Effects of Subinhibitory Concentrations of Ceftaroline on Methicillin-Resistant Staphylococcus aureus (MRSA) Biofilms

Ceftaroline (CPT) is a novel cephalosporin with in vitro activity against Staphylococcus aureus. Ceftaroline exhibits a level of binding affinity for PBPs in S. aureus including PBP2a of methicillin-resistant S. aureus (MRSA). The aims of this study were to investigate the morphological, physiological and molecular responses of MRSA clinical strains and MRSA biofilms to sub-MICs (1/4 and 1/16 MIC) of ceftaroline by using transmission, scanning and confocal microscopy. We have also used quantitative Real-Time PCR to study the effect of sub-MICs of ceftaroline on the expression of the staphylococcal icaA, agrA, sarA and sasF genes in MRSA biofilms. In one set of experiments, ceftaroline was able to inhibit biofilm formation in all strains tested at MIC, however, a strain dependent behavior in presence of sub-MICs of ceftaroline was shown. In a second set of experiments, destruction of preformed biofilms by addition of ceftaroline was evaluated. Ceftaroline was able to inhibit biofilm formation at MIC in all strains tested but not at the sub-MICs. Destruction of preformed biofilms was strain dependent because the biofilm formed by a matrix-producing strain was resistant to a challenge with ceftaroline at MIC, whereas in other strains the biofilm was sensitive. At sub-MICs, the impact of ceftaroline on expression of virulence genes was strain-dependent at 1/4 MIC and no correlation between ceftaroline-enhanced biofilm formation and gene regulation was established at 1/16 MIC. Our findings suggest that sub-MICs of ceftaroline enhance bacterial attachment and biofilm formation by some, but not all, MRSA strains and, therefore, stress the importance of maintaining effective bactericidal concentrations of ceftaroline to fight biofilm-MRSA related infections.     

Effects of growth in the presence of subinhibitory concentrations of dicloxacillin on Staphylococcus epidermidis and Staphylococcus haemolyticus biofilms

Low concentrations of antibiotics can inhibit microbial adherence to medical device surfaces. However, little is known about the changes that occur in the physiology of bacteria within biofilms formed in the presence of subinhibitory (sub-MIC) concentrations of antibiotics. In this study, the densities and matrix compositions of biofilms formed by two coagulase-negative Staphylococcus species in the absence and in the presence of sub-MIC concentrations of dicloxacillin were evaluated. Biofilms formed in the presence of sub-MIC concentrations of dicloxacillin contained less biomass, and there were notable changes in the composition of the biofilm matrix. Changes in the spatial structure were also verified by confocal scanning laser microscopy, indicating that biofilms grown in the presence of sub-MIC concentrations of dicloxicilln had a lower cell density. Physiological alterations in the bacteria within biofilms grown in the presence of subinhibitory concentrations of the antibiotic were also evaluated. The results showed that there were differences in bacterial surface characteristics when cultures were grown in the presence of sub-MIC concentrations of dicloxacillin, including decreased hydrophobicity and decreased expression of the exopolysaccharide poly-N-acetylglucosamine. The elemental composition of the cell surface was also analyzed, and whereas in Staphylococcus epidermidis there were decreases in the oxygen and nitrogen contents, in Staphylococcus haemolyticus there were increases in these two parameters. Additionally, increases in resistance to several antibiotics were observed for the cells within biofilms formed in the presence of dicloxacillin.     

The evaluation of lipolytic activity of strains of Staphylococcus epidermidis and Staphylococcus haemolyticus

A total of 103 isolates of CNS (66 strains of S. epidermidis and 37 strains of S. haemolyticus) were investigated. Lipolytic activity of staphylococcal strains was determined by Tryptic Soy Agar containing Tween 20 or Tween 60. The 95.4% strains of staphylococci demonstrated the lipolytic activity on Tween 20 agar and the 89.4% of strains of staphylococci degradation ester of fatty acids on Tweens 60 agar. We detected that S. epidermidis strains (respectively 95,4%, 89,4%) produced lipases more frequently than S. haemolyticus strains (respectively 72,9%, 59,4%). Studies suggest that source of isolation from clinical materials (blood, wound and pus) does not have an influence on the ability hydrolysis esters.     

Removal and Inactivation of Staphylococcus epidermidis Biofilms by Electrolysis

Bacterial biofilms were exposed to electrolysis by making the steel substratum an electrode in a circuit including a 6-V battery. These treatments resulted in killing (2.1-log reduction) and removal (4.0-log reduction) of viable cells at the anode and cathode, respectively, within a few minutes.     

Small-Cell Formation of Staphylococcus aureus by Subinhibitory Concentrations of Nitrofuran Derivatives

Small-cell formation of Staphylococcus aureus by subinhibitory concentration of nitrofuran derivatives was examined by scanning and transparent electron microscopy.     

Differential Roles of Poly-N-Acetylglucosamine Surface Polysaccharide and Extracellular DNA in Staphylococcus aureus and Staphylococcus epidermidis Biofilms

Staphylococcus aureus and Staphylococcus epidermidis are major human pathogens of increasing importance due to the dissemination of antibiotic-resistant strains. Evidence suggests that the ability to form matrix-encased biofilms contributes to the pathogenesis of S. aureus and S. epidermidis. In this study, we investigated the functions of two staphylococcal biofilm matrix polymers: poly-N-acetylglucosamine surface polysaccharide (PNAG) and extracellular DNA (ecDNA). We measured the ability of a PNAG-degrading enzyme (dispersin B) and DNase I to inhibit biofilm formation, detach preformed biofilms, and sensitize biofilms to killing by the cationic detergent cetylpyridinium chloride (CPC) in a 96-well microtiter plate assay. When added to growth medium, both dispersin B and DNase I inhibited biofilm formation by both S. aureus and S. epidermidis. Dispersin B detached preformed S. epidermidis biofilms but not S. aureus biofilms, whereas DNase I detached S. aureus biofilms but not S. epidermidis biofilms. Similarly, dispersin B sensitized S. epidermidis biofilms to CPC killing, whereas DNase I sensitized S. aureus biofilms to CPC killing. We concluded that PNAG and ecDNA play fundamentally different structural roles in S. aureus and S. epidermidis biofilms.     

Electric Current-Induced Detachment of Staphylococcus epidermidis Biofilms from Surgical Stainless Steel

Biomaterial-centered infections of orthopedic percutaneous implants are serious complications which can ultimately lead to osteomyelitis, with devastating effects on bone and surrounding tissues, especially since the biofilm mode of growth offers protection against antibiotics and since removal frequently is the only ultimate solution. Recently, it was demonstrated that as a possible pathway to prevent infections of percutaneous stainless steel implants, electric currents of 60 to 100 μA were effective at stimulating the detachment of initially adhering staphylococci from surgical stainless steel. However, initially adhering bacteria are known to adhere more reversibly than bacteria growing in the later stages of biofilm formation. Hence, the aim of this study was to examine whether a growing Staphylococcus epidermidis biofilm can be stimulated to detach from surgical stainless steel by the use of electric currents. In separate experiments, four currents, i.e., 60 and 100 μA of direct current (DC) and 60 and 100 μA of block current (50% duty cycle, 1 Hz), were applied for 360 min to stimulate the detachment of an S. epidermidis biofilm that had grown for 200 min. A 100-μA DC yielded 78% detachment, whereas a 100-μA block current under the same experimental conditions yielded only 31% detachment. The same trend was found for 60 μA, with 37% detachment for a DC and 24% for a block current. Bacteria remaining on the surface after the current application were less viable than they were prior to the current application, as demonstrated by confocal laser scanning microscopy. In conclusion, these results suggest that DCs are preferred for curing infections.     

五倍子水煎剂对表皮葡萄球菌生物膜抑制的研究

通过五倍子水煎剂对表皮葡萄球菌MIC测定和生物膜形成干预的研究,为表皮葡萄球菌引起感染提供新的治疗途径。用微量肉汤稀释法分别测定五倍子水煎剂对表皮葡萄球菌的MIC;刚果红及刚果红红霉素、五倍子水煎剂琼脂平板测定表皮葡萄球菌PIA生成与抑制;五倍子水煎剂、红霉素干预表皮葡萄球菌生物膜形成,于光镜和电镜下观察其生物膜形态。134株表皮葡萄球菌五倍子水煎剂的MIC50为0.488 mg/mL,MIC90为0.977 mg/mL。134株表皮葡萄球菌中有50株为PIA阳性,PIA阳性的50株菌全部产生生物膜,红霉素对表皮葡萄球菌生物膜形成有抑制,而五倍子水煎剂则无。表皮葡萄球菌PIA的相互作用在其生物膜的生成中起主要作用;五倍子水煎剂对表皮葡萄球菌生长有明显的抑制但对生物膜形成无干预作用。     

The Effects of Low-Frequency Ultrasound on Staphylococcus epidermidis

Low frequency ultrasound (LFUS) significantly enhances skin permeability to a variety of drugs; however, its bacterial effects have not been well studied. Staphylococcus epidermidis organisms were grown and standardized to 10⁵ cfu/ml 24 h prior to investigation and suspended in normal saline. LFUS was applied with two probes immersed in the bacterial suspensions over a range of suspension volumes, intensities, and exposure times. The suspension temperature was measured, and a sample was removed, streaked onto blood agar plates, and incubated at 37°C for 24 h. Quantitative bacterial counts were then obtained. LFUS resulted in significant reductions in bacterial counts that correlated with fluid temperature. Probe size and ultrasound intensity appeared to affect bacterial counts, but were also correlated with temperature. Bacterial growth was minimal with temperatures exceeding 45°C. While LFUS can reduce bacterial counts, these conditions have the potential to cause burns in humans. Received: 21 August 1998 / Accepted: 29 September 1998     

表皮葡萄球菌对大鼠变应原敏感性的影响

目的 筛选具有较好抑制金黄色葡萄球菌效果的表皮葡萄球菌,研究通过鼻腔进行表皮葡萄球菌调节观察大鼠对变应原敏感性的效果。方法 由鼻腔拭子稀释液分离筛选得到的9株表皮葡萄球菌（Staphylococcus epidermidis）,使用上清液抑制法选取抑制金黄色葡萄球菌效果最好的表皮葡萄球菌做体内实验。将Wistar大鼠分为阴性组、模型组、表葡调节组。模型组、表葡调节组使用OVA建立呼吸道炎症模型,使用表皮葡萄球菌DMSA16001进行滴鼻调节2周,同时用PBS进行滴鼻对照。统计抓鼻和喷嚏数,用ELISA进行肺灌洗液（BALF）和外周血IgE的检测。结果 （1）不同株的表皮葡萄球菌均对金黄色葡萄球菌有抑制作用,表皮葡萄球菌DMSA16001对金黄色葡萄球菌的抑制率最高。（2）与模型组相比,表葡调节组的抓鼻数显著下降（t=2.173,P=0.049）,喷嚏数有下降趋势,表葡调节组的肺灌洗液和外周血血清的IgE水平均呈现下降趋势。结论 对金黄色葡萄球菌有抑制作用的表皮葡萄球菌能使OVA造模的哮喘大鼠对变应原敏感性降低。     

372株溶血葡萄球菌的临床分布及耐药性

目的了解医院4年来溶血葡萄球菌的临床分布特征及耐药性变化,为临床合理用药提供参考依据。方法回顾性分析2010年1月至2013年12月住院患者标本分离的372株溶血葡萄球菌,采用美国BD Phoenix 100全自动细菌鉴定药敏分析仪及其配套GP检测卡进行药敏分析。结果分离的372株溶血葡萄球菌主要分布于ICU、呼吸内科和外科,主要来源于痰液、尿液和伤口分泌物;未检测到万古霉素耐药菌株,对阿莫西林/克拉维酸、氨苄西林、苯唑西林、红霉素、青霉素、头孢西丁均保持90%以上的高耐药性,对其它抗生素均存在不同程度耐药性。结论溶血葡萄球菌耐药现象日益严重,且多重耐药现象呈上升趋势,临床在治疗此类细菌感染时,应根据药敏试验结果制定用药方案,减少经验用药。     

Prostaglandin E2 from Candida albicans Stimulates the Growth of Staphylococcus aureus in Mixed Biofilms

Background

Previous studies showed that Staphylococcus aureus and Candida albicans interact synergistically in dual species biofilms resulting in enhanced mortality in animal models.

Methodology/Principal Findings

The aim of the current study was to test possible candidate molecules which might mediate this synergistic interaction in an in vitro model of mixed biofilms, such as farnesol, tyrosol and prostaglandin (PG) E₂. In mono-microbial and dual biofilms of C.albicans wild type strains PGE₂ levels between 25 and 250 pg/mL were measured. Similar concentrations of purified PGE₂ significantly enhanced S.aureus biofilm formation in a mode comparable to that observed in dual species biofilms. Supernatants of the null mutant deficient in PGE₂ production did not stimulate the proliferation of S.aureus and the addition of the cyclooxygenase inhibitor indomethacin blocked the S.aureus biofilm formation in a dose-dependent manner. Additionally, S. aureus biofilm formation was boosted by low and inhibited by high farnesol concentrations. Supernatants of the farnesol-deficient C. albicans ATCC10231 strain significantly enhanced the biofilm formation of S. aureus but at a lower level than the farnesol producer SC5314. However, C. albicans ATCC10231 also produced PGE₂ but amounts were significantly lower compared to SC5314.

Conclusion/Significance

In conclision, we identified C. albicans PGE₂ as a key molecule stimulating the growth and biofilm formation of S. aureus in dual S. aureus/C. albicans biofilms, although C. albicans derived farnesol, but not tyrosol, may also contribute to this effect but to a lesser extent.     

Staphylococcus epidermidis role in the skin microenvironment

Wound healing is a complex dynamic physiological process in response to cutaneous destructive stimuli that aims to restore the cutaneous’ barrier role. Deciphering the underlying mechanistic details that contribute to wound healing will create novel therapeutic strategies for skin repair. Recently, by using state‐of‐the‐art technologies, it was revealed that the cutaneous microbiota interact with skin immune cells. Strikingly, commensal Staphylococcus epidermidis‐induced CD8+ T cells induce re‐epithelization of the skin after injury, accelerating wound closure. From a drug development perspective, the microbiota may provide new therapeutic candidate molecules to accelerate skin healing. Here, we summarize and evaluate recent advances in the understanding of the microbiota in the skin microenvironment.     

Comparative Genotypes,Staphylococcal Cassette Chromosome mec (SCCmec) Genes and Antimicrobial Resistance amongst Staphylococcus epidermidis and Staphylococcus haemolyticus Isolates from Infections in Humans and Companion Animals

This study compares the characteristics of Staphylococcus epidermidis (SE) and Staphylococcus haemolyticus (SH) isolates from epidemiologically unrelated infections in humans (Hu) (28 SE-Hu; 8 SH-Hu) and companion animals (CpA) (12 SE-CpA; 13 SH-CpA). All isolates underwent antimicrobial susceptibility testing, multilocus sequence typing and DNA microarray profiling to detect antimicrobial resistance and SCCmec-associated genes. All methicillin-resistant (MR) isolates (33/40 SE, 20/21 SH) underwent dru and mecA allele typing. Isolates were predominantly assigned to sequence types (STs) within a single clonal complex (CC2, SE, 84.8%; CC1, SH, 95.2%). SCCmec IV predominated among MRSE with ST2-MRSE-IVc common to both Hu (40.9%) and CpA (54.5%). Identical mecA alleles and nontypeable dru types (dts) were identified in one ST2-MRSE-IVc Hu and CpA isolate, however, all mecA alleles and 2/4 dts detected among 18 ST2-MRSE-IVc isolates were closely related, sharing >96.5% DNA sequence homology. Although only one ST-SCCmec type combination (ST1 with a non-typeable [NT] SCCmec NT9 [class C mec and ccrB4]) was common to four MRSH-Hu and one MRSH-CpA, all MRSH isolates were closely related based on similar STs, SCCmec genes (V/V_T or components thereof), mecA alleles and dts. Overall, 39.6% of MR isolates harbored NT SCCmec elements, and ACME was more common amongst MRSE and CpA isolates. Multidrug resistance (MDR) was detected among 96.7% of isolates but they differed in the prevalence of specific macrolide, aminoglycoside and trimethoprim resistance genes amongst SE and SH isolates. Ciprofloxacin, rifampicin, chloramphenicol [fexA, cat-pC221], tetracycline [tet(K)], aminoglycosides [aadD, aphA3] and fusidic acid [fusB] resistance was significantly more common amongst CpA isolates. SE and SH isolates causing infections in Hu and CpA hosts belong predominantly to STs within a single lineage, harboring similar but variable SCCmec genes, mecA alleles and dts. Host and staphylococcal species-specific characteristics were identified in relation to antimicrobial resistance genes and phenotypes, SCCmec and ACME.     

Significance of Staphylococcus epidermidis in the Clinical Laboratory

Susceptibility to 11 antibiotics was determined for 63 cultures of Staphylococcus aureus and 63 cultures of Staphylococcus epidermidis obtained at random from the clinical laboratory. The incidence of resistance to nine of these antibiotics was greater for S. epidermidis than for S. aureus. Studies of the minimal inhibitory concentration of these cultures to clindamycin showed that 61 cultures of S. aureus were susceptible whereas only 46 cultures of S. epidermidis were susceptible to this antibiotic. Although cultures of S. aureus were more active in the production of seven virulence factors, some cultures of S. epidermidis produced virulence factors. By successive cultivation in increasing concentrations of clindamycin, resistant variants were obtained for 10 cultures of S. aureus and 3 cultures of S. epidermidis. The presence of subinhibitory concentrations of clindamycin inhibited the production of some virulence factors by the resistant variants. In view of the greater resistance of S. epidermidis to antibiotics and its ability to produce virulence factors, its isolation in the clinical laboratory should not be regarded lightly.