Susceptibility of Brazilian Staphylococcal Strains to Glycopeptides Evaluated by Different Testing Methods

Reports of staphylococci with reduced susceptibility to glycopeptides are cause for concern. This study evaluated the susceptibility of 84 staphylococci clinical isolates to glycopeptides by the disk diffusion, agar dilution, E-test, and BHIA screening methods. Vancomycin agar dilution showed all strains presented minimum inhibitory concentration (MIC) ranging from 0.5 to 2 μg/ml, and the E-test showed similar results. Teicoplanin agar dilution test showed MICs ranging from ≤ 0.5 to 2 μg/ml for Staphylococcus aureus and MICs ranging from <0.25 to 32 μg/ml for coagulase-negative Staphylococcus (CNS). Ten CNS isolates presented MICs ranging from 8 to 32 μg/ml for agar dilution and/or E-test. All the staphylococci were susceptible to vancomycin by the disk diffusion test (DDT), but two CNS isolates presented intermediate resistance to teicoplanin by the DDT and MICs of susceptibility, with two other CNS strains, teicoplanin-susceptible by the DDT, presented MICs of intermediate resistance. On the vancomycin-containing agar, 20 CNS isolates were able to grow, but no S. aureus strain. All these isolates showed MICs to teicoplanin (4–32 μg/ml) higher than those isolates that did not grow on the agar screen plate. PFGE of chromosomal SmaI digests showed a wide diversity of these CNS strains, without any predominance of a single PFGE pattern. Received: 25 May 2001 / Accepted: 9 July 2001     

Erythromycin resistance genes in Streptococcus pyogenes isolates in Kanagawa, Japan

The susceptibility of 224 Streptococcus pyogenes isolates obtained from children in Japan from 1981 to 1997 to treatment with erythromycin was determined by the agar dilution method. A total of 17 isolates belonging to serotype M12T12 were resistant (MICs>1 microg/ml). Fourteen of the 17 resistant strains obtained from 1982 to 1985 harbored ermB and showed an identical pulsed-field gel electrophoresis pattern, indicating the spread of a single clone. Two ermTR-containing isolates were obtained in 1983. mefA gene was found in a strain obtained in 1994 in the present study, although this gene is predominantly associated with recent erythromycin resistance among S. pyogenes strains in many countries.     

Antibiotic Susceptibility Patterns of Recent Isolates of Corynebacterium diphtheriae

A variety of factors which might affect zone sizes were studied with strains of Corynebacterium diphtheriae; a standard disc method for antimicrobial sensitivity testing was used. Moderate variations in inoculum size, inoculum preparation, and pH of Mueller Hinton agar (MHA) did not appreciably affect zone sizes. The addition of blood to MHA was necessary to insure the growth of all C. diphtheriae strains on all lots of MHA. Zone diameters on MHA with blood were consistently 4 to 9 mm smaller than on plain MHA; however, zone diameters were within the sensitive range for seven antibiotic discs used on both media. Minimal inhibitory concentration (MIC) values for penicillin, erythromycin, and rifampin were determined by a plate dilution method. The geographical source, toxigenicity, and type of the strains showed no significant correlation with MIC values or zone diameters for eight antibiotic discs. When MIC values were compared to obtainable blood levels, all of the strains appeared to be sensitive with MIC values of 相似文献   

In vitro activity of linezolid and eperezolid, two novel oxazolidinone antimicrobial agents, against anaerobic bacteria

Linezolid (formerly U-100766) and eperezolid (formerly U-100592) are novel oxazolidinone antimicrobial agents that are active against multi-drug-resistant staphylococci, streptococci, enterococci, corynebacteria, and mycobacteria. Preliminary studies also demonstrated that the compounds inhibited some test strains of anaerobic bacteria. Therefore, we extended the in vitro evaluation of these agents to include a total of 54 different anaerobic species. Minimal inhibitory concentration (MIC) values were determined using a standard agar dilution method for 143 anaerobic bacterial isolates. Eperezolid and linezolid demonstrated potent activity against the anaerobic Gram-positive organisms with most MIC values in the range of 0.25-4 microg/mL. Viridans streptococci demonstrated MICs of 1-2 microg/mL; Peptostreptococcus species and Propionibacterium species were inhibited by 相似文献   

Comparative analysis and validation of different assays for glycopeptide susceptibility among methicillin-resistant Staphylococcus aureus strains

Detection of methicillin-resistant Staphylococcus aureus (MRSA) isolates exhibiting intermediate susceptibility to glycopeptides (GISA) is challenging for clinical microbiology laboratories. We compared three different screening assays for evaluating trends in decreased glycopeptide susceptibility during two periods. Ninety four and ninety five consecutive MRSA blood isolates from 189 bacteremic patients collected during periods A (1989-1994) and B (1999-2001), respectively, were screened in parallel for vancomycin or teicoplanin susceptibility by glycopeptide-containing brain-heart infusion agar (BHIA) tests, Etest MICs performed at a standard (0.5 McFarland) or high (2.0 McFarland) inoculum on Mueller-Hinton (MHA) or BHIA, respectively. Any MRSA isolate yielding <50 CFU (representing <10(-6) of the plated inoculum) on either BHIA containing 2 mg/l of vancomycin (V2-BHIA) or 5 mg/l of teicoplanin (T5-BHIA) was considered as fully susceptible to vancomycin or teicoplanin, respectively. The proportion of MRSA isolates yielding>50 CFU on either V2-BHIA or T5-BHIA significantly (P<0.01) increased from 7/94 (7.4 %) or 8/94 (8.5%) in period A to 16/95 (16.8 %) or 14/95 (14.7%) in period B, respectively. Vancomycin Etests MICs on MHA were of lower sensitivity (<30% and<65%), but high specificity (100% and 99%) on periods A and B isolates, respectively, compared to those on V2-BHIA. Vancomycin Etests MICs on BHIA were of a higher sensitivity (57% and 81%) but lower specificity (91% and 65%) compared to those on V2-BHIA, on periods A and B isolates, respectively, reflecting an unexpectedly high number of false positive isolates on period B isolates (28/95). Screening of decreased susceptibility to vancomycin or teicoplanin on V2-BHIA or T5-BHIA, respectively, may represent simple low-cost alternatives to Etest MICs, minimising the risk of missing potential GISA isolates.     

Development of a fluorogenic 5' nuclease PCR assay for detection of the ail gene of pathogenic Yersinia enterocolitica

In this report we describe the development and evaluation of a fluorogenic PCR assay for the detection of pathogenic Yersinia enterocolitica. The assay targets the chromosomally encoded attachment and invasion gene, ail. Three primer-probe sets (TM1, TM2, and TM3) amplifying different, yet overlapping, regions of ail were examined for their specificity and sensitivity. All three primer-probe sets were able to detect between 0.25 and 0.5 pg of purified Y. enterocolitica DNA. TM1 identified all 26 Y. enterocolitica strains examined. TM3 was able to detect all strains except one, whereas TM2 was unable to detect 10 of the Y. enterocolitica strains tested. None of the primer-probe sets cross-reacted with any of the 21 non-Y. enterocolitica strains examined. When the TM1 set was utilized, the fluorogenic PCR assay was able to detect 相似文献   

Analysis of the rdxA gene in high-level metronidazole-resistant clinical isolates confirms a limited use of rdxA mutations as a marker for prediction of metronidazole resistance in Helicobacter pylori

Metronidazole (Mtz) resistance in the gastric pathogen Helicobacter pylori is closely associated with inactivation of the nitroreductase gene rdxA. In order to identify respective mutations for diagnostic purposes we analyzed the rdxA gene in a collection of high-level Mtz-resistant clinical H. pylori isolates. Size alterations in the rdxA gene region were found in only two out of 45 and one out of 40 isolates showing lower-level (minimal inhibitory concentrations (MICs) 32-192 microg ml(-1)) and high-level (MIC>/=256 microg ml(-1)) Mtz resistance, respectively. Point mutations that interrupt the rdxA reading frame were detected in two out of eight high-level resistant isolates (MICs>/=256 microg ml(-1)). Most remarkably, the rdxA gene sequence was found to be identical in four out of five high-level Mtz-resistant and -susceptible paired H. pylori isolates from the same patients each. Taken together, these results demonstrate that although some isolates carry classical resistance-associated rdxA mutations, as described earlier, the use of rdxA mutations as a marker for prediction of Mtz resistance is limited.     

Influence of incubation time in the in vitro antifungal activity of terbinafine against Trichophyton rubrum.

Antifungal susceptibility tests are influenced by a number of technical variables, including inoculum size, temperature, medium formulation and duration of incubation. In this study, we have compared the in vitro susceptibility of 20 strains de Trichophyton rubrum against clotrimazole and terbinafine, and studied the influence of incubation time on MICs of both drugs. The assay was performed by agar dilution, the medium used was Saboraud glucose agar without an antibiotic. The MIC was evaluated at 15, 30 and 45 days' incubation. The MICs ranges of terbinafine were 0.002 to 0.0975 microg/ml, 0.0975 to 0.39 microg/ml and 0.195 to 0.39 microg/ml at 15, 30 and 45 days' incubation, respectively. The MICs ranges of clotrimazole at 15, 30 and 45 days' incubation were 3.125 to 50 microg/ml. T. rubrum was markedly more susceptible to terbinafine than to clotrimazole (p<0.001). In addition, we observed that an increase of incubation time causing an increase in the MIC value of terbinafine (p<0.001), but MIC values for clotrimazole remained constant with time (p=0.464). In conclusion, the MIC is dependent on reading time and the antifungal compound.     

Expression of Mycobacterium smegmatis pyrazinamidase in Mycobacterium tuberculosis confers hypersensitivity to pyrazinamide and related amides

A pyrazinamidase (PZase)-deficient pncA mutant of Mycobacterium tuberculosis, constructed by allelic exchange, was used to investigate the effects of heterologous amidase gene expression on the susceptibility of this organism to pyrazinamide (PZA) and related amides. The mutant was highly resistant to PZA (MIC, >2,000 microg/ml), in accordance with the well-established role of pncA in the PZA susceptibility of M. tuberculosis (A. Scorpio and Y. Zhang, Nat. Med. 2:662-667, 1996). Integration of the pzaA gene encoding the major PZase/nicotinamidase from Mycobacterium smegmatis (H. I. M. Boshoff and V. Mizrahi, J. Bacteriol. 180:5809-5814, 1998) or the M. tuberculosis pncA gene into the pncA mutant complemented its PZase/nicotinamidase defect. In both pzaA- and pncA-complemented mutant strains, the PZase activity was detected exclusively in the cytoplasm, suggesting an intracellular localization for PzaA and PncA. The pzaA-complemented strain was hypersensitive to PZA (MIC, /=20 microg/ml) and was also sensitive to benzamide (MIC, 20 microg/ml), unlike the wild-type and pncA-complemented mutant strains, which were highly resistant to this amide (MIC, >500 microg/ml). This finding was consistent with the observation that benzamide is hydrolyzed by PzaA but not by PncA. Overexpression of PzaA also conferred sensitivity to PZA, nicotinamide, and benzamide on M. smegmatis (MIC, 150 microg/ml in all cases) and rendered Escherichia coli hypersensitive for growth at low pH.     

A novel method using micropig stratum corneum in vitro for the evaluation of anti-Trichophyton mentagrophytes activity

Antifungal susceptibility testing under conditions close to clinical status is expected to provide more helpful information than that obtained by a conventional microdilution method. For this purpose, we developed a novel method to evaluate anti-Trichophyton mentagrophytes activity of antifungal agents in vitro by using disks of micropig stratum corneum epidermis (SCE). Basal agar medium containing K2HPO4, MgSO4, CaCl2 and three kinds of antibiotics. Bifonazole (BFZ), lanoconazole (LCZ) or terbinafine (TBF) was added to the basal agar medium to give serially doubling dilutions ranging from 0.0006 to 10 microg/ml. Five-hundred-microl portions of the agar media thus prepared were solidified in wells of flat-bottomed plates. SCE disks (6 mm in diameter) were placed on surfaces of the agar medium and 10(4) conidia of T. mentagrophytes were inoculated on each SCE disk. There was very good correlation between the initial concentration of the antifungal agents added to the basal agar medium (microg/ml) and the concentration of the agents impregnated into the SCE disks (microg/g) (r2>0.99). The minimum inhibitory concentration (MIC) values of BFZ, LCZ and TBF were respectively 26-, 10- and 78-times higher than those measured by the standard microdilution method. From the correlation between the concentration of the agents in the basal medium and that in the SCE disks, the above MIC values corresponded to the concentrations in SCE disks (microg/g), 832.95 for BFZ, 1.42 for LCZ and 8.87 for TBF. This novel method of antidermatophytic susceptibility testing using SCE would be useful as an in vitro screening of proper antimycotics for topical treatment of dermatophytosis.     

Response of tomato genotypes to bacterial speck (Pseudomonas syringae pv. tomato)

Two genotypes of tomato A 100 and Ontario 7710 which were inoculated separately with four strains of Pseudomonas syringae pv. tomato differed significantly in disease severity (susceptibility) to bacterial speck. At both concentrations of inoculum of each strain used (10⁷ and 10⁸ cfu/ml) A 100 appeared to be highly susceptible whereas Ontario 7710 showed very low or no susceptibility. The significant differences in virulence between strains and in response of tomato plants in three replicate experiments were found. Generally, concentration of inoculum 10⁷ cfu/ml was too low to induce consistent level of disease severity. The obtained results indicate the importance of consistent and favorable conditions for disease development in screening of tomato resistance to bacterial speck.     

Effects of disinfectants on Campylobacter jejuni

Because of the increasing recognition that Campylobacter jejuni is an important enteric pathogen of humans, we studied the effects of widely used disinfectants on the viability of this organism. At an inoculum size of 10(3) to 10(4) CFU/ml, 1.25 mg of hypochlorite per liter killed three strains within 1 min. At an inoculum size of 10(6) to 10(7) CFU/ml, 5 mg of hypochlorite per liter killed three strains within 15 min. Killing of similar concentrations of C. jejuni and Escherichia coli by hypochlorite was approximately the same. At the high inoculum, 0.15% phenolic compound, 10 mg of iodophor per liter, 1:50,000 quaternary ammonium compound, 70% ethyl alcohol, and 0.125% glutaraldehyde killed all three strains within 1 min. These studies demonstrate that, under the conditions we tested (pH 7.0; 24 to 26 degrees C), the recommended standard concentrations of disinfecting agents are adequate to destroy C. jejuni.     

Effects of disinfectants on Campylobacter jejuni.

Because of the increasing recognition that Campylobacter jejuni is an important enteric pathogen of humans, we studied the effects of widely used disinfectants on the viability of this organism. At an inoculum size of 10(3) to 10(4) CFU/ml, 1.25 mg of hypochlorite per liter killed three strains within 1 min. At an inoculum size of 10(6) to 10(7) CFU/ml, 5 mg of hypochlorite per liter killed three strains within 15 min. Killing of similar concentrations of C. jejuni and Escherichia coli by hypochlorite was approximately the same. At the high inoculum, 0.15% phenolic compound, 10 mg of iodophor per liter, 1:50,000 quaternary ammonium compound, 70% ethyl alcohol, and 0.125% glutaraldehyde killed all three strains within 1 min. These studies demonstrate that, under the conditions we tested (pH 7.0; 24 to 26 degrees C), the recommended standard concentrations of disinfecting agents are adequate to destroy C. jejuni.     

Quantitative disk diffusion as a convenient method for determining minimum inhibitory concentrations of oxacillin for staphylococci strains

In this study the susceptibility of 58 coagulase-negative staphylococci (CoNS) strains and 58 Staphylococcus aureus strains to oxacillin was evaluated by a novel method called quantitative disk diffusion (DD) method. The results obtained were compared to phenotypic methods as agar dilution (AD) for oxacillin, disk diffusion (DD) for cefoxitin, and related to the presence of the mecA gene detected by PCR. Minimum inhibitory concentrations (MIC) determined by the quantitative DD method were equivalent to MICs determined in the AD method for S. aureus (Student's t test, p=0.99) and CoNS (Student's t test, p=0.97). Incongruent results between PCR mecA gene determinations and the quantitative DD method were obtained in 8 strains (5 S. aureus and 3 CoNS) where the mecA gene expression was blocked. However, oxacillin resistance was detected by the proposed method even in staphylococci strains showing low-level or heterogeneous resistance to the antibiotic while other phenotypic methods failed. The single quantitative DD method is not expensive, it can be performed in any laboratory and permits accurate identification of oxacillin resistant staphylococci.     

Synergism of azlocillin, mezlocillin, piperacillin in combination with tobramycin against Klebsiella and Pseudomonas

Fifty-three clinical isolates of Klebsiella and fifty-one clinical isolates of Pseudomonas aeruginosa, twenty-six of which were carbenicillin-(CARB) resistant, were tested for susceptibility to mezlocillin (MEZ), azlocillin (AZL), and piperacillin (PIP), both alone and in combination with tobramycin (TOB) using the microtiter broth diluent method and an inoculum density of 10(6) CFU/ml. The Klebsiella were highly resistant to TOB, MEZ, and PIP (MIC90: 8, greater than 256, greater than 128 micrograms/ml, respectively). Synergy was demonstrated in 53 percent (PIP/TOB) and 51 percent (MEZ/TOB). An indifferent response was observed in 47 percent (PIP/TOB) and 49 percent MEZ/TOB of the Klebsiella. PIP, MEZ, and AZL in combination with TOB showed synergism against CARB-resistant Pseudomonas in less than 10 percent of the strains tested. Synergy could be demonstrated against CARB-susceptible Pseudomonas with the combinations PIP/TOB, AZL/TOB, and MEZ/TOB in 12 percent, 12 percent, and 24 percent, respectively, of the twenty-five strains tested. Indifferent effects were observed in 84 percent, 88 percent, and 76 percent, respectively, of these same CARB-susceptible strains. These data suggest that there is no significant difference in the incidence of synergy with these new penicillins and tobramycin against either Pseudomonas or Klebsiella.     

Immobilization in alginate as a technique for the preservation of Bacillus thuringiensis var. israelensis for long-term preservation

Technique for immobilization using sodium alginate as the matrix to preserve Bacillus thuringiensis var. israelensis isolates for long time storage was developed. Two strains of B. thuringiensis var. israelensis viz., VCRC B-17 and WHO standard strain IPS-82 were immobilized in alginate matrix and preserved at 4 degrees C and when tested both were found to have maintained excellent viability and mosquito larvicidal activity for 10 years. Mosquito larvicidal activity of B-17 and IPS-82 alginate beads, in term of LC(50) values before storage was 72.07 ng/ml and 47.07 ng/ml, respectively and after storage at 4 degrees C for a period of 1 to 10 years the values ranged from 69.88 to 73.86 ng/ml with a mean of 72.38 ng/ml and 45.32 to 48.60 ng/ml with a mean of 47.49 ng/ml, respectively. Similarly spore count of the beads of the respective strains was 4.37 x 10(8) and 3.33 x 10(10) CFU/mg before storage. After storage at 4 degrees C for a period of 1 to 10 years the counts of the beads of the respective strains ranged from 4.23 x 10(8) to 4.83 x 10(8) CFU/mg (mean of 4.49 x 10(8) CFU/mg) and 3.2 x 10(10) to 3.87 x 10(10) CFU/mg (mean of 3.54 x 10(10) CFU/mg). The alginate matrix immobilization technique has many advantages over free cells are that they enhance the stability of both spores and toxin against several physicochemical conditions and confer reduced susceptibility to contamination.     

Antibiotic resistance of staphylococci isolated from outpatients

The aim of the study was to determine susceptibility of 587 strains of S. aureus and 85 strains of coagulase-negative staphylococci isolated from outpatients in Poznań to co-trimoxazole, amoxycillin/clavulanic acid, erythromycin, gentamycin, doxycycline, ampicillin, oxacillin, cephradine, clindamycin and neomycin. Also methicillin-resistant strains were determined as well as strains ability to produce beta-lactamases. Susceptibility testing and examination of methicillin-resistant strains were performed by the disc diffusion techniques according to recommendation of NCCLS. Methicillin-resistant strains were additionally examined to their sensitivity to vankomycin and teicoplanin. beta-lactamase production was detected using nitrocefin impregnated discs and iodometric method. Amoxacillin/clavulanic acid, gentamycin, co-trimoxazole, cephradin, oxacillin and clindamycin occurred to be very active against both, S. aureus and coagulase-negative staphylococci. 84.7% to 100% of examined strains were sensitive to these drugs. Doxycyclin, erythromycin and ampicillin were less effective. Nine strains (1.5%) of 587 strains of S. aureus as well as 7 strains (8.7%) of coagulase-negative staphylococci were methicillin-resistant. All of methicillin-resistant strains were sensitive to vancomycin and teicoplanin. More than 75% of S. aureus and close to 50% of coagulase-negative staphylococci were able to produce beta-lactamases.     

Time-resolved fluorescence immunoassay (TRFIA) for the detection of Escherichia coli O157:H7 in apple cider.

An immunoassay based on immunomagnetic separation and time-resolved fluorometry was developed for the detection of E. coli O157:H7 in apple cider. The time-resolved fluorescent immunoassay (TRFIA) uses a polyclonal antibody bound to immunomagnetic beads as the capture antibody and the same antibody labeled with europium as the detection antibody. Cell suspensions of 10(1) to 10(8) E. coli O157:H7 and K-12 organisms per ml were used to test the sensitivity and specificity of the assay. The sensitivity of the assay was 10(3) E. coli O157:H7 cells with no cross-reaction with K-12. Pure cultures of E. coli O157:H7 (10(1) to 10(5) CFU/ml) in apple cider could be detected within 6 h, including 4 h for incubation in modified EC broth with novobiocin and 2 h for the immunoassay. When apple cider was spiked with 1 to 10(3) CFU/ml of E. coli O157:H7 and 10(6) CFU/ml of K-12, our data show that the high level of K-12 in apple cider did not impede the detection of low levels of O157:H7. The minimum detectable numbers of cells present in the initial inoculum were 10(2) and 10(1) CFU/ml after 4- and 6-h enrichment. The TRFIA provides a rapid and sensitive means of detecting E. coli O157:H7 in apple cider.     

Augmentation of Soil with Sporangia of Actinoplanes spp. for Biological Control of Pythium Damping-off

A method was developed for applying strains of Actinoplanes spp. that are hyper-parasites of oospores of Pythium ultimum to soil for reducing Pythium damping-off of plants. The method is based on the augmentation of soil with sporangia of a strain of Actinoplanes spp. borne on clay granules. In vitro sporulation of strains K30, W57, W257 and 25844 was: (1) greater for most strains on dilute Czapek-Dox agar than on four other agar media; (2) inhibited by continuous exposure to fluorescent light of intensity 4-150 μEm^-2s^-1, but not by exposure to 1 μEm^-2s^-1 or darkness; (3) greater at 20-307deg;C than at 10°C;and (4) greater at pH 6-7 than at pH 5 or 8. On solid carriers treated with dilute Czapek-Dox broth (pH 7) and incubated in the dark at 30°C for 3 weeks, strains sporulated poorly or not at all on vermiculite, perlite and rice hulls, but sporulated abundantly (10⁷-10⁹ colony-forming units (CFU) g^-1 of granules) on montmorillonite clay granules. When strains 25844, W57 and W257 were applied as granules (4 10⁷ - 4 × 10⁸ CFU g^-1) at 5% (w/w) to field plots infested with 750-1000 oospores of P. ultimum g^-1 of soil, only strain 25844 consistently increased emergence and reduced root rot of table beets 8- 1 at 24-28 days after planting compared with controls. Strain 25844 (10⁸ CFU g^-1 of granules) at 1% (w/w) also increased the emergence of bush beans at 28 days after planting in P. ultimum-infested plots, but lower rates were ineffective. The inoculum viability of strain 25844 on clay granules declined 100-fold during 2 months of storage at 5-35°C, but thereafter remained stable for another 4 months. Strain 25844 on 6-month-old granules retained a high degree of hyper-parasitic activity toward oospores of P. ultimum. Augmentation of field soil with sporangia of Actinoplanes spp. is a valid approach to the biological control of pythium damping-off.