Activity of Gallidermin on Staphylococcus aureus and Staphylococcus epidermidis Biofilms

Due to their abilities to form strong biofilms, Staphylococcus aureus and Staphylococcus epidermidis are the most frequently isolated pathogens in persistent and chronic implant-associated infections. As biofilm-embedded bacteria are more resistant to antibiotics and the immune system, they are extremely difficult to treat. Therefore, biofilm-active antibiotics are a major challenge. Here we investigated the effect of the lantibiotic gallidermin on two representative biofilm-forming staphylococcal species. Gallidermin inhibits not only the growth of staphylococci in a dose-dependent manner but also efficiently prevents biofilm formation by both species. The effect on biofilm might be due to repression of biofilm-related targets, such as ica (intercellular adhesin) and atl (major autolysin). However, gallidermin''s killing activity on 24-h and 5-day-old biofilms was significantly decreased. A subpopulation of 0.1 to 1.0% of cells survived, comprising “persister” cells of an unknown genetic and physiological state. Like many other antibiotics, gallidermin showed only limited activity on cells within mature biofilms.     

Effects of Azithromycin in Combination with Vancomycin,Daptomycin, Fosfomycin,Tigecycline, and Ceftriaxone on Staphylococcus epidermidis Biofilms

Staphylococcal biofilms on surgical implants are the underlying cause of a lack of response to antimicrobial treatment. We investigated the effects of vancomycin (VAN), daptomycin (DAP), fosfomycin (FOS), tigecycline (TGC), and ceftriaxone (CRX), alone and in combination with azithromycin (AZI), on established biofilms of Staphylococcus epidermidis. Biofilms were studied using the static microtiter plate model with established S. epidermidis biofilms, with an initial inoculum of 10⁶/ml in 96-well polystyrene flat-bottom microtiter plates. Biofilms were inoculated with VAN, DAP, FOS, TGC, or CRX at two concentrations, alone or in combination with AZI (2, 512, or 1,024 mg/liter). To assess the reduction in biomass, the optical density ratio (ODr), calculated as (optical density [OD] of the treated biofilm)/(OD of the untreated biofilm, taken as 1), was used. For antibacterial efficacy, the viable bacterial count was used. Reductions in the biofilm ODr were observed for VAN (15 and 40 mg/liter) and FOS (200 mg/liter) only (ODr [mean ± standard deviation] for VAN at 15 and 40 mg/liter, 0.77 ± 0.32 and 0.8 ± 0.35, respectively; ODr for FOS at 200 mg/liter, 0.78 ± 0.26; P < 0.05), but not for DAP (2 and 5 mg/liter), TGC (0.2 and 2 mg/liter), or CRX (600 and 2,400 mg/liter). The addition of AZI had no further effect on the ODr, but a significant reduction of bacterial growth was achieved with high doses of AZI plus TGC or AZI plus CRX (a 3-log count reduction for AZI at 1,024 mg/liter plus CRX at 600 mg/liter and for AZI at 512 or 1,024 mg/liter plus CRX at 2,400 mg/liter; a 2-log count reduction for AZI at 512 or 1,024 mg/liter plus TGC at 2 mg/liter [P < 0.05]). No significant reduction in bacterial growth was observed for FOS (50 and 200 mg/liter), DAP (2 and 5 mg/liter), or TGC (0.2 mg/liter) in combination with AZI. None of the antibiotics at either concentration reduced the bacterial count of the biofilms when used alone. Thus, the use of a combination of AZI plus TGC, FOS, or CRX at high concentrations has little effect on biofilm density but significantly reduces bacterial growth.Staphylococcus epidermidis is the leading pathogen causing infections of surgical implants. Given the high incidence of fracture fixation devices, 2 million per annum, the number of implant infections amounts to 100,000 per year in the United States (10). Many of these infections are associated with biofilms formed by staphylococci on implant surfaces (9, 26, 34). The biofilm consists of a structured community of bacterial cells enclosed in a self-produced polymeric matrix and adherent to an inert or living surface. The two consequences of biofilm formation on implant surfaces are increased resistance to antimicrobial agents and frequent failure of conventional antimicrobial therapy. This resistance of bacteria within biofilms is attributed to a possible barrier function of the biofilm, binding of the antimicrobial agents within the matrix, and the metabolic change in the bacterial cells. Thus, infection of medical implants is associated with considerable morbidity and costs due to loss of mobility, nonproductive time, and health care (10, 18, 35).The antimicrobial agents most widely used for staphylococcal infections are beta-lactam antibiotics, primarily cephalosporins, and in the case of beta-lactam resistance, vancomycin (23, 28). Alternative agents are intravenous fosfomycin, a small-molecule antibiotic with a wide antibacterial spectrum and excellent tissue penetration, and two newer antibiotics, the glycylcycline tigecycline and the cyclic lipopeptide daptomycin (14). Daptomycin, which is highly active against gram-positive cocci resistant to commonly used antibiotics, including methicillin (meticillin)-resistant staphylococci, causes membrane changes in the bacteria, whereas tigecycline inhibits protein synthesis (20). Although macrolides are not commonly used in the treatment of staphylococcal infections, clinical and experimental data suggest that azithromycin decreases biofilm formation and enhances the efficacy of other antibiotics for patients with cystic fibrosis and Pseudomonas infections (17, 19).The MICs of antimicrobial agents tested on bacterial biofilms are dramatically increased, up to concentrations >1,000 times the MICs for staphylococci under planktonic conditions (5). However, there is some evidence that higher antibiotic concentrations reduce biofilms and bacterial growth, whereas standard concentrations are not effective (24, 30). In a previous study, we demonstrated that antibiotic concentrations equivalent to 100 times the MIC under planktonic conditions did not decrease established staphylococcal biofilms (16). To investigate if even higher concentrations will overcome the bacterial resistance within biofilms and reduce biofilm thickness and bacterial growth, static biofilms of clinical S. epidermidis isolates causing implant infections and catheter-associated bacteremia were incubated with vancomycin, daptomycin, fosfomycin, tigecycline, or ceftriaxone at two concentrations. To explore the additional effect of the macrolide azithromycin (22) on S. epidermidis biofilms, we treated the biofilms with vancomycin, daptomycin, fosfomycin, tigecycline, or ceftriaxone in combination with azithromycin at three concentrations (2, 512, and 1,024 mg/liter).(Part of this research was presented as a poster at the 47th Interscience Conference on Antimicrobial Agents and Chemotherapy, Chicago, IL, 2007 [29a].)     

In Vitro Studies of Pharmacodynamic Properties of Vancomycin against Staphylococcus aureus and Staphylococcus epidermidis

The bactericidal activities of vancomycin against two reference strains and two clinical isolates of Staphylococcus aureus and Staphylococcus epidermidis were studied with five different concentrations ranging from 2× to 64× the MIC. The decrease in the numbers of CFU at 24 h was at least 3 log₁₀ CFU/ml for all strains. No concentration-dependent killing was observed. The postantibiotic effect (PAE) was determined by obtaining viable counts for two of the reference strains, and the viable counts varied markedly: 1.2 h for S. aureus and 6.0 h for S. epidermidis. The determinations of the PAE, the postantibiotic sub-MIC effect (PA SME), and the sub-MIC effect (SME) for all strains were done with BioScreen C, a computerized incubator for bacteria. The PA SMEs were longer than the SMEs for all strains tested. A newly developed in vitro kinetic model was used to expose the bacteria to continuously decreasing concentrations of vancomycin. A filter prevented the loss of bacteria during the experiments. One reference strain each of S. aureus and S. epidermidis and two clinical isolates of S. aureus were exposed to an initial concentration of 10× the MIC of vancomycin with two different half-lives (t_1/2s): 1 or 5 h. The post-MIC effect (PME) was calculated as the difference in time for the bacteria to grow 1 log₁₀ CFU/ml from the numbers of CFU obtained at the time when the MIC was reached and the corresponding time for an unexposed control culture. The difference in PME between the strains was not as pronounced as that for the PAE. Furthermore, the PME was shorter when a t_1/2 of 5 h (approximate terminal t_1/2 in humans) was used. The PMEs at t_1/2s of 1 and 5 h were 6.5 and 3.6 h, respectively, for S. aureus. The corresponding figures for S. epidermidis were 10.3 and less than 6 h. The shorter PMEs achieved with a t_1/2 of 5 h and the lack of concentration-dependent killing indicate that the time above the MIC is the parameter most important for the efficacy of vancomycin.     

Spatial and Temporal Patterns of Biocide Action against Staphylococcus epidermidis Biofilms

The dynamic antimicrobial action of chlorine, a quaternary ammonium compound, glutaraldehyde, and nisin within biofilm cell clusters of Staphylococcus epidermidis was investigated using time-lapse confocal scanning laser microscopy. The technique allowed for the simultaneous imaging of changes in biofilm structure and disruption of cellular membrane integrity through the loss of an unbound fluorophore loaded into bacterial cells prior to antimicrobial challenge. Each of the four antimicrobial agents produced distinct spatial and temporal patterns of fluorescence loss. The antimicrobial action of chlorine was localized around the periphery of biofilm cell clusters. Chlorine was the only antimicrobial agent that caused any biofilm removal. Treatment with the quaternary ammonium compound caused membrane permeabilization that started at the periphery of cell clusters, then migrated steadily inward. A secondary pattern superimposed on the penetration dynamic suggested a subpopulation of less-susceptible cells. These bacteria lost fluorescence much more slowly than the majority of the population. Nisin caused a rapid and uniform loss of green fluorescence from all parts of the biofilm without any removal of biofilm. Glutaraldehyde caused no biofilm removal and also no loss of membrane integrity. Measurements of biocide penetration and action time at the center of cell clusters yielded 46 min for 10 mg liter⁻¹ chlorine, 21 min for 50 mg liter⁻¹ chlorine, 25 min for the quaternary ammonium compound, and 4 min for nisin. These results underscore the distinction between biofilm removal and killing and reinforce the critical role of biocide reactivity in determining the rate of biofilm penetration.The action of a biocide or antibiotic against microorganisms in biofilms varies in time and space. There is insight to be gained into the phenomena important in this process by watching, through a microscope, the antimicrobial attack. Here we describe the application of a recently developed technique for visualizing antimicrobial action (29) to biofilms formed by Staphylococcus epidermidis. We describe distinct behaviors for the four antimicrobial agents examined, which were chlorine, glutaraldehyde, a quaternary ammonium compound (QAC), and an antimicrobial peptide, nisin.S. epidermidis, a commensal resident of the skin and an opportunistic pathogen, is a common culprit in nosocomial infections (13, 15, 31). In particular, this microorganism is known to form biofilms on indwelling devices such as catheters, prosthetic joints, and contact lenses. There is therefore interest in understanding the efficacy of biocides against biofilms formed by this organism in such applications as control of contamination on hospital countertops and in catheter lock solutions, skin disinfectants, and contact lens storage case disinfection.Numerous evaluations of biocide activity against S. epidermidis biofilms have been reported for such agents as chlorhexidine, hydrogen peroxide, povidone-iodine, alcohols, and chlorine (4, 6, 10, 17, 19, 21, 32). These data support the accepted paradigm that bacteria in biofilms are more difficult to kill than are the same microorganisms when in free aqueous suspension. Analytical techniques such as colony formation assays (plating), regrowth assays, crystal violet staining for total biomass, tetrazolium salt reduction, and ATP content measurement have been employed. These measurements provide an indication of overall efficacy, in terms of either viability reduction or biofilm removal, but do not shed light on fundamental phenomena important in the interaction of the biocide with the biofilm. In other words, there is little or no mechanistic insight that can be gleaned from these conventional testing data.The technique described in this article affords information about the time scale for penetration of biologically active concentrations of the antimicrobial agent into the biofilm interior, the failure of most of the antimicrobials to induce any release of biomass from the biofilm, the important role of hydrodynamics outside the biofilm in removing biofilm weakened by reaction with chlorine, the lack of correlation of antimicrobial molecular weight with observed penetration time, and the presence of a subpopulation of cells that are less susceptible to an antimicrobial.     

Vancomycin and Daptomycin Pharmacodynamics Differ against a Site-Directed Staphylococcus epidermidis Mutant Displaying the Small-Colony-Variant Phenotype

Catheter-related bloodstream infections due to slow-growing Staphylococcus epidermidis small-colony variants (SCVs) are extremely difficult to treat. Daptomycin and vancomycin pharmacodynamics were evaluated against a site-directed hemB mutant of S. epidermidis displaying the SCV phenotype and compared to that of the parental strain. The maximal killing effect decreased by 7.7-fold for vancomycin and 1.5-fold for daptomycin against the SCV mutant and were well characterized by a Hill-type mathematical model (R² > 0.97).Staphylococcus epidermidis is the most common pathogen involved in catheter-related bloodstream infections (CRBSIs) (8). Clinical experience demonstrates that agents with proven in vitro activity are often unable to cure these infections when infected-device salvage is attempted. The persistent and often recurrent course of device-associated infections has been linked, in part, to the ability of S. epidermidis to establish adherent, multilayered biofilms on surfaces of inserted or implanted foreign bodies (15-17). In addition, infections caused by these pathogens are extremely difficult to treat due to the emergence of multidrug resistance and reduced susceptibility to vancomycin (5, 8).A number of recurrent foreign-body-associated infections due to coagulase-negative staphylococcal small-colony variants (SCVs), including several pacemaker-related infections, have been described (2, 9, 13, 16). Reduced susceptibility and tolerance to a variety of antimicrobials, including aminoglycosides, trimethoprim-sulfamethoxazole, and vancomycin, have been described and may complicate management of infections due to phenotypic variants. While antibiotic killing activity and pharmacodynamic parameters have been studied in detail for Staphylococcus aureus SCVs (3, 9, 12), data on such phenotypic variants of Staphylococcus epidermidis are missing. Since SCVs recovered from clinical specimens have been genetically undefined and exhibit a high rate of reversion to the large-colony form, a clinically derived, site-directed S. epidermidis mutant was constructed by interrupting one of the hemin-biosynthetic genes, hemB, in S. epidermidis by inserting an ermB cassette into hemB (1). Therefore, this S. epidermidis mutant along with the corresponding parent strain with normal phenotype was utilized as a tool to compare vancomycin and daptomycin pharmacodynamic parameters.The bacterial strains utilized in this study were S. epidermidis O-47 and its respective hemB mutant O-47 hemB::ermB, which exhibits a stable SCV phenotype (1, 6). The construction of the mutant and its characteristics were previously described (1). Analytical-grade daptomycin powder was obtained from Cubist Pharmaceuticals, Lexington, MA. Analytical-grade vancomycin powder was obtained from Sigma Chemical Co., St. Louis, MO. Fresh working solutions of daptomycin and vancomycin were made prior to each experimental run. Mueller-Hinton broth (Difco, Detroit, MI) supplemented with 25 mg/liter calcium and 12.5 mg/liter magnesium was utilized for all experiments involving vancomycin; Mueller-Hinton broth supplemented with 50 mg/liter calcium and 12.5 mg/liter magnesium was utilized for all experiments involving daptomycin. MICs were determined by quadruplicate broth microdilution techniques in accordance with standards of the Clinical and Laboratory Standards Institute (4). Time-kill experiments were performed as previously described (12). The following concentrations for daptomycin and vancomycin were evaluated: 0, 0.5, 1, 2, 4, 8, 16, 32, 64, and 128 mg/liter against a starting inoculum of S. epidermidis of approximately 10⁷ CFU/ml. Samples were withdrawn for determination of bacterial counts at 0, 2, 4, 8, and 24 h. All time experiments were completed in duplicate.An integrated pharmacokinetic-pharmacodynamic area measure (log ratio area) was applied to all CFU data as previously described (13), using equation 1. The traditional approach (log ratio change) was also used (equation 2). See reference 13 for more details. (1) (2) A four-parameter concentration-effect Hill-type model was fit to the effect parameter using Systat (version 12; Systat Software Inc., San Jose, CA) as previously described (12) and equation 3. (3) AU CFU_drug is the number of CFU of the culture with drug (in arbitrary units), AU CFU_{growth control} is the number of CFU of the culture with controlled growth, the dependent variable (E) is either the log ratio area or the log ratio change, E₀ is the measured effect at a zero drug concentration, E_max is the maximal effect, C is the concentration of drug, EC₅₀ is the concentration for which there is a 50% maximal effect, and H is the Hill or sigmoidicity constant.Against S. epidermidis strain O-47 and its hemB mutant, which displays the SCV phenotype, the MICs of vancomycin were 2.0 and 4.0 mg/liter and of daptomycin were 0.25 and 0.25 mg/liter, respectively. The antibacterial activities and pharmacodynamics of vancomycin and daptomycin against the O-47 strain and the hemB mutant are displayed in Fig. ​Fig.1.1. Vancomycin achieved bactericidal activity against the parent strain with a normal phenotype at concentrations of >4 mg/liter, which occurred at 24 h. At higher concentrations, vancomycin demonstrated concentration-independent killing, with apparent thresholds of 4 and 8 mg/liter, after which increases in drug concentration did not result in subsequent increases in killing activity. Daptomycin displayed bactericidal activity against the parent strain at concentrations of >2 mg/liter. However, with daptomycin, a greater concentration-dependent trend was observed, with increasing concentrations resulting in greater reduction in bacterial colonies. In contrast, vancomycin achieved little activity against the hemB mutant, which displayed the SCV phenotype, even at a concentration of 128 mg/liter, with maximal reductions of less than 1 log in bacterial counts after 24 h. However, for daptomycin, although early killing of the mutant was attenuated, with concentrations of >16 mg/liter achieving bactericidal activity by 8 h, all concentrations of >4 mg/liter were able to achieve bactericidal activity at 24 h.Open in a separate windowFIG. 1.Vancomycin (Vanco) and daptomycin (Dapto) time-kill experiments evaluating their bactericidal activities versus the S. epidermidis strain O-47, displaying the normal phenotype (N), and the corresponding hemB mutant, displaying the SCV phenotype (SCV). (A) Vancomycin versus O-47; (B) vancomycin versus the hemB mutant; (C) daptomycin versus O-47; (D) daptomycin versus the hemB mutant. The pharmacodynamic relationships between concentration (mg/liter) and log ratio change for vancomycin versus O-47 (E), vancomycin versus the hemB mutant (F), daptomycin versus O-47 (G), and daptomycin versus the hemB mutant (H) and the log ratio areas for vancomycin versus O-47 (I), vancomycin versus the hemB mutant (J), daptomycin versus O-47 (K), and daptomycin versus the hemB mutant (L) are shown.Model-fitted parameter estimates for vancomycin and daptomycin against both strains are shown in Table ​Table1.1. Model fits for the Hill-type model to the data were excellent, with all coefficients of determination (R²) being >0.97. There was a difference in the pharmacodynamic activities of both drugs against the strains with the two different phenotypes, with the E_max and EC₅₀ of vancomycin and daptomycin decreasing for the mutant with the SCV phenotype. Additionally, there was a greater discrepancy for E_max between vancomycin and daptomycin when S. epidermis displaying the SCV phenotype was compared to the normal phenotype: the vancomycin E_max for the strain with the SCV phenotype was approximately 7.7 times lower than that for the strain with the normal phenotype, and the daptomycin E_max was 1.5 times lower (Table ​(Table11).

TABLE 1.

Pharmacodynamic-model-fitted parameter estimates for vancomycin and daptomycin versus S. epidermidis strains^a

Extracellular DNA Acidifies Biofilms and Induces Aminoglycoside Resistance in Pseudomonas aeruginosa

Biofilms consist of surface-adhered bacterial communities encased in an extracellular matrix composed of DNA, exopolysaccharides, and proteins. Extracellular DNA (eDNA) has a structural role in the formation of biofilms, can bind and shield biofilms from aminoglycosides, and induces antimicrobial peptide resistance mechanisms. Here, we provide evidence that eDNA is responsible for the acidification of Pseudomonas aeruginosa planktonic cultures and biofilms. Further, we show that acidic pH and acidification via eDNA constitute a signal that is perceived by P. aeruginosa to induce the expression of genes regulated by the PhoPQ and PmrAB two-component regulatory systems. Planktonic P. aeruginosa cultured in exogenous 0.2% DNA or under acidic conditions demonstrates a 2- to 8-fold increase in aminoglycoside resistance. This resistance phenotype requires the aminoarabinose modification of lipid A and the production of spermidine on the bacterial outer membrane, which likely reduce the entry of aminoglycosides. Interestingly, the additions of the basic amino acid l-arginine and sodium bicarbonate neutralize the pH and restore P. aeruginosa susceptibility to aminoglycosides, even in the presence of eDNA. These data illustrate that the accumulation of eDNA in biofilms and infection sites can acidify the local environment and that acidic pH promotes the P. aeruginosa antibiotic resistance phenotype.     

Subinhibitory concentrations of antibiotics alter fibronectin binding to Staphylococcus aureus.

Fibronectin, a high-molecular-weight glycoprotein, is found in plasma and on mammalian cell surfaces. Recent reports have suggested that bacterial-fibronectin interactions play a role in bacterial attachment to host cells. Subinhibitory concentrations of lincosamines, erythromycin, and chloramphenicol decreased fibronectin binding to Staphylococcus aureus, whereas beta-lactam antibiotics enhanced this interaction.     

Susceptibility of Staphylococcus aureus and Staphylococcus epidermidis to 65 Antibiotics

The susceptibilities of 36 recent isolates of Staphylococcus aureus and 35 recent isolates of Staphylococcus epidermidis were determined against each of 65 antimicrobial agents and against two of them in combination. Rifampin was the most active of all the agents tested against both S. aureus and S. epidermidis. Among the penicillins, cloxacillin, dicloxacillin, and nafcillin were most active, although benzylpenicillin and phenoxymethyl penicillin were more active against susceptible strains. Cephaloridine was the most active of the cephalosporins, and sisomicin was the most active aminoglycoside. Minocycline was more active than the other tetracycline analogues tested. Among the macrolide-lincomycin compounds in clinical use, clindamycin was more active, and lincomycin was less active than erythromycin. The synergy of trimethoprim-sulfamethoxazole was more striking against S. aureus than against S. epidermidis. The median minimal inhibitory concentrations of the penicillins, cephalosporins, and aminoglycosides were lower against S. aureus, whereas the minimal inhibitory concentrations of the tetracyclines were lower against S. epidermidis.     

头孢曲松对表皮葡萄球菌生物膜形成的抑制作用

目的观察头孢曲松等4种抗生素抑制表皮葡萄球菌生物膜的作用,探讨抗生素防治葡萄球菌感染的作用环节。方法用半定量粘附实验筛选出形成生物膜的表皮葡萄球菌15株,观察不同浓度的药物对生物膜形成的抑制作用。结果发现头孢曲松能抑制生物膜形成,而头孢他啶、红霉素无抑制葡萄球菌生物膜的作用,万古霉素对葡萄球菌有很强的杀菌作用,但无抑制生物膜的作用。结论头孢曲松能抑制葡萄球菌生物膜形成,可利用头孢曲松抑制生物膜的作用,通过阻止粘附来阻断葡萄球菌引起的粘附相关感染,达到预防感染的目的。     

头孢曲松对表皮葡萄球菌生物膜形成的抑制作用

目的观察头孢曲松等4种抗生素抑制表皮葡萄球菌生物膜的作用,探讨抗生素防治葡萄球菌感染的作用环节。方法用半定量粘附实验筛选出形成生物膜的表皮葡萄球菌15株,观察不同浓度的药物对生物膜形成的抑制作用。结果发现头孢曲松能抑制生物膜形成,而头孢他啶、红霉素无抑制葡萄球菌生物膜的作用,万古霉素对葡萄球菌有很强的杀菌作用,但无抑制生物膜的作用。结论头孢曲松能抑制葡萄球菌生物膜形成,可利用头孢曲松抑制生物膜的作用,通过阻止粘附来阻断葡萄球菌引起的粘附相关感染,达到预防感染的目的。     

Strain differences in the opsonisation of Staphylococcus epidermidis

Ten isolates of coagulase-negative staphylococci, collected from patients receiving treatment with continuous ambulatory peritoneal dialysis (CAPD), exhibited marked differences in the degree of opsonisation when incubated in 10% and 1% pooled human serum, 10% and 1% heat-treated serum, Hanks' Balanced Salt Solution, and timed peritoneal dialysis (PD) effluent. The addition of exogenous IgG to PD effluent results in a greater increase in opsonisation in those fluids with the weakest inherent opsonic activity, but is ineffective against the majority of isolates in the absence of heat-labile opsonic activity. The results of this in vitro study suggest that host resistance to CAPD peritonitis due to coagulase-negative staphylococci may be determined as much by the characteristics of the contaminating strain, as by the opsonising activity of PD effluent.     

表皮葡萄球菌icaADBC操纵子检出与细菌耐氨基糖苷类抗菌药物的关系

目的分析临床分离表皮葡萄球菌对不同类型氨基糖苷类抗菌药物的耐药特征;分析icaADBC操纵子在临床分离的表皮葡萄球菌中的检出及与细菌耐药的关系。方法收集2013年1~10月上海市东方医院临床分离的表皮葡萄球菌77株,采用纸片扩散法检测细菌4种氨基糖苷类抗菌药物(庆大霉素、链霉素、奈替米星和阿米卡星)药敏试验特征;构建icaADBC特异性引物,采用聚合酶链反应扩增技术检测77株细菌中icaADBC的分布状况。结果结果77株表皮葡萄球菌对氨基糖苷类抗菌药物耐药率较高,分别为庆大霉素:62.3%,链霉素:61.0%,阿米卡星:22.1%,奈替米星:23.4%。icaADBC操纵子的检出率为20.8%,ica阳性菌株与阴性菌株对氨基糖苷类抗菌药物耐药性比较差异有统计学意义(P0.05)。结论临床分离的icaADBC操纵子阳性表皮葡萄球菌对氨基糖苷类抗菌药物耐药率较高。     

Comparison of the Antibiotic Activities of Daptomycin,Vancomycin, and the Investigational Fluoroquinolone Delafloxacin against Biofilms from Staphylococcus aureus Clinical Isolates

Biofilm-related infections remain a scourge. In an in vitro model of biofilms using Staphylococcus aureus reference strains, delafloxacin and daptomycin were found to be the most active among the antibiotics from 8 different pharmacological classes (J. Bauer, W. Siala, P. M. Tulkens, and F. Van Bambeke, Antimicrob. Agents Chemother. 57:2726–2737, 2013, doi:10.1128/AAC.00181-13). In this study, we compared delafloxacin to daptomycin and vancomycin using biofilms produced by 7 clinical strains (S. aureus epidemic clones CC5 and CC8) in order to rationalize the differences observed between the antibiotics and strains. The effects of the antibiotics on bacterial viability (resazurin reduction assay) and biomass (crystal violet staining) were measured and correlated with the proportion of polysaccharides in the matrix, the local microenvironmental pH (micro-pH), and the antibiotic penetration in the biofilm. At clinically meaningful concentrations, delafloxacin, daptomycin, and vancomycin caused a ≥25% reduction in viability against the biofilms formed by 5, 4, and 3 strains, respectively. The antibiotic penetration within the biofilms ranged from 0.6 to 52% for delafloxacin, 0.2 to 10% for daptomycin, and 0.2 to 1% for vancomycin; for delafloxacin, this was inversely related to the polysaccharide proportion in the matrix. Six biofilms were acidic, explaining the high potency of delafloxacin (lower MICs at acidic pH). Norspermidine and norspermine (disassembling the biofilm matrix) drastically increased delafloxacin potency and efficacy (50% reduction in viability for 6 biofilms at clinically meaningful concentrations) in direct correlation with its increased penetration within the biofilm, while they only modestly improved daptomycin efficacy (50% reduction in viability for 2 biofilms) and penetration, and they showed marginal effects with vancomycin. Delafloxacin potency and efficacy against biofilms are benefited by its penetration into the matrix and the local acidic micro-pH.     

Effects of Subinhibitory Concentrations of Antibiotics on Alpha-Toxin (hla) Gene Expression of Methicillin-Sensitive and Methicillin-Resistant Staphylococcus aureus Isolates

Concentrations of antibiotics below the MIC are able to modulate the expression of virulence-associated genes. In this study, the influence of subinhibitory doses of 31 antibiotics on the expression of the gene encoding the staphylococcal alpha-toxin (hla), a major virulence factor of Staphylococcus aureus, was investigated with a novel gene fusion protocol. The most striking observation was a strong induction of hla expression by subinhibitory concentrations of β-lactams and an almost complete inhibition of alpha-toxin expression by clindamycin. Whereas glycopeptide antibiotics had no effect, the macrolide erythromycin and several aminoglycosides reduced and fluoroquinolones slightly stimulated hla expression. Furthermore, Northern blot analysis of hla mRNA and Western blot (immunoblot) analysis of culture supernatants of both methicillin-sensitive and methicillin-resistant S. aureus strains revealed that methicillin-induced alpha-toxin expression is a common phenomenon of alpha-toxin-producing strains. Some methicillin-resistant S. aureus isolates produced up to 30-fold more alpha-toxin in the presence of 10 μg of methicillin per ml than in its absence. The results indicate that the novel gene fusion technique is a useful tool for studying the modulation of virulence gene expression by antibiotics. Moreover, the results suggest that the effects of certain antibiotics on virulence properties may be relevant for the management of S. aureus infections.     

Some Effects of Subinhibitory Concentrations of Penicillin on the Structure and Division of Staphylococci

A strain of Staphylococcus aureus was planted on filter membranes placed on Trypticase soy agar (BBL). After incubation, the membranes with growing staphylococci were transferred to Trypticase soy agar containing a subinhibitory concentration of penicillin (one-third minimal inhibitory concentration) and again incubated. The membranes were then returned to drug-free agar and incubated once more. Counts of the colony-forming units and electron microscopy were carried out at several time intervals. When grown on agar containing penicillin, the staphylococci formed what appeared to be abnormally large cells with multiple and unusually thick septa. Examination of a number of sections showed that these large cells were in reality clusters of staphylococci that had divided but failed to separate. When these large cells were subsequently grown on drug-free agar, smaller cells and normal staphylococci emerged. Subinhibitory concentrations of penicillin do not kill staphylococci; they seem to inhibit lysis of cross walls, preventing the separation of otherwise divided cells.     

Transduction of Tetracycline Resistance in Staphylococcus epidermidis

Fourteen Staphylococcus epidermidis strains were biotyped according to the scheme of Baird-Parker and tested for their sensitivities to S. epidermidis typing phages. Tetracycline-susceptible strains were tested for the capacity to be transduced to resistance. Five strains, all of biotype 1, were lysed by one or more phages. Five of eight biotype 1 strains were transduced to tetracycline resistance with rates of 10(-8) to 10(-5). Three strains of other biotypes were not transduced, nor was PS 73, an unusual coagulase-negative strain. The transducing phage phi367 was isolated from the lysogenic tetracycline-resistant donor strain. The phage morphologically resembles serological group B S. aureus phages. Restriction and modification were indicated from the efficiency of plating on phage-sensitive strains.