Comparison of BCYEα+AB agar and MWY agar for detection and enumeration of Legionella spp. in hospital water samples.

BACKGROUND: This study illustrates for the first time the performance (sensitivity and selectivity) of the selective medium BCYEα +AB suggested by the new edition of ISO 11731 for legionella isolation and enumeration. We compared the efficacy of the selective BCYEα +AB medium with that of the highly selective MWY medium. RESULTS: Legionella spp. was detected in 48.2 and 47.1% of the samples by BCYEα +AB and MWY agar, respectively. For optimal detection of Legionella spp., most protocols recommend using selective media to reduce the number of non-Legionella bacteria. Agreement between the two media was 86.7%. CONCLUSIONS: According to the results, both media have a very similar performance and they both have advantages and disadvantages over each other. In AB medium there is the risk of being less selective so more interfering microbiota may grow but in MWY medium there is the risk of being too selective. The low selectivity of the AB medium could be resolved if other treatments are applied after filtration, e.g. acid and/or heat treatment, but it must be taken into account that these treatments still reduce the number of viable Legionella. In conclusion, we recommend using MWY as a selective medium for the detection of Legionella spp. as it is easier discern suspected colonies and facilitate the final Legionella spp.

CHROMagarTM Candida Plus: A novel chromogenic agar that permits the rapid identification of Candida auris.

Candida auris is a serious nosocomial health risk, with widespread outbreaks in hospitals worldwide. Successful management of such outbreaks has depended upon intensive screening of patients to identify those that are colonized and the subsequent isolation or cohorting of affected patients to prevent onward transmission. Here we describe the evaluation of a novel chromogenic agar, CHROMagarTM Candida Plus, for the specific identification of Candida auris isolates from patient samples. Candida auris colonies on CHROMagarTM Candida Plus are pale cream with a distinctive blue halo that diffuses into the surrounding agar. Of over 50 different species of Candida and related genera that were cultured in parallel, only the vanishingly rare species Candida diddensiae gave a similar appearance. Moreover, both the rate of growth and number of colonies of C. auris recovered from swabs of pure and mixed Candida species were substantially increased on CHROMagarTM Candida Plus agar when compared with growth on the traditional mycological isolation medium, Sabouraud dextrose agar. Taken together, the present data suggest that CHROMagarTM Candida Plus agar is an excellent alternative to current conventional mycological media for the screening of patients who are potentially colonized/infected with Candida auris, can be reliably used to identify this emerging fungal pathogen, and should be tested in a clinical setting. LAY ABSTRACT: Candida auris is a novel pathogenic yeast that has been associated with large hospital outbreaks across several continents. Affected patients become colonized, predominantly on the skin, with large quantities of C. auris which they then shed into the hospital environment. Identification of C. auris is challenging using routine laboratory methods, and time consuming when patients are colonized with a mixture of different Candida species. Here we demonstrate that a novel chromogenic agar, CHROMagarTM Candida Plus, permits the rapid differentiation of C. auris from a wide range of other yeast species and is potentially ideally suited to screening of patients that are suspected of being colonized or infected with this medically important yeast.

Comparison of four agar media for the enumeration of the Mycobacterium abscessus complex.

Background: Traditional culture of nontuberculous mycobacteria (NTMs) has involved egg-based formulations (Lowenstein-Jensen medium) or defined media (Middlebrook formulations), which have disadvantages of composition complexity, availability, and cost. This study quantitatively compared three non-selective, non-blood based basal agars with Columbia blood agar (CBA), to enumerate Mycobacterium abscessus complex organisms in pure culture. Methods: M. abscessus subsp. massiliense, M. abscessus subsp. bolletii, and M. abscessus subsp. abscessus were employed. Inocula of each of these were counted on three basal agar media, including (i) standard plate count agar (SPCA), (ii) tryptone soya agar (TSA), and (iii) Mueller-Hinton agar (MHA) and compared to counts on CBA. Results: All NTM isolates of all subspecies grew successfully on all four media examined. The growth was most profuse on SPCA, with a mean colony diameter of 3 mm, whereas the mean colony diameter on all other media was 1 mm. Statistically, there was no significant difference in counts when comparing CBA with SPCA or MHA (P > 0.05), whereas there was a statistically significant difference between CBA and TSA (P = 0.01). There was no statistically significant difference between SPCA and MHA (P = 0.53). Conclusion: This study indicates that SPCA and MHA are equally effective as CBA, when enumerating of M. abscessus complex organisms. Employment of TSA gave significantly lower counts than CBA (P = 0.01) and therefore should not be employed when enumerating these organisms. SPCA yielded the most profuse growth of all media examined. In addition to these advantages, given that SPCA does (i) not require blood as a medium constituent, (ii) is simple to reconstitute, (iii) is relatively cheap, and (iv) is widely available commercially, this study endorses employment of SPCA for the nonselective culture of M. abscessus complex organisms, including enumeration.

Development of a selective and differential media for the isolation and enumeration of Bacillus cereus from food samples.

AIM: Identification and enumeration of foodborne pathogens in food stuffs are valuable concerns. In the present study, starch-blood-egg yolk-polymyxin B-trimethoprim-ceftazidime (SBYPTC) agar was established to isolate and specify the number of Bacillus cereus in food products. METHODS AND RESULTS: The effectiveness of the developed medium in selecting for B. cereus from pure cultures and food matrixes naturally contaminated by high levels of microbiota was estimated, and the results were compared with that of two commercially available MYPA and PMBA media. In pure cultures, there were no significant differences in the recoverability of B. cereus among the three media, however, SBYPTC agar showed a greater exclusivity. To examine SBYPTC performance in food, B. cereus were artificially inoculated into lettuce and potato samples with high background microbiota in two separated experiments. There were no significant differences between MYPA and PEMBA. However, SBYPTC manifested greater selectivity and exclusivity and made the differentiation easier by allowing growth of B. cereus in separated colonies and inhibiting competing microflora. CONCLUSION: Our results showed that SBYPTC has high selective properties in comparison with MYPA and PEMBA. Thus, it can be considered as a useful tool to monitor the existence and the number of B. cereus in foods especially those contaminated with high levels of microflora. SIGNIFICANCE AND IMPACT OF THE STUDY: In the food industry, SBYPTC can be employed for food quality assurance to monitor B. cereus in food products contaminated with high levels of microbiota.

Discolored Red Seaweed Pyropia yezoensis with Low Commercial Value Is a Novel Resource for Production of Agar Polysaccharides.

The red seaweed Pyropia yezoensis has been demonstrated to be a novel resource for the production of high-quality agar. P. yezoensis is grown for the food industry in large-scale Japanese mariculture operations. However, discolored P. yezoensis is mostly discarded as an industrial waste, although it has some kind of utility values. Here, we evaluated the utility of discolored P. yezoensis as a resource for agar production. The quality of agar from the discolored seaweed was comparable to that from normal seaweed. In addition, as a distinguishing characteristic, agar yield was higher from discolored seaweeds than from normal types. Moreover, we successfully used agar from discolored P. yezoensis for bacterial plate media and DNA electrophoresis gels without agarose purification. Thus, our results demonstrate that discolored P. yezoensis is suitable for agar production and use in life science research. Diverting discolored P. yezoensis from disposal to agar production provides a solution to the current industrial waste problem in mariculture, as well as a secure source of agar for research purposes.

Mupirocin-mucin agar for selective enumeration of Bifidobacterium bifidum.

Bifidobacterium bifidum is a bacterial species exclusively found in the human intestinal tract. This species is becoming increasingly popular as a probiotic organism added to lyophilized products. In this study, porcine mucin was used as the sole carbon source for the selective enumeration of B. bifidum in probiotic food additives. Thirty-six bifidobacterial strains were cultivated in broth with mucin. Only 13 strains of B. bifidum utilized the mucin to produce acids. B. bifidum was selectively enumerated in eight probiotic food supplements using agar (MM agar) containing mupirocin (100 mg/L) and mucin (20 g/L) as the sole carbon source. MM agar was fully selective if the B. bifidum species was presented together with Bifidobacterium animalis subsp. lactis, Bifidobacterium breve, and Bifidobacterium longum subsp. longum species and with lactic acid bacteria (lactobacilli, streptococci). Isolated strains of B. bifidum were identified using biochemical, PCR, MALDI-TOF procedures and 16S rRNA gene sequencing. The novel selective medium was also suitable for the isolation of B. bifidum strains from human fecal samples.

Evaluation of CP Chromo Select Agar for the enumeration of Clostridium perfringens from water.

The European Directive on drinking water quality has included mCP agar as the reference method for recovering Clostridium perfringens from drinking waters. In the present study, three media (mCP, TSCF and CP Chromo Select Agar) were evaluated for recovery of C. perfringens in different surface water samples. Out of 139 water samples, using a membrane filtration technique, 131 samples (94.2%) were found to be presumptively positive for C. perfringens in at least one of the culture media. Green colored colonies on CP Chromo Select Agar (CCP agar) were counted as presumptive C. perfringens isolates. Out of 483 green colonies on CCP agar, 96.3% (465 strains, indole negative) were identified as C. perfringens, and 15 strains (3.1%) were indole positive and were identified as Clostridium sordellii, Clostridium bifermentans or Clostridium tetani. Only 3 strains (0.6%) gave false positive results and were identified as Clostridium fallax, Clostridium botulinum, and Clostridium tertium. Variance analysis of the data obtained shows statistically no significant differences in the counts obtained between media employed in this work. The mCP method is very onerous for routine screening and bacterial colonies could not be used for further biochemical testing. The colonies on CCP and TSCF were easy to count and subculture for confirmation tests. TSCF detects sulfite-reducing clostridia, including species other than C. perfringens, and in some cases excessive blackening of the agar frustrated counting of the colonies. If the contamination was too high, TSCF did not consistently produce black colonies and as a consequence, the colonies were white and gave false negative results. On the other hand, the identification of typical and atypical colonies isolated from all media demonstrated that CCP agar was the most useful medium for C. perfringens recovery in water samples.

Development of blood-yolk-polymyxin B-trimethoprim agar for the enumeration of Bacillus cereus in various foods.

Blood-yolk-polymyxin B-trimethoprim agar (BYPTA) was developed by the addition of egg yolk, laked horse blood, sodium pyruvate, polymyxin B, and trimethoprim, and compared with mannitol-yolk-polymyxin B agar (MYPA) for the isolation and enumeration of Bacillus cereus (B. cereus) in pure culture and various food samples. In pure culture, there was no statistical difference (p>0.05) between the recoverability and sensitivity of MYPA and BYPTA, whereas BYPTA exhibited higher specificity (p<0.05). To evaluate BYPTA agar with food samples, B. cereus was experimentally spiked into six types of foods, triangle kimbab, sandwich, misugaru, Saengsik, red pepper powder, and soybean paste. No statistical difference was observed in recoverability (p>0.05) between MYPA and BYPTA in all tested foods, whereas BYPTA exhibited higher selectivity than MYPA, especially in foods with high background microflora, such as Saengsik, red pepper powder, and soybean paste. The newly developed selective medium BYPTA could be a useful enumeration tool to assess the level of B. cereus in foods, particularly with high background microflora.

[Evaluation of the ChromID ESBL agar for the detection of ESBL-positive Enterobacteriaceae and vancomycin-resistant enterococcus isolates from urine cultures]. / GSBL Pozitif Enterobacteriaceae ve Vankomisine Dirençli Enterokoklarin Idrar Kültürlerinden Erken Tespitinde ChromID ESBL Agarin Degerlendirilmesi.

Extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae strains are frequent causative agents both in community-acquired infections and in nosocomial infections. The newly developed ChromID ESBL agar (bioMerieux, Marcy I'Etoile, France) is a chromogenic medium that helps rapid identification of ESBL-positive Enterobacteriaceae species from the clinical samples. The aim of this study was to evaluate the performance of ChromID ESBL agar in the rapid identification of ESBL-positive pathogens from the urine samples of the patients with urinary tract infections. A total of 672 urine samples (437 outpatients, 235 inpatients) were included in the study. All of the samples were inoculated simultaneously to 5% sheep blood agar, McConkey agar and ChromID ESBL agar media, and evaluated after incubation at 37°C for 18-24 hours. Gram-negative pathogens were tested for ESBL both by the standard combined double-disk diffusion (CDD) method using ceftazidime and cefotaxime disks and by doubledisk synergy (DDS) test. Among 672 urine cultures, 199 yielded microbial growth in routine media (sheep blood agar and/or McConkey agar), whereas 57 yielded bacterial growth in ChromID ESBL agar. When CDD method was accepted as the reference method according to Clinical and Laboratory Standards Institute (CLSI) recommendations, the sensitivity, specificity, positive and negative predictive values for ChromID ESBL agar for the detection of ESBL-positive bacteria in urinary tract infections were estimated as 97%, 92.9%, 89.1%, and 98.1%, respectively. Additionally, we also discovered that Chrom ID ESBL agar could detect vancomycin-resistant enterococci (VRE) as well as ESBL-positive bacteria, in our study. In order to investigate this observation we inoculated a total of 203 stock strains of Enterococcus spp. (118 vancomycin-sensitive, 85 vancomycin-resistant) to this medium. None of the vancomycinsensitive Enterococcus spp. did grow in ChromID ESBL medium, while 83 of the 85 resistant isolates (97.6%) did grow in the medium. As a result, it was concluded that ChromID ESBL agar medium was advantageous since it led to the growth of VRE and ESBL-positive Enterobacteriaceae isolates in different colors and helped in early identification of these two problematic bacteria. We thought that especially early detection of VRE will accelerate the establishment of necessary measures to prevent the nosocomial spread of this microorganism.

[Evaluation of oxacillin resistance screening agar and chromogenic MRSA agar media for the detection of methicillin resistance in Staphylococcus aureus clinical isolates]. / Staphylococcus aureus klinik izolatlarinda metisilin direncinin saptanmasinda oksasilin direnci tarama agar ve kromojenik MRSA agar besiyerlerinin degerlendirilmesi.

Methicillin-resistant Staphylococcus aureus (MRSA) strains which are the most frequent causes of hospital acquired infections, are also currently encountered with increasing frequency in community acquired infections. Therefore rapid and accurate identification of MRSA strains is essential in both implementation of infection control measures and prevention of the nosocomial spread of this microorganism. The aim of this study was to determine the specifisity, sensitivity, positive and negative predictive values of two commercial media, one was Oxacillin Resistance Screening Agar Base (ORSAB; Oxoid, England) and the other was chromogenic MRSA agar (BBL CHROMagar MRSA; BD, Paris, France), for the identification of MRSA strains. A total of 175 clinical S. aureus isolates, of which 45 were MRSA, and 130 were methicillin-susceptible S. aureus (MSSA), whose susceptibility to methicillin were determined by disk diffusion method using oxacillin and cefoxitin disks in Mueller-Hinton agar medium, were included in the study. When oxacillin disk diffusion test was accepted as the reference method, the specificity, sensitivity, positive and negative predictive values of ORSAB were found as 97.7%, 40%, 36.5% and 98.1%, respectively; while these values were detected as 95.5%, 37.6%, 35.7% and 96.1% for CHROMagar MRSA, respectively. These results indicated that both media may be used in laboratories where work load is high and the number of personnel is inadequate especially in screening studies together or in addition to another medium (mannitol-salt agar). However, since these methods exhibit low specifity (high false positive results), positive results should be confirmed using other methods such as disk diffusion, E-test or microdilution susceptibility testing.

Agars of Gelidiella acerosa of west and southeast coasts of India.

Seventeen agar samples were extracted from Gelidiella acerosa (Forsskal) Feldmann and Hamel (Rhodophyta, Gelidiales) specimens collected from nine different sites on the Indian coast-five from southeast coast and four from the west coast. The agar samples were analysed. The stability characteristics of the gels of selected agar samples were studied by rheometry under applied stress conditions, i.e. variation of the storage (G') and loss moduli (G'') were studied under varying frequency and duration (time) of the stress applied. Yield, apparent and dynamic viscosities, gelling and melting temperatures, 3,6-anhydrogalactose (3,6-AG), sulphate contents and TGA (Thermogravimetric Analysis) measurements of the products were done. It was observed that the best quality agar was produced by G. acerosa occurring in the Gulf of Mannar region in the southeast coast. The gel strengths and the viscosities of agars extracted from Gelidiella acerosa occurring in the Gulf of Mannar ranged from 500 to 700gcm(-2) and 33 to 45cP for 2001 collections and for 2002 collections the corresponding values were 450 to 845gcm(-2) and 55 to 67cP respectively. On the other hand, for the agar samples extracted from the west coast of India, the gel strength and viscosities values ranged from 225 to 400gcm(-2) and from 15 to 30cP, respectively. The agars obtained from G. acerosa collected from southeast coast have been found to be suitable for bacterial culture and molecular biology. This is the first report of superior quality of agar from the Indian agarophytes.

'Spiking' as a rapid method for differentiation of Candida albicans from other yeast species.

This paper describes a simple and rapid method for the differentiation of Candida albicans from other yeast species in primary cultures based on colonial morphology following incubation in carbon dioxide. The technique has superior sensitivity to the traditional germ-tube method and requires no additional laboratory tests. In a busy laboratory, this can result in significant savings in cost and time, as well as improvements in patient care.

Cost-effectiveness and efficacy of CHROMagar Candida medium in clinical specimens.

CHROMagar Candida is a new medium for the differential isolation and identification of certain clinically important Candida species. This study evaluated the cost-effectiveness of this medium compared with conventional methods. Thirty reference strains, 158 clinical specimens and 105 stock cultures were investigated. Specimens were cultured on CHROMagar Candida medium and on Sabouraud chloramphenicol agar. Identification was by conventional methods on Sabouraud agar and appearance of colonies on CHROMagar Candida medium. CHROMagar Candida correctly identified isolates of C. albicans, C. tropicalis and C. krusei. It was superior in detecting mixed cultures. A comparison of time and cost was carried out. CHROMagar Candida provides a simple, accurate and cost-effective method for identifying some clinically important Candida species.

[The results of a comparative study of different series of commercial casein-carbon agar]. / Rezul'taty sravnitel'nogo izucheniia razlichnykh serii kommercheskogo kazeinovo-uglo'nogo agara.

Comparative assessment of the diagnostic properties of 30 lots of commercial casein-carbon agar used in practical bacteriology for the diagnosis of pertussis has shown essential differences in the efficacies of various lots of this medium. Only 11 of the 30 tested lots provided the formation of B. pertussis colonies in the periods established by the instruction. Addition of antibiotics to the medium reduced its diagnostic characteristics still more.

Growth study of a nonpathogenic strain of Leishmania donovani with different nutrients.

The studies on growth pattern of a nonpathogenic Leishmania donovani, strain UR6, in different media showed that it can be regularly cultivated amd maintained in modified Ray's Medium (Agar) and three other liquid media, namely DME Medium, Medium 199 and RPMI-1640 which are manufactured in India. The well known N.N.N. Medium provided quantitatively poorer growth in comparison to these medium. Measurement of L. donovani cell concentration by optical density in a spectrophotometer has been worked out for expressing immunochemical observations on antigens in terms of cells per mililitre.

Selection of a reference lot of Mueller-Hinton agar.

A collaborative study was undertaken to evaluate the performance of currently marketed Mueller-Hinton agars from seven manufacturers by replicate disk diffusion tests with standard quality control strains. Identification of the manufacturers was concealed, and the resulting data were evaluated for the selection of a physical reagent standard against which the performance of future production lots would be tested and made to conform. A medium was selected which was sufficiently close to existing National Committee for Clinical Laboratory Standards quality control limits that current interpretive criteria would require minimum modification. Two of the seven lots were eliminated from further consideration because the final pHs were outside acceptable limits. The remaining four lots had 96% of mean zone diameters less than or equal to 2 mm from those of the chosen lot and 65% of the means were less than or equal to 1 mm from those of the chosen lot for all 28 antimicrobial agent-organism combinations. Manufacturers then attempted to produce new lots of Mueller-Hinton agar which performed within the prescribed limits of the chosen lot. One lot performed in close conformity with the selected standard, but the overall performance of the media was essentially the same as that of the randomly chosen lots in the initial study. It was concluded that one of the original seven lots demonstrated properties which made it a tentative candidate for a physical reagent standard and that the use of a physical reagent standard in evaluating production lots might aid in stabilizing the performance of Mueller-Hinton agar.

Evaluation of the effect of agar on the results obtained in the L5178Y mouse lymphoma assay.

The L5178Y TK+/- mouse lymphoma assay is widely used in mutagenicity testing. Trifluorothymidine-resistant (TFTr) mutants are quantitated following growth in agar-supplemented cloning medium. In an attempt to evaluate the effect of agar on plating efficiency, we have tested several lots of Difco Noble agar (cat. No. 0142-01-8; normally used in this assay) and compared it with Baltimore Biological Laboratory (BBL) agar (cat No. 11849). We find that BBL agar gives a higher and less variable plating efficiency than any of the Noble lots tested. Colonies plated in BBL agar tend to appear significantly earlier on the plates than those cloned in Noble agar. The absolute mutant number and the induced mutant frequency quantitated from a treated culture is generally higher in BBL compared to Noble agar. To determine if this higher frequency is due to increased mutant recovery rather than "sneak through" of nonmutant cells, we isolated 97 mutants from treated cultures (44 large colonies and 53 small colonies) and 69 mutants from untreated cultures (24 large colonies and 45 small colonies) and tested them for TFT resistance. All but one (a large colony from an untreated culture) were found to be TFTr, indicating that the mutant frequency is due to an increased mutant recovery. The spontaneous mutant frequency was quantitated for 122 untreated cultures. Showing little variation within and between experiments, the spontaneous mutant frequency yielded a mean of 57.7, with a standard deviation of 14.4. Under our laboratory conditions, BBL agar gave reliable results, and we prefer it for use in cloning L5178Y mouse lymphoma cells.