A new antimicrobial susceptibility testing method of Escherichia coli against ampicillin by MSPQC

A new antimicrobial susceptibility testing method by multi-channel series piezoelectric quartz crystal (MSPQC) was proposed. This method was used to test susceptibility of clinical Escherichia coli isolates against ampicillin. Both the minimum inhibitory concentrations (MICs) and interpretive categorization of clinical E. coli isolates were determined by proposed method. Comparing tests were run at the same time by the agar dilution method and the disk diffusion method. The experimental results showed that MSPQC method had a good agreement with the reference methods. Compared with those methods, the MSPQC method is simple, rapid, and convenient to perform. It can offer both a minimum inhibitory concentration (MIC) and an interpretive category result.     

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.     

Cost-effectiveness of blood agar for isolation of mycobacteria

Background

Mycobacterium species are grown using specific media that increase laboratory cost, thus hampering their diffusion in resource-limited countries. Preliminary data suggested that versatile blood agar may be also used for mycobacterial culture.

Methodology

We examined the growth of 41 different Mycobacterium species on 5% blood agar. Over a 24-month period we analysed isolation of mycobacteria after parallel inoculation of clinical specimens into both a reference automated system (BACTEC 9000 MB broth) and 5% blood agar slant tubes, after NaOH decontamination, and compared the cost of performing 1,000 analyses using these two techniques.

Conclusions

Mycobacterium reference species cultured on blood agar, with the exception of Mycobacterium ulcerans. Inoculation of 1,634 specimens yielded 95 Mycobacterium isolates. Blood agar performed significantly more efficiently than BACTEC 9000 MB broth (94 vs 88 isolates, P = 0.03). Decontamination of Candida albicans in 5 specimens by addition of amphotericin B in blood agar yielded one more M. tuberculosis isolate that could not be isolated in BACTEC broth. Uneven distribution of time to culture positivity for M. tuberculosis had a median (range) of 19±5 days using blood agar and 26±6 days using BACTEC 9000 MB broth. Cost for 1,000 analyses in France was estimated to be of 1,913 euros using the blood agar method and 8,990 euros using the BACTEC 9000 MB method. Blood agar should be regarded as a first-line medium for culturing Mycobacterium species. It saves time, is cost-effective, is more sensitive than, and at least as rapid as the automated method. This is of particular importance for resource-limited countries in which the prevalence of tuberculosis is high.     

Recommendation for a Standardised Method of Broth Microdilution Susceptibility Testing for Porcine Bordetella bronchiseptica

The objective was to establish and standardise a broth microdilution susceptibility testing method for porcine Bordetella (B.) bronchiseptica. B. bronchiseptica isolates from different geographical regions and farms were genotyped by macrorestriction analysis and subsequent pulsed-field gel electrophoresis. One reference and one type strain plus two field isolates of B. bronchiseptica were chosen to analyse growth curves in four different media: cation-adjusted Mueller-Hinton broth (CAMHB) with and without 2% lysed horse blood, Brain-Heart-Infusion (BHI), and Caso broth. The growth rate of each test strain in each medium was determined by culture enumeration and the suitability of CAMHB was confirmed by comparative statistical analysis. Thereafter, reference and type strain and eight epidemiologically unrelated field isolates of B. bronchiseptica were used to test the suitability of a broth microdilution susceptibility testing method following CLSI-approved performance standards given in document VET01-A4. Susceptibility tests, using 20 antimicrobial agents, were performed in five replicates, and data were collected after 20 and 24 hours incubation and statistically analysed. Due to the low growth rate of B. bronchiseptica, an incubation time of 24 hours resulted in significantly more homogeneous minimum inhibitory concentrations after five replications compared to a 20-hour incubation. An interlaboratory comparison trial including susceptibility testing of 24 antimicrobial agents revealed a high mean level of reproducibility (97.9%) of the modified method. Hence, in a harmonization for broth microdilution susceptibility testing of B. bronchiseptica, an incubation time of 24 hours in CAMHB medium with an incubation temperature of 35°C and an inoculum concentration of approximately 5 x 10⁵ cfu/ml was proposed.     

Use of the Chrome Azurol S Agar Plate Technique To Differentiate Strains and Field Isolates of Rhizobium leguminosarum biovar trifolii

Identification of Rhizobium and Bradyrhizobium strains and especially of indigenous isolates continues to be one of the major difficulties associated with competition studies. Because there is no universally accepted method, the method of choice depends on preference, experience, and equipment. Here, an agar plate technique was used to distinguish strains and field isolates of Rhizobium leguminosarum biovar trifolii to provide a basis for identifying nodule occupants in further competition studies. A rapid plate technique, based on differential growth characteristics, complements other techniques such as serological reactions, particularly when antisera cross-react with nonhomologous strains. The technique involves culturing strains and isolates on chrome azurol S agar. Although similar responses were observed among some strains, the response was highly reproducible and was considered an ideal complementary technique used in conjunction with serological procedures. Strains with similar responses could often be differentiated by varying media components, such as the source of carbon.     

Development of pooled suppression subtractive hybridization to analyze the pangenome of Staphylococcus aureus

We describe the development and application of a Pooled Suppression Subtractive Hybridization (PSSH) method to describe differences between the genomic content of a pool of clinical Staphylococcus aureus isolates and a sequenced reference strain. In comparative bacterial genomics, Suppression Subtractive Hybridization (SSH) is normally utilized to compare genomic features or expression profiles of one strain versus another, which limits its ability to analyze communities of isolates. However, a PSSH approach theoretically enables the user to characterize the entirety of gene content unique to a related group of isolates in a single reaction. These unique fragments may then be linked to individual isolates through standard PCR. This method was applied to examine the genomic diversity found in pools of S.aureus isolates associated with complicated bacteremia infections leading to endocarditis and osteomyelitis. Across four pools of 10 isolates each, four hundred and twenty seven fragments not found in or significantly divergent from the S. aureus NCTC 8325 reference genome were detected. These fragments could be linked to individual strains within its pool by PCR. This is the first use of PSSH to examine the S. aureus pangenome. We propose that PSSH is a powerful tool for researchers interested in rapidly comparing the genomic content of multiple unstudied isolates.     

Differential susceptibility of aeromonads and coliforms to cefsulodin.

Cefsulodin was evaluated as a potential selective agent for aeromonads. Resistance of Aeromonas and coliform isolates was determined by using a standard disk diffusion technique. A total of 119 Aeromonas and 78 coliform strains were isolated. For 102 of 130 [corrected] Aeromonas isolates (environmental and reference strains), the MIC of cefsulodin was < 8 micrograms/ml. Results of MIC tests by the agar dilution method showed that a concentration of cefsulodin of 10 micrograms/ml or less inhibited the growth of 96% of isolates. In comparison, for 81 of 94 coliform isolates (environmental and reference strains), the MIC of cefsulodin was > 32 micrograms/ml. Because cefsulodin suppresses growth of Aeromonas and other oxidase-positive organisms, total coliform (TC) and Escherichia coli counts on Chromocult Coliform agar (CC agar) without cefsulodin and on CC agar with 10 mg of cefsulodin per liter (CC-CFS) were compared. Variance analysis of data from 14 sewage-polluted irrigation water specimens did not demonstrate any statistically significant difference in the enumeration of E. coli with CC and CC-CFS media. On average, the CC agar recovered 2.46 times as many TCs as CC-CFS. However, Aeromonas colonies made up an average of 58.6% of the TC counts on CC agar. Because no Aeromonas spp. were recovered on CC-CFS, background interference was eliminated and the counts that were obtained reflected more accurately the number of TCs. Results of this study suggest that cefsulodin may be a useful selective agent against Aeromonas spp. which should be included in coliform chromogenic media when high levels of accompanying flora are expected.     

Isolation and Partial Characterization of Bacterial Strains on Low Organic Carbon Medium from Soils Fertilized with Different Organic Amendments

A total of 720 bacterial strains were isolated from soils with four different organic amendment regimes on a low organic carbon (low-C) agar medium (10 µg C ml^?1) traditionally used for isolation of oligotrophs. Organic amendments in combination with field history resulted in differences in dissolved organic carbon contents in these soils. There were negative correlations between total and dissolved organic carbon content and the number of isolates on low-C agar medium, whereas these correlations were absent for bacterial strains isolated from the same soil on high-C agar medium (1,000 µg C ml^?1). Repeated transfers (up to ten times) of the isolates from low-C agar medium to fresh low- and high-C agar media were done to test for exclusive growth under oligotrophic conditions. The number of isolates exclusively growing under oligotrophic conditions dropped after each subsequent transfer from 241 after the first to 98 after the third transfer step. Identification on the basis of partial 16S rRNA gene sequences revealed that most of the 241 isolates (as well as the subset of 98 isolates) belong to widespread genera such as Streptomyces, Rhizobium, Bradyrhizobium, and Mesorhizobium, and the taxonomic composition of dominant genera changed from the first transfer step to the third. A selected subset of 17 isolates were further identified and characterized for exclusive growth on low-C agar medium. Two isolates continued to grow only on low-C agar medium up to the tenth transfer step and matched most closely with Rhizobium sullae and an uncultured bacterium on the basis of the almost full-length 16S rRNA gene. It was concluded that the vast majority of strains which are isolated on low-C agar media belong to the trophic group of microorganisms adapted to a “broad range” of carbon concentrations, including well-known and widespread bacterial genera. Oligotrophy is a physiological, not a taxonomic property, and can only be identified by cultural means so far. We showed that true oligotrophs that are unable to grow on high carbon media are rare and belong to genera that also contain broad-range and copiotrophic strains.     

Evaluation of Etest Performed in Mueller–Hinton Agar Supplemented with Glucose for Antifungal Susceptibility Testing of Clinical Isolates of Filamentous Fungi

Although reference broth microdilution protocol is currently available for filamentous fungi antifungal susceptibility testing (AFST), simpler alternatives as Etest^® tend to be favoured in clinical routine, making their validation of utmost importance. In this study, Etest^® method using 2 % glucose supplemented Muller–Hinton agar was compared to the Clinical and Laboratory Standards Institute (CLSI) M38-A2 protocol for filamentous fungi AFST. The echinocandins, caspofungin and anidulafungin, the azoles voriconazole and posaconazole, and the polyene amphotericin B were tested against 48 Aspergillus spp., seven Fusarium spp., one Beauveria bassiana and three Paecilomyces lilacinus isolates. The majority of the isolates were susceptible to the antifungals tested, and the overall level of agreement between the CLSI and Etest methods was 71.9 % for one dilution and 99.7 % when using two dilutions. Since interpretative breakpoints for filamentous fungi employing the CLSI or Etest methods are not available yet, the established epidemiological cut-off values for Aspergillus spp. were used to distinguish wild-type isolates from those with acquired resistance mechanisms. Forty-five Aspergillus strains did not evidence resistance mutations.     

Evaluation of the selectivity of white rot isolates using near infrared spectroscopic techniques

Seventeen isolates from white rotted beech wood and six strains from a local culture collection were evaluated for their capability to delignify beech and spruce wood selectively. Six peroxidase-positive isolates were found using a colorimetric agar plate test (Poly R-478), and genetically identified by their internal transcribed spacer (ITS1) or 28S rDNA sequences. Colonised on beech and spruce wood veneers, some of the peroxidase-positive isolates caused selective white rot on both wood species. Weight loss and lignin content of the degraded veneers were estimated from FT-NIR spectra with established linear regression models and multivariate models based on partial least squares regression (PLSR). Weight loss of the samples was also determined gravimetrically. A measure for the relative selectivity of the strains for lignin degradation was formulated and the values were calculated. Two strains that were identified as Oxyporus latemarginatus and Trametes cervina exhibited high selectivity on spruce wood, but the lignin content of the decayed wood was higher than that degraded by the reference strain Ceriporiopsis subvermispora. One strain – identified as Phlebia tremellosa – led to a lower lignin content of beech wood but caused also comparably high weight loss and thus exhibited an overall lower selectivity. The NIR spectroscopic method proved to be convenient for the quick screening of selective white rot fungi. Furthermore, the results revealed that high selectivity for lignin degradation is much more pronounced in early degradation stages.     

Comparison of Real-Time PCR with Disk Diffusion, Agar Screen and E-test Methods for Detection of Methicillin-Resistant Staphylococcus aureus

Methicillin-resistant Staphylococcus aureus (MRSA) is a nosocomial pathogen. Our main objective was to compare oxacillin disk test, oxacillin E-test, and oxacillin agar screen for detection of methicillin resistance in S. aureus, using real-time PCR for mecA as the “gold standard” comparison assay. 196 S. aureus isolates were identified out of 284 Staphylococcus isolates. These isolates were screened for MRSA with several methods: disk diffusion, agar screen (6.0 μg/ml), oxacillin E-test, and real-time PCR for detection of mecA gene. Of the 196 S. aureus isolates tested, 96 isolates (49%) were mecA-positive and 100 isolates (51%) mecA-negative. All methods tested had a statistically significant agreement with real-time PCR. E-test was 100% sensitive and specific for mecA presence. The sensitivity and specificity of oxacillin agar screen method were 98 and 99%, respectively and sensitivity and specificity of oxacillin disk diffusion method were 95 and 93%, respectively. In the present study, oxacillin E-test is proposed as the best phenotypic method. For economic reasons, the oxacillin agar screen method (6.0 μg/ml), which is suitable for the detection of MRSA, is recommended due to its accuracy and low cost.     

Interspecific Interactions Among Members of Family Halobacteriaceae from Natural Solar Salterns

Solar salterns are crystallizer ponds with highly diverse extremophilic microbial flora living individually or in consortium. Twenty-four culturable haloarchaeal isolates were obtained from solar salterns of Goa, which were grouped under Halococcus, Haloferax, Haloarcula and Halorubrum. Cell-free supernatants of different isolates were checked against each other by pour plate technique combined with agar well diffusion method. This resulted in a zone of growth inhibition or stimulation around wells, indicating that some isolates had antagonistic and/or a beneficial effect on the other genera. Thus, members of family Halobacteriaceae were found to secrete extracellular metabolites, which can act as growth enhancers or repressors.     

The production of ginsenosides in hairy root cultures of American Ginseng, Panax quinquefolium L. and their antimicrobial activity

Panax quinquefolium, American ginseng, is valued for its triterpene saponins, known as ginsenosides. These constituents possess a number of pharmacological properties and hairy root cultures can synthesize similar saponins to those of field-cultivated roots. The antibacterial activity of extracts from three hairy root clones of P. quinquefolium L. was tested against a range of standard bacterial and yeast strains. The agar diffusion method was used to evaluate inhibition of microbial growth at various extract concentrations. Commercial antibiotics were used as positive reference standards to determine the sensitivity of the strains. Susceptibility testing to antibiotics was also tested using the disk diffusion method. The minimal inhibitory concentration values of the extracts, obtained by agar diffusion, ranged from 0.8 to 1.4 mg/ml. The results showed that extracts from hairy root cultures inhibited the growth of bacteria and yeast strains and suggest that they may be useful in the treatment of infections caused by pathogenic microorganisms.     

Influence of the culture medium on antibiotic susceptibility testing of food-associated lactic acid bacteria with the agar overlay disc diffusion method

AIMS: To investigate the influence of the culture medium on antibiotic susceptibility testing of food-associated lactic acid bacteria (LAB) with the agar overlay disc diffusion (DD) method. METHOD: The antibiotic resistance profile of 39 food-associated lactobacilli and enterococci was determined with the agar overlay DD method using a defined medium (i.e. Iso-sensitest agar; ISA) or an undefined medium (i.e. de Man, Rogosa, Sharpe or MRS agar). RESULTS: The study revealed that ampicillin discs and, although to a lesser extent, also tetracycline discs consistently produced larger zones on MRS medium compared to ISA medium. For the antibiotics gentamicin, bacitracin and erythromycin, the radius of the inhibition zones produced on MRS medium was significantly smaller in relation to ISA. For categorizing LAB isolates into resistant, intermediate and susceptible groups, it was demonstrated that major errors can occur in determining bacitracin and gentamicin resistance if MRS medium instead of ISA medium is used. On the other hand, the performance of both media was found to be equivalent for testing tetracycline resistance. CONCLUSIONS, SIGNIFICANCE AND IMPACT OF THE STUDY: Despite the fact that MRS medium generally supports the growth of lactic acid bacteria much better than the nutrient-poor ISA medium, the present study clearly demonstrates that both media are not compatible in susceptibility testing against various classes of antibiotics. These results may stimulate future discussions on a generally recommended DD method for susceptibility testing of food LAB strains.     

Tryptic Soy Bile-Kanamycin Test for the Identification of Bacteroides fragilis

A simple and practical test for the identification of Bacteroides fragilis is described. It utilizes two well-known properties of this species, i.e., stimulation of growth by bile and resistance to kanamycin. The test media are a tryptic-soy bile agar plate and a supplemented blood agar plate on which a kanamycin 1,000-mug/ml disk is placed. Incubation is for 24 h at 37 C in GasPak. The results of screening 190 strains, mostly clinical isolates, indicate that B. fragilis can be easily and reliably distinguished from other Bacteroides and from Fusobacterium species by its growth on tryptic-soy bile agar and resistance to kanamycin.     

Roche Susceptibility Test (RST) medium,a defined formulation for susceptibility testing: I. Nutrient interaction and optimization studies

Roche Susceptibility Test (RST) Medium represents the most completely optimized and convenient fully defined medium described. It requires no post-autoclaving supplementation with vitamins, supports good growth of all common aerobic and anaerobic pathogens and may be used as a broth or agar gel on which the swarming of Proteus spp. is virtually eliminated. The broth as a superior buffering capacity to most complex media and an osmolality and pH close to those of human serum. RST is highly satisfactory for the susceptibility testing of commonly used antibiotics and meets the requirements of the National Committe for Clinical Laboratory Standards of the U.S.A. in almost every respect.     

In vitro activity of clindamycin and other antimicrobials against gram-positive bacteria and Hemophilus influenzae.

Summary: Seven antimicrobials--clindamycin, penicillin, ampicillin, cloxacillin, erythromycin, lincomycin and cephalexin--were found to have a high degree of activity in vitro against 256 isolates of gram-positive bacteria and Hemophilus influenzae. Clindamycin was clearly superior against staphylococci and 3.12 mug/ml or less of clindamycin inhibited all 35 isolates of H. influenzae. Synergism was not demonstrated when clindamycin was tested in combination with sulfisoxazole or sulfamethoxazole by either the agar dilution or 24-hour growth curve method. This was true for penicillin as well, and the effect was independent of sulfonamide sensitivity. The erythromycin-sulfonamide combination was synergistic against 6 of 10 strains studied by the growth curve method; this effect was not demonstrable by the agar dilution method.     

Minimal inhibitory concentrations of Candida species to five antifungals by using the reference dilution micromethod and the E-test.

It is accepted that the frequency of candidosis has increased during the last decade, specially in hospitalized patients. The more frequent use of azole antifungals and the recognition of isolates of Candida sp resistant to these and other drugs such as 5-fluorocytosine constitute a great need for a reproducible and useful C. albicans in vitro susceptibility testing method for monitoring antifungal therapy in clinical mycological laboratories. The E-test is a novel agar diffussion technique for testing the susceptibility of yeasts against a defined continous gradient of drug and could be used by most clinical laboratories. In this study the E-test and the NCCLS reference microbroth method (M27-P guidelines) were used to determine the MICs of amphotericin B, 5-flucytosine, itraconazole, fluconazole and ketoconazole for 50 clinical isolates of Candida albicans, Torulopsis glabrata, C. tropicalis and Hansenula anomala and five reference ATCC strains. The main purpose of the study was to compare the results obtained by the two methods. In general good agreement (+/- 1 dilution) was otained between both methods, despite differences observed for some species-antifungal combinations in which the MICs were lower by the E-test than by the microbroth method. MICs for C. albicans and T. glabrata to amphotericin B were < 0.50 microg/mL. Two isolates of C. albicans and two others of H. anomala, showed MIC < 8 microg/mL for 5- flucytosine. All isolates of T. glabrata and 40% of C. albicans showed MICs > 16 microg/mL for fluconazole. The results of this study indicate that E-test is an alternative for susceptibility testing to the NCCLS reference method. Because its simplicity it seems to be an easier test for routine clinical laboratories.     

Use of breakpoint combination sensitivity testing as a simple and convenient method to evaluate the combined effects of ceftazidime and tobramycin on Pseudomonas aeruginosa and Burkholderia cepacia complex isolates in vitro

An in vitro method of determining the activity of antibiotics in combination which is simple and convenient to perform and which could be used routinely in clinical microbiology laboratories is desirable. We investigated the activity, against Pseudomonas aeruginosa and Burkholderia cepacia complex clinical isolates, of ceftazidime and tobramycin in combination using a broth macrodilution sensitivity method based on breakpoint minimum inhibitory concentrations and compared the results obtained using this method with those obtained using the microtitre checkerboard method. There was good agreement in interpretation of results between the two methods for both P. aeruginosa (90%) and B. cepacia complex isolates (70%) with tobramycin and for P. aeruginosa isolates (70%) with ceftazidime. As the breakpoint combination sensitivity testing method employs only four tubes and does not require initial determination of individual antibiotic minimum inhibitory concentrations, it is simpler and more convenient for determining the activity of antibiotics in combination than the microtitre checkerboard method. The use of this method in routine microbiology laboratories to determine the activity of antibiotic combinations against clinical isolates should optimise treatment of infection by ensuring that appropriate antibiotic combinations are prescribed.     

Isolation of New Aureobasidium Strains That Produce High-Molecular-Weight Pullulan with Reduced Pigmentation

New isolates of Aureobasidium pullulans were obtained from plant leaf surfaces gathered in San Diego County. The new fungal isolates were identified as A. pullulans on the basis of the appearance of polymorphic colonies formed on agar plates, the electrophoretic profiles of repeated genomic DNA sequences, and the production of pullulan in shake flask cultures. The isolates showed different degrees of pigmentation. One of the natural isolates was nonpigmented under mock production conditions in liquid culture, but was still able to synthesize a reduced amount of pigment on agar plates at late times. A mutagenic treatment with ethidium bromide produced derivatives of normally pigmented natural isolates that exhibited an increased tendency toward yeastlike growth and reduced pigmentation. Additionally, some of the new isolates and mutant derivatives accumulated pullulan of relatively high molecular weight in the culture broths.