Reduced susceptibility to triclosan in methicillin-resistant Staphylococcus aureus

Susceptibility to triclosan in Staphylococcus aureus was determined. The study was carried out on 200 strains, including 100 resistant (MRSA) and 100 susceptibile (MSSA) to methicillin. The examined strains were isolated from varied clinical samples and patients in 18 medical centers, in majority from hospitals in the region of Gdansk. The susceptibility was estimated by the MIC (minimal inhibitory concentration) using dilution test in Mueller-Hinton agar. The antimicrobial resistance patterns were determined, including resistance to methicillin and mupirocin. The most of MRSA strains (62%) demonstrated reduced susceptibility to triclosan (MIC 2mg/L), while 93% of MSSA strains were highly sensitive to this antibacterial agent (MIC 0,031mg/L). The majority (66,1%) of MRSA strains with reduced susceptibility to triclosan demonstrated the same antimicrobial resistance pattern. Reduced susceptibility to triclosan was observed in 8 from 9 high - level mupirocin resistant strains, but the most of MRSA strains with reduced triclosan susceptibility (91,5%) were found among fusidic acid resistant strains.     

Bioluminescent assay as a potential method of rapid susceptibility testing

A method of rapid susceptibility testing by bioluminescent assay was developed. Correlation between the 50% inhibition dose of antimicrobics for bacterial adenosine triphosphate measured by bioluminescent assay and the minimum inhibitory concentration obtained by the broth dilution method was satisfactory. In the bioluminescent assay the incubation time required was only 90 min.     

Different antibacterial actions of isoflavones isolated from Erythrina poeppigiana against methicillin-resistant Staphylococcus aureus

AIMS: To screen six isoflavones isolated from Erythrina poeppigiana (Leguminosae) for their antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA). METHODS AND RESULTS: Stem bark of E. poeppigiana was macerated with acetone and the methylene chloride-soluble fraction of the residue was applied to repeated silica gel column chromatography and eluted. Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were determined by a broth dilution method. Inactive compounds that failed inhibiting bacterial growth at 25 microg ml(-1) were further investigated for their combination effects with methicillin and oxacillin. Of the isolated isoflavones, 5,7,4'-trihydroxy-8,3'-di(gamma,gamma-dimethylallyl)isoflavone (isolupalbigenin) exhibited the highest anti-MRSA activity (MICs: 1.56-3.13 microg ml(-1); MBCs: 6.25-12.5 microg ml(-1)), followed by 5,7,4'-trihydroxy-6-gamma,gamma-dimethylallylisoflavone (erythrinin B). Inactive compounds were combined with methicillin or oxacillin, 5,4'-dihydroxy-(3',4'-dihydro-3'-hydroxy)-2',2'-dimethylpyrano[5',6':6,7]isoflavone (M-Wi-2) intensifying the susceptibility of MRSA strains to these antibiotics. In all but one strain, the MIC values of methicillin were reduced from > or =100 to 6.25-12.5 microg ml(-1) in the presence of M-Wi-2 (25 microg ml(-1)). CONCLUSIONS: Isoflavones from E. poeppigiana showed two different antibacterial activities against MRSA: direct growth inhibition and intensification of methicillin sensitivity. SIGNIFICANCE AND IMPACT OF THE STUDY: Isolupalbigenin and M-Wi-2 could lead to the development of compounds for new approaches against MRSA infection.     

Luciferin-luciferase assay of adenosine triphosphate from bacteria: a comparison of dimethylsulphoxide (DMSO) and acetone with other solvents

The extraction of adenosine triphosphate (ATP) from six strains of bacteria, chosen for differences in cell-wall composition and habitat, was performed. The solvents were two in common use, Tris-ethylenediaminetetraacetate (Tris-EDTA) and trichloroacetic acid (TCA), and two promising, though less utilized solvents, dimethylsulphoxide (DMSO) and acetone. ATP was determined by the luciferin-luciferase reaction. Of the solvents used, DMSO and acetone were the most effective considering the different kinds of bacteria tested and, of these two, DMSO was the most convenient to use. Tris-EDTA was not as effective as the other solvents tested and TCA, which was effective with most strains, gave low yields when used with cultures grown in artificial seawater broth. Internal standards were used to determine if there were substances present that could inhibit the reaction of released ATP with the luciferin-luciferase reagent. Extracts of ATP, stored at -20 degrees C, were stable for up to 3 weeks.     

Light Induced Concentration Changes of Adenosine-Triphosphate in Phycomyces Sporangiophores

The concentrations of extractable adenosine triphosphate (ATP) following the induction of positive light-growth responses in Phycomyces sporangiophores by blue light stimuli have been measured by means of the luciferin-luciferase assay. The ATP concentration in the light-sensitive growing zone increases 30 to 50% within 30 seconds after the start of a light stimulus and returns to the normal adapted level within 1 minute after stimulation. The ATP concentration is constant for any level of light adaptation and is uniform along the length of sporangiophores even though the light sensitivity is confined to a growing zone less than 5 mm long. These results suggest that one of the initial biochemical steps after a light stimulus is the production of extractable ATP.     

Evaluation of methicillin-resistance in Staphylococcus aureus by the agar disk diffusion method and PCR

Staphylococcus aureus is a widespread human pathogen. One the most striking characteritics of this bacterium is resistance to methicillin and all beta-lactam antibiotics. The agar disk diffusion method is the most widely used in vitro susceptibility test, but recently molecular methods, e.g. Polymerase Chain Reaction, have been also introduced. We compared the detection of methicillin resistant coagulase positive Staphylococcus aureus isolated from clinical materials in Silesian microbiological laboratories by diffusion method and PCR through the detection of nuc and mec A genes. Our results show that PCR used for the detection of mec A gene increases the detection of methicillin-resistant Staphylococcus aureus strains by 10% as compared to the agar disk diffusion method. Among Staphylococcus aureus strains, detected as methicillin-resistant, 17% of organisms showed no presence of mec A gene.     

微生物分析仪法与常规纸片扩散法在细菌耐药表型检测中的比较

为了比较VITEK 2Compact微生物分析仪法和常规纸片扩散法在细菌耐药表型检测中的差异,随机选取我院细菌室2012年7月至2013年6月分离保存的金黄色葡萄球菌、大肠杆菌、肺炎克雷伯杆菌各100株,分别以常规纸片扩散法和VITEK 2Compact微生物分析仪检测其耐药表型,并分析比较结果。检测结果显示,VITEK 2Compact检测耐药表型结果与常规纸片扩散法检测结果无显著性差异(p>0.05)。本研究表明VITEK 2Compact微生物分析仪能对致病菌的耐药表型进行快速、准确地测定,且灵敏度、特异度较高,能够及时、准确地为临床提供药敏结果,大大提高抗感染治疗的疗效。     

Susceptibility and frequency of selected groups of bacteria isolated in 2001 from patients at the Holy Cross Cancer Center

The aim of the presented study was the analysis of microbiological data obtained from patients hospitalized in The Holly Cross Cancer Center in Kielce in 2001. The frequency of important nosocomial pathogens in selected specimens and their susceptibility to antibiotics were determined. The strains were identified by using commercial tests (bioMerieux) and their antibiotic susceptibility patterns were performed by disc diffusion technique. The most prevalent bacteria were Gram-negative rods of Enterobacteriaceae family (43%), mainly Escherichia coli. Only 2.7% strains of isolated Escherichia coli isolated from clinical specimens collected from hospitalized patients were beta-lactamase--positive (ESBL+). The second important group of microorganisms were Staphylococci, followed by Enterococcus spp., Pseudomonas aeruginosa and Candida spp. About twenty eight percent of Staphylococcus aureus isolates were resistant to methicillin.     

Diverse responses of Staphylococcus aureus NCTC 10442 and some of its variants to methicillin.

The methicillin resistant strain of Staphylococcus aureus NCTC 10442 when aged in a broth culture at 42 degrees C yielded variants showing responses of sensitivity, dependence and indifference to the antibiotic. These responses and that of resistance in the parent cells were displayed in agar dilution and agar diffusion experiments at 30 degrees C. At 42 degrees C, all four organisms were sensitive to methicillin.     

MRSA中mecA及femB基因的检测与耐药相关性

研究femB、mecA基因在耐甲氧西林金黄色葡萄球菌(MRSA)中的表达与耐药的关系.运用PCR对MRSA的femB、mecA基因进行检测,MRSA耐药检测采用头孢西丁纸片法.40 株金黄色葡萄球菌(下简称金葡菌)通过头孢西丁纸片法,检出 30 株耐头孢西丁的菌株,通过PCR检测这 40 株金葡菌mecA基因,30 株MRSA全部为阳性, femB基因在 30 株MRSA中全部表达,而甲氧西林敏感的金黄色葡萄球菌(MSSA)的未表达.结果可见,PCR能快速准确地鉴定MRSA, mecA基因是MRSA的耐药基因,femB基因是MRSA的耐药相关基因.     

Comparison of methods for the detection of methicillin resistance in Staphylococcus aureus isolates from food products

AIMS: To compare several methods for detection of methicillin resistance in Staphylococcus aureus isolates from food. METHODS AND RESULTS: Two hundred S. aureus isolates from food of animal origin were screened for methicillin resistance by a PCR assay specific for the mecA gene, an oxacillin agar screen test and a cefoxitin disk diffusion test. Six out of 200 strains (3%) were found to be methicillin-resistant Staphylococcus aureus (MRSA) by PCR. The oxacillin agar screen test detected only one of the MRSA isolates (sensitivity of 16.7%) and mischaracterized three additional strains as MRSA (specificity of 98.45%). None of the MRSA strains was detected by the cefoxitin test (sensitivity of 0%), while 15 methicillin-susceptible S. aureus (MSSA) strains were misclassified as resistant (specificity of 92.3%). Fifteen MSSA strains displayed a beta-lactamase hyperproducer-like phenotype. The six MRSA (mecA-positive) strains resembled the characteristics of heteroresistant strains. CONCLUSIONS: As MRSA of animal origin may display atypical phenotypes, PCR appears to be more reliable for detection of methicillin resistance in animal strains. SIGNIFICANCE AND IMPACT OF THE STUDY: The study stresses the need for implementing the methods of screening S. aureus from food of animal origin for methicillin resistance.     

mecA基因PCR扩增法检测耐甲氧西林金黄色葡萄球菌

目的 应用mecA基因PCR扩增法检测耐甲氧西林金黄色葡萄球菌（methicillin resistant staphylococcus aureus,MRSA）。方法 临床分离的70株金黄色葡萄球菌,应用mecA基因PCR扩增法鉴定MRSA,并与苯唑西林纸片扩散法进行比较。结果 70株金黄色葡萄球菌用PCR扩增法和纸片扩散法有6株鉴定有差异,4株。mecA基因阳性而纸片扩散法鉴定为敏感,1株mecA基因阳性纸片扩散法鉴定为临界耐药,1株mecA基因阴性却表现为苯唑西林耐药,2种方法符合率为91．43％。结论 mecA基因PCR扩增法可以准确、快速判定MRSA,特别是对隐匿型或低水平耐药菌株的检出有重要的价值。     

Comparative study of in vitro methods to analyse the antifungal activity of propolis against yeasts isolated from patients with superficial mycoses

AIM: To test a total of 15 strains belonging to four species of yeasts by different in vitro methods against propolis and itraconazole (ITC). METHODS AND RESULTS: Three methods were compared for susceptibility testing of yeast isolates to propolis: disc diffusion method, agar dilution method and National Committee for Clinical Laboratory Standards (NCCLS, M27A) broth microdilution method. ITC was selected as the antifungal agent for comparison study. Using the broth microdilution method, the geometric mean for MIC (microg ml(-1)) with regard to all isolates was < or =0.06 for propolis and < or =0.35 for ITC. The broth microdilution and the agar dilution methods were in good agreement (75%) for propolis against yeasts isolated from patients with superficial mycoses. Using the diffusion method, all strains showed a broad zone of inhibition at the first available reading time (24 or 48 h). An increase of MIC values was accompanied by a decrease of growth inhibition zone diameter. A favourable correlation was found between MIC and inhibition zone around the disc for propolis sample and the correlation coefficient was: r = -0.626 (P < 0.01). CONCLUSIONS: This study suggests the potential value of the agar dilution and disc diffusion method as a convenient alternative method for testing of yeasts to propolis. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrated that propolis and ITC were very active against yeasts from patients with superficial mycoses. The other prominent finding in this study is that RPMI 1640 with L-glutamine was the available broth for the in vitro susceptibility testing of yeasts.     

Sensitivity of Certain Porcine and Bovine Mycoplasmas to Antimicrobial Agents in a Liquid Medium Test Compared to a Disc Assay

The sensitivity of some porcine and bovine mycoplasmas to potent antimicrobial agents was examined. Minimal inhibitory concentration (MIC) values were estimated for M. hyosynoviae, M. hyopneumoniae, M. dispar and M. bovis against enrofloxacin, lincomycin, tetracycline, tiamulin and tylosin, in a liquid medium test and in a disc assay. All 6 examined strains of each species and the respective type strains were significantly inhibited. The greatest sensitivity was noted for tiamulin against strains of M. hyosynoviae with a final MIC50 broth value of 0.025 µg ml−1 and disc value of 0.03 µg per disc. Enrofloxacin was found very potent against M. hyopneumoniae with a final MIC50 of 0.025 µg ml−1 and 0.1 µg per disc, and for M. dispar with 0.05 µg ml−1 and 0.03 µg per disc. Most disc assay estimates in ug per disc were similar to or moderately greater than corresponding final broth figures in µg ml−1. It may be possible to convert observed disc assay values into representative final broth MIC values for use in the clinic.     

The enumeration of Leptospira interrogans serovar pomona by a bioluminescence ATP assay

A rapid method for enumerating viable Leptospira interrogans serovar pomona cells was investigated using a bacterial adenosine triphosphate (ATP) assay. The ATP was assayed by the luciferin-luciferase bioluminescence reaction. Samples of serovar pomona grown in liquid polysorbate 80-bovine albumin (P80-BA) medium for 1-3 days were analysed for ATP content, culture density (nephelometry), direct cell count and most probable number of viable cells (MPNVC) as determined by the dilution tube technique. A linear relationship was found between ATP content and the number of viable cells over the range of 4 X 10(8) to 8 X 10(9) leptospires/ml. Over this range the correlation coefficient for ATP content versus viable cells (0.96) was similar to the coefficient for culture density versus the number of viable cells. The coefficient for direct counts versus the number of viable cells was smaller. The bioluminescence assay of bacterial ATP is a promising method for enumerating viable leptospires in pure culture.     

Effect of subinhibitory levels of selected antibiotics on susceptibility of Staphylococcus aureus strains to phagocytosis and killing by rabbit granulocytes

The aim of the study was to assess the of chosen antibiotics in subinhibitory concentrations (sub-MIC) on the sensitive of Staphylococcus aureus cells to phagocytosis and killing by rabbit granulocytes. The following antibiotics were used: cloxacillin, cefadroxil, cefuroxim, cefotaxim, gentamicin, netilmicin, lincomicin, doxycycline and riphamicin. A total of 144 S. aureus strains with varied sensitivity to these antibiotics were selected for the study. The experiment used granulocytes isolated from rabbit blood and S. aureus strains incubated for 18 h in TSB broth containing antibiotics in the concentrations of 0.1 MIC, 0.2 MIC and 0.5 MIC, and in the antibiotics-free medium. Phagocytosis was assessed by the method of differential staining with acridine orange and crystal violet, allowing simultaneous determination of phagocytised and killed S. aureus cell counts. The findings revealed that the culture of S. aureus in the presence of all the antibiotics used in subinhibitory concentrations increased significantly the susceptibility of most S. aureus strains to phagocytosis and killing by granulocytes. The above effect usually occurred in the concentrations of 0.1 MIC (54.2%), more seldom in 0.2 MIC (13%) and 0.5 MIC (15% of strains). Each group of S. aureus contained some which showed no change in susceptibility following culture with the chemotherapeutic agents in subinhibitory concentrations (26.3%). Insensitive strains to the subinhibitory effects were equally common among susceptible (27%), intermediate (23%) and resistant (26%) strains of S. aureus to the antibiotics used. No statistically significant reduction was noted in phagocytosis or killing by rabbit granulocytes. No correlation was observed between the susceptibility to the subinhibitory effects of the antibiotics involved and their biochemical mechanisms.     

Evaluation of susceptibility to antibiotics of Staphylococcus aureus strains resistant to methicillin]

Methicillin-resistant strains of Staphylococcus aureus (MRSA) constitute a serious diagnostic and therapeutic problem. Over 500 strains of Staphylococcus aureus were tested for susceptibility to methicillin. By application of a screening method, 13.7% of these strains were classified as methicillin-resistant. Over 95% of these strains were isolated from hospital infections. Applying criteria of belonging of these strains to methicillin-resistance classes it was found that 49.3% belonged to class II, 31.2% to class III and 19.5% to class IV. Analysis of susceptibility to antibiotics of MRSA strains demonstrated significant differences between class II and between class III and IV in resistance to imipenem, gentamycin, erythromycin and tetracycline. All tested strains were susceptible to ciprofloxacin, ofloxacin, vancomycin and teicoplanin. The screening method (25 mg methicillin/l of TSA medium) results in obtaining of univocal results of determination of methicillin-resistance in S. aureus.     

Susceptibility to selected chemotherapeutics of Staphylococcus aureus strains resistant to methycyline isolated from clinical materials in the years 1991-1992 and 1997

The susceptibility to selected chemotherapeutic agents was determined in 100 strains of Staphylococcus aureus methicillin-resistant (MRSA) isolated from clinical materials in 1991-1992 (50 strains) and in 1997 (50 strains). Two methods were used for the determination: disc method and antibiotic dilution in agar. The minimal inhibitory concentration (MIC) was determined for vancomycin, teicoplanin, furazolidone, nitrofurantoin, ofloxacin, gentamicin, netilmicin and trimethoprim. The concentrations of the chemotherapeutics in the substrate ranged from 0.125 to 512 mg/l. The obtained results served for drawing of the following conclusions: all studied MRSA strains isolated in 1991-1992 and in 1997 were sensitive to glycopeptide antibiotics: vancomycin and teicoplanin, to nitrofurans: nitrofurantoin and furazolidone, and to fusidic acid. MRSA strains isolated in 1991-1992 were sensitive to ofloxacin, but in 1997 about 80% of the strains were resistant to that antibiotic, and this resistance was noted in S. aureus strains with homogeneous resistance to methicillin. Increasing frequency of resistance to mupirocin was found, in 1991-1992 4% of the strains were resistant, and in 1997 the resistance of MRSA to that antibiotic was found in 12%. No changes occurred in the sensitivity of staphylococci to trimethoprim/sulfamethoxazole (cotrimoxazole). About 94% of strains in 1991-1992 and 1997 were sensitive to that drug. The sensitivity to cotrimoxazole is connected with one of its components (trimethoprim), with 94% of MRSA strains sensitive to it.     

Molecular epidemiology of methicillin-resistant Staphylococcus aureus isolates from clinical specimens of patients with nosocomial infection: are there unnoticed silent outbreaks?

Bacteriological and epidemiological studies were carried out on 90 isolates of methicillin-resistant Staphylococcus aureus (MRSA) at Turgut Ozal Medical Center of In?nü University, (Malatya/Turkey). MRSA isolates were obtained from patients with nosocomial infections. Staphylococcus aureus clinical isolates were collected between May 2004-May 2005. Isolates were tested for resistance to methicillin. Antimicrobial susceptibility testing and slime production evaluation was performed. Genotype studies were carried out by arbitrarily primed polymerase chain reaction (AP-PCR) and consequent cluster analysis. All of the isolates were mecA-positive in a PCR-based assay; all exhibited resistance to oxacillin, by agar dilution (MICs > or = 4 mg/L) and disc diffusion methods, and multiple antibiotics. Most MRSA isolates were collected in intensive care units. Of 90 samples, 53 were found to be unrelated to the others while the remaining 37 strains were either identical or closely related. Dendrogram analysis identified nine major clusters. These data support the opinion that MRSA are significant nosocomial pathogens in intensive care units and that resistant clones may be transmitted between patients. Molecular epidemiological tools are helpful for understanding transmission patterns and sources of infection, and are useful for measuring outcomes of intervention strategies implemented to reduce nosocomial MRSA.     

Involvement of multiple genetic determinants in high-level methicillin resistance in Staphylococcus aureus.

A methicillin-susceptible, novobiocin-resistant strain of Staphylococcus aureus (RN2677; methicillin MIC, 0.8 micrograms/ml) was transformed with DNA prepared from highly and homogeneously methicillin-resistant S. aureus strains (methicillin MIC, greater than or equal to 400 micrograms/ml) or from heterogeneous strains in which the majority of cells had a low level of resistance (methicillin MIC, 6.3 micrograms/ml). All methicillin-resistant transformants showed low and heterogeneous resistance (methicillin MIC, 3.1 micrograms/ml) irrespective of the resistance level of DNA donors. All transformants examined produced normal amounts of the low-affinity penicillin-binding protein (PBP) 2a, and methicillin resistance and the capacity to produce PBP 2a showed the same degree of genetic linkage to the novobiocin resistance marker with both homogeneous and heterogeneous DNA donors. Next, we isolated a methicillin-susceptible mutant from a highly and homogeneously resistant strain which had a Tn551 insertion near or within the PBP 2a gene and thus did not produce PBP 2a. With this mutant used as the recipient, genetic transformation of the methicillin resistance gene was repeated with DNA isolated either from highly and homogeneously resistant strains or from heterogeneous (low-resistance) strains. All transformants obtained expressed high and homogeneous resistance and produced PBP 2a irrespective of the resistance level of the DNA donors. Our findings suggest that (i) the methicillin resistance locus is identical to the structural gene for PBP 2a, (ii) although the ability to produce PBP 2a is essential for resistance, the MICs for the majority of cells are not related to the cellular concentration of PBP 2a, and (iii) high MICs and homogeneous expression of resistance require the products of other distinct genetic elements as well.