salicylate leads to reduced ciprofloxacin and ethidium accumulation and increased resistance to ethidium. Salicylate induced reduction in ciprofloxacin accumulation is energy-independent while salicylate induced alterations in ethidium accumulation and efflux is proton motive force-dependent. NorA is an intrinsic multidrug efflux pump that contributes to intrinsic levels of fluoroquinolone and ethidium resistance in S. aureus. The NorA inhibitor reserpine did not dramatically affect the ability of salicylate to induce increased ciprofloxacin and ethidium resistance. Inactivation of norA did not alter the ability of salicylate to induce increased ciprofloxacin and ethidium resistance levels and a reduction in ciprofloxacin accumulation. These data demonstrate that NorA is not absolutely required for the salicylate-inducible multidrug resistance mechanism of S. aureus.

The bacterial multiple antibiotic resistant (Mar) phenotype leads to increased tolerance to tea tree oil.

Mutants of Escherichia coil strain AG100 exhibiting the multiple antibiotic resistance (Mar) phenotype demonstrated a greater level of tolerance to tea tree oil (TTO) compared with the parent strain. The ability of TTO to kill all E. coil strains studied was greater at 37 than at 30 degrees C. Growth of parent strain AG100 in the presence of salicylate, which induces the mar operon leading to the Mar phenotype, also increased tolerance to TTO. Escherichia coli Mar mutant YL1 demonstrated greater tolerance to antimicrobial terpenes found in TTO and did not leak K+ as rapidly in the presence of TTO when compared with its parent strain AG100. Attempts to isolate Mar mutants of Staphylococcus aureus using tetracycline gradients proved unsuccessful. However, when grown in the presence of salicylate, S. aureus strain BB255 demonstrated greater tolerance to TTO and did not leak K+ as rapidly in the presence of TTO compared with this strain grown without additions. This evidence demonstrates that bacterial Mar phenotypes increase tolerance to the killing action of TTO. This work also adds indirect evidence that the target of TTO is the cell membrane.

The effects of salicylate on bacteria.

Salicylate and related compounds, such as aspirin, have a variety of effects in eucaryotic systems and are well known for their medicinal properties. Salicylate also has numerous effects on bacteria, yet only a handful of individuals within the scientific community appreciate these findings. From a bacterial viewpoint, growth in the presence of salicylate can be both beneficial and detrimental. On one hand, growth of certain bacteria in the presence of salicylate can induce an intrinsic multiple antibiotic resistance phenotype. On the other hand, growth in the presence of salicylate can reduce the resistance to some antibiotics and affect virulence factor production in some bacteria. This review provides an overview of the effects salicylate has on various bacterial species.

The mode of antimicrobial action of the essential oil of Melaleuca alternifolia (tea tree oil).

The essential oil of Melaleuca alternifolia (tea tree) exhibits broad-spectrum antimicrobial activity. Its mode of action against the Gram-negative bacterium Escherichia coli AG100, the Gram-positive bacterium Staphylococcus aureus NCTC 8325, and the yeast Candida albicans has been investigated using a range of methods. We report that exposing these organisms to minimum inhibitory and minimum bactericidal/fungicidal concentrations of tea tree oil inhibited respiration and increased the permeability of bacterial cytoplasmic and yeast plasma membranes as indicated by uptake of propidium iodide. In the case of E. coli and Staph. aureus, tea tree oil also caused potassium ion leakage. Differences in the susceptibility of the test organisms to tea tree oil were also observed and these are interpreted in terms of variations in the rate of monoterpene penetration through cell wall and cell membrane structures. The ability of tea tree oil to disrupt the permeability barrier of cell membrane structures and the accompanying loss of chemiosmotic control is the most likely source of its lethal action at minimum inhibitory levels.

Effects of salicylate and related compounds on fusidic acid MICs in Staphylococcus aureus.

Salicylate, acetyl-salicylate, benzoate and ibuprofen increased fusidic acid MICs for fusidic acid-resistant and -susceptible strains of Staphylococcus aureus representing six genetic lineages. The effects of these substances on fusidic acid resistance levels occurred in a strain-dependent manner. The weak acid acetate, and acetaminophen did not alter fusidic acid resistance levels, while the addition of saligenin, the alcohol of salicylate, reduced gradient plate MICs for all strains studied. These findings indicate that a benzoic acid structure is required for the induction of increased intrinsic fusidic acid resistance levels. When 2 mM salicylate was added to media used in population analyses, the number of cells able to survive on high concentrations of fusidic acid increased. This increase in cell survival was observed in two unrelated fusidic acid-resistant strains, with chromosomal (WBG8287) or plasmid (WBG1576) mediated resistance determinants and two unrelated susceptible strains. The salicylate-induced increase in fusidic acid resistance was phenotypic at low fusidic acid concentrations (relative to resistance phenotype) for WBG8287 and a fusidic acid-susceptible strain. On media containing salicylate and high fusidic acid concentrations, the mutation frequency to higher fusidic acid resistance levels was greater for WBG8287, compared with unsupplemented fusidic acid-containing media. These experiments provide evidence for a novel salicylate inducible fusidic acid resistance mechanism in S. aureus.

Growth in the presence of salicylate increases fluoroquinolone resistance in Staphylococcus aureus.

Salicylate and acetylsalicylate slightly increased fluoroquinolone resistance in ciprofloxacin-susceptible and -resistant Staphylococcus aureus. Salicylate allowed a greater number of cells from ciprofloxacin-susceptible and -resistant strains to survive on high fluoroquinolone concentrations. Salicylate also increased the frequency with which a susceptible strain mutated to become more resistant to ciprofloxacin.

Heterogeneous expression of fusidic acid resistance in Staphylococcus aureus with plasmid or chromosomally encoded fusidic acid resistance genes.

Fusidic acid resistance expression in a methicillin susceptible Staphylococcus aureus strain (WBG1576), which carries fusidic acid resistance on plasmid pUB101, and a prevalent Western Australian methicillin-fusidic acid resistant strain (WBG8287) were compared. WBG8287 carries fusidic acid resistance on the chromosome and its plasmid content has no effect on the levels of this resistance. WBG1576 and WBG8287 exhibited similar heterogeneous populations in respect to fusidic acid resistance levels in population analyses. A high-level fusidic acid resistant mutant of WBG1576 (BE8) had alterations in Smal chromosomal profiles, but not in plasmid size or resistance expression. Mutations causing increased fusidic acid resistance in WBG1576 are chromosomally located. A high-level fusidic acid resistant mutant of WBG8287 (BE3) had no alterations in Smal chromosomal profiles, or plasmid content and resistances. Comparison of resistance levels to kanamycin and spectinomycin, between high-level resistant colonies of WBG8287 and WBG8287, indicate that mutations in the chromosomal gene fusA, which encodes elongation factor-G, are probably the cause of the increased resistance levels observed in these mutant strains.

Conditions that induce Staphylococcus aureus heat shock proteins also inhibit autolysis.

When Staphylococcus aureus strain 8325 was grown at 30 degrees C and heat shocked at 40 degrees C the rate of cell autolysis in buffer with or without Triton X-100 was reduced. Treatment of growing cells with other agents (CdCl2, ethanol, NaCl) known to induce heat shock proteins also resulted in cells that showed a decreased rate of autolysis. Heat shocked cells showed lower rates of freeze-thaw autolysin activity on purified cell walls, and isolated crude cell walls from heat shocked cells had lower rates of autolytic activity compared to controls. No differences in the peptidoglycan hydrolase activity profiles of control and heat shocked cells were detected by renaturing sodium dodecyl sulfate polyacrylamide gel electrophoresis. It is proposed that autolysins are damaged by heat shock and their targeting to the cell wall is impaired, possibly by complexing with heat shock proteins, which may also inhibit autolysin activity. Heat shock also inhibited the autolytic activity of methicillin-resistant and related-susceptible strains, and the possible relationship of this to the expression of methicillin resistance is discussed.

Tea tree oil causes K+ leakage and inhibits respiration in Escherichia coli.

Concentrations of tea tree oil (TTO) which inhibit or decrease growth of Escherichia coli also inhibit glucose-dependent respiration and stimulate the leakage of intracellular K+. Stationary phase cells are more tolerant to these TTO effects than exponential phase cells.

Effects of tea tree oil on Escherichia coli.

Tea tree oil (TTO) stimulates autolysis in exponential and stationary phase cells of Escherichia coli. Electron micrographs of cells grown in the presence of TTO showed the loss of electron dense material, coagulation of cell cytoplasm and formation of extracellular blebs. Stationary phase cells demonstrated less TTO-stimulated autolysis and also had greater tolerance to TTO-induced cell death, compared to exponentially grown cells. It was also revealed that subpopulation of stationary phase cells demonstrated increased tolerance to TTO-bactericidal effects.

Antitumor activity of bacteria and bacterial products: enhancement of the tumor-protective effect of bacteria by lipoteichoic acid.

The ability of some microbial agents and/or their products to affect local tumor growth was assessed in the D-12 DA rat ascites tumor model. Various bacteria and bacterial products markedly enhanced tumor resistance when injected i.p. several days before tumor cell challenge. The tumor-protective effect of these compounds was amplified further by lipoteichoic acid (LTA) inoculated i.p. a few days after tumor cell challenge. Under these conditions, the majority of animals did not exhibit progressive tumor growth.

The macrophage response to bacteria. Modulation of macrophage functional activity by peptidoglycan from Moraxella (Branhamella) catarrhalis.

Moraxella (Branhamella) catarrhalis organisms have been shown to be particularly efficient in inducing in a pure population of bone marrow-derived mononuclear phagocytes secretory and cellular activities. In the present study, the ability of peptidoglycan from this Gram-negative organism to trigger a macrophage response was compared with that elicited by peptidoglycan from Staphylococcus aureus and Bacillus subtilis. The results show that the three peptidoglycans were similarly active in triggering the secretion of tumour necrosis factor and tumouricidal activity but differed considerably in their ability to induce the generation of nitrite in macrophages; in this respect, peptidoglycan from M. catarrhalis was particularly potent. The impressive capacity of M. catarrhalis peptidoglycan to induce in low concentration the secretion of tumour necrosis factor and nitrite and tumouricidal activity may, in addition to its lipopolysaccharide, contribute to the extraordinary potential of this organism to trigger the functional activities of macrophages.

Mapping and characterization of multiple chromosomal factors involved in methicillin resistance in Staphylococcus aureus.

Chromosomal factors, termed fem or aux factors, are needed for the expression of methicillin resistance in methicillin-resistant (Mcr) Staphylococcus aureus; also needed is the mec-encoded low-affinity penicillin-binding protein PBP 2'. These factors make up part of the normal set of genes present in susceptible and resistant strains of S. aureus and can be identified by Tn551-mediated insertional inactivation of the methicillin resistance. In this study, we characterized different Tn551 inserts and mapped them into four distinct loci on the SmaI chromosomal map of S. aureus NCTC 8325, thereby identifying two new loci which code for fem factors. The largest fragment, SmaI-A, carries three loci, two coding for both closely linked factors femA and femB and a novel third locus (femC) that is not linked to the other two. An additional, fourth, locus, femD, was identified in fragment SmaI-I. femA and femB inactivation reduced overall methicillin resistance, whereby femB had less of an influence on the resistance level. femC and femD inactivation reduced mainly the basal resistance level in heterogeneously Mcr strains and had less of an impact on the subpopulation with high-level resistance. Inactivation of either of these factors was shown to have no influence on the production of PBP 2', the main factor mediating methicillin resistance. In addition, no changes were observed in the banding patterns of the major autolysins in whole-cell extracts of the fem mutants, suggesting that the reduced cell wall turnover and autolysis observed in some of the insertionally inactivated strains were due to changes either of the substrate or in the autolysin control.

Autolysis of methicillin-resistant and -susceptible Staphylococcus aureus.

The autolytic activities, including unstimulated, Triton X-100-stimulated, and daptomycin-induced, of various sets of methicillin-resistant and related methicillin-susceptible strains were compared. Faster rates of autolysis were noted in two heterogeneous methicillin-resistant transductants than in their methicillin-susceptible parental recipients, in a heterogeneous resistant strain than in a susceptible derivative created by chemical mutagenesis, and in a homogeneous resistant strain than in a derivative that had decreased methicillin resistance and was created by transposon Tn551 mutagenesis. These results suggest that the presence of the methicillin resistance region, mec, either directly or indirectly through an interaction with other host genes, confers a faster rate of autolysis on strains. Various auxilliary genes are known to affect methicillin resistance expression, and one of these genes, femA, was necessary for the expression of this faster rate of autolysis. These differences in autolytic activities were not observed in isolated crude cell walls retaining autolytic activities, suggesting different modes of regulation of autolysins in intact cells and isolated walls. In contrast, one homogeneous, highly resistant strain, DU4916, had a lower autolytic activity than did derived heterogeneous resistant and susceptible strains created by chemical mutagenesis and a strain that had decreased resistance and was created by transposon mutagenesis. Our observations suggest that methicillin resistance expression is associated with an enhanced rate of autolysis, in heterogeneous resistant strains at least.

Lower autolytic activity in a homogeneous methicillin-resistant Staphylococcus aureus strain compared to derived heterogeneous-resistant and susceptible strains.

It has been proposed that in addition to production of a penicillin-binding protein with low affinity for beta-lactam antibiotics, control of autolysin activity is involved in the mechanism of staphylococcal methicillin resistance. A homogeneous methicillin-resistant Staphylococcus aureus strain (DU4916) had lower rates of unstimulated, NaCl- and Triton X-100-stimulated autolysis, and daptomycin (LY146032)-induced lysis than a heterogeneous methicillin-resistant strain (DU4916-K7) and a methicillin-susceptible strain (DU4916S) derived from DU4916.

Contraindications to the repositioning of fractured or dislocated limbs in the fields.

Supracondylar fractures of the femor or humerus and fractures of the radius, ulna, tibia or fibula, plus crush injuries of the limbs, most likely should not be manipulated in the field because of the potential for traumatizing adjacent nerves, veins and arteries. An emergency splinting system that allows immobilization without repositioning has been developed. Furthermore, fractures in or about the elbow, wrist, knee, or ankle should not be subjected to reduction in the field, unless there are compelling reasons to justify the risk of producing significant additional trauma. Conservative immobilization without repositioning may yield a more positive prognosis for the patient.