Comparison of ChromID Agar and Clostridium difficile Selective Agar for Effective Isolation of C. difficile from Stool Specimens

BACKGROUND: ChromID Clostridium difficile agar (IDCd; bioMerieux SA, France) is a recently developed chromogenic medium for rapid and specific isolation of C. difficile. We compared the performance of IDCd with that of Clostridium difficile Selective Agar (CDSA). METHODS: A total of 530 fresh stool specimens were collected from patients with clinical signs compatible with C. difficile infection, and cultures for C. difficile were performed on IDCd and CDSA. C. difficile colonies were identified by spore staining, odor, use of an ANI identification test kit (bioMerieux SA), and multiplex PCR for tcdA, tcdB, and tpi. RESULTS: The concordance rate between IDCd and CDSA was 90.6% (480/530). The positivity rates on IDCd on days 1 and 2 (55.6% and 85.0%, respectively) were significantly higher than those on CDSA (19.4% and 75.6%, respectively) (P<0.001 for day 1 and P=0.02 for day 2), but the detection rates on IDCd and CDSA on day 3 were not different (89.4% vs. 82.8%, P=0.0914). On day 3, the recovery rates for non-C. difficile isolates on IDCd and CDSA were 30.2% (160/530) and 22.1% (117/530), respectively (P=0.0075). Clostridium spp. other than C. difficile were the most prevalent non-C. difficile isolates on both media. CONCLUSIONS: The culture positivity rates on IDCd and CDSA were not different on day 3 but IDCd may allow for rapid and sensitive detection of C. difficile within 2 days of cultivation.

Performance of chromID Clostridium difficile Agar Compared with BBL C. difficile Selective Agar for Detection of C. difficile in Stool Specimens

We evaluated the performance of a new chromogenic medium for detection of Clostridium difficile, chromID C. difficile agar (CDIF; bioMerieux, France), by comparison with BBL C. difficile Selective Agar (CDSA; Becton Dickinson and Company, USA). After heat pre-treatment (80degrees C, 5 min), 185 diarrheal stool samples were inoculated onto the two media types and incubated anaerobically for 24 hr and 48 hr for CDIF and for 48 hr and 72 hr for CDSA. All typical colonies on each medium were examined by Gram staining, and the gram-positive rods confirmed to contain the tpi gene by PCR were identified as C. difficile. C. difficile was recovered from 36 samples by using a combination of the two media. The sensitivity with CDIF 48 hr was highest (100%) and was significantly higher than that with CDIF 24 hr (58.3%; P<0.001), because samples with a low burden of C. difficile tended to require prolonged incubation up to 48 hr (P<0.001). The specificity of CDIF 24 hr and CDIF 48 hr (99.3% and 90.6%, respectively) was significantly higher than that of CDSA 48 hr and CDSA 72 hr (72.5% and 67.1%, respectively; P<0.001). CDIF was effective for detecting C. difficile in heat-pretreated stool specimens, thus reducing unnecessary testing for toxin production in non-C. difficile isolates and turnaround time.

Usefulness of a Chromogenic Selective Agar for the Identification of Bacillus cereus Isolated from Blood Cultures / 대한진단검사의학회지

BACKGROUND: The incidence of Bacillus cereus bacteremia is increasing, but the identification of Bacillus species remains difficult. Brilliance Bacillus cereus agar (BBC agar; Oxoid, UK) is a new CHROMagar medium that allows selective isolation and identification of B. cereus; however, its clinical usefulness is seldom studied. We evaluated the usefulness of BBC agar to identify B. cereus isolates recovered from blood cultures. METHODS: We analyzed a total of 53 blood isolates that showed a Bacillus-like morphology on Gram staining. All isolates were identified by using both the API Coryne (bioMerieux, France) and API 50CH/B (bioMerieux) systems. They were subsequently subcultured on BBC agar, incubated for 24 hr, and then examined for characteristic blue-green colonies. The clinical characteristics of patients whose isolates were identified as B. cereus were assessed. RESULTS: Of the 53 isolates, 18 were identified as B. cereus by API 50CH/B. With the API 50CH/B system used as gold standard, the sensitivity and specificity for the identification of B. cereus were 100% (18/18) and 100% (35/35), respectively, using BBC agar, and 67% (12/18) and 100% (35/35), respectively, using the API Coryne system. Of the 18 patients with B. cereus bacteremia, 15 showed infectious signs, and 3 had more than 2 blood cultures positive for B. cereus on separate days. CONCLUSIONS: Our study shows, for the first time, that BBC agar, with its good agreement and ease of use, is a valuable alternative to the API 50CH/B system for the presumptive identification of B. cereus isolates from blood cultures.

Evaluation of the Usefulness of Selective Chromogenic Agar Medium (ChromID VRE) and Multiplex PCR Method for the Detection of Vancomycin-resistant Enterococci / 대한진단검사의학회지

BACKGROUND: Accurate and early detection of vancomycin-resistant enterococci (VRE) is critical for controlling nosocomial infection. In this study, we evaluated the usefulness of a selective chromogenic agar medium and of multiplex PCR for detection of VRE, and both these techniques were compared with the conventional culture method for VRE detection. METHODS: We performed the following 3 methods for detecting VRE infection in stool specimens: the routine culture method, culturing in selective chromogenic agar medium (chromID VRE, bioMerieux, France), and multiplex PCR using the Seeplex(R) VRE ACE Detection kit (Seegene Inc., Korea) with additional PCR for vanC genes. RESULTS: We isolated 109 VRE strains from 100 stool specimens by the routine culture method. In chromID VRE, all the isolates showed purple colonies, including Enterococcus gallinarum and E. raffinosus, which were later identified using the Vitek card. All VRE isolates were identified by the multiplex PCR method; 100 were vanA-positive E. faecium, 8 were vanA- and vanC-1-positive E. gallinarum, and 1 was vanA-positive E. raffinosus. CONCLUSIONS: For VRE surveillance, culturing the isolates in chromID VRE after broth enrichment appears to be an accurate, rapid, and easy method for routine screening test. Multiplex PCR is relatively expensive and needs skilled techniques for detecting VRE, but it can be an auxiliary tool for rapid detection of genotype during a VRE outbreak.

The Effect of Lactic Acid Bacteria Isolates on the Urinary Tract Pathogens to Infants In Vitro

Urinary tract infections are common clinical problems in children, even though lots of treatment strategies have been tried. Many studies of the application of probiotics for urinary tract infection in female adults exist, but there is a lack of studies in children. The aims of this study were to screen probiotic strains for inhibiting the uropathogens in vitro, to find candidates for in vivo study. Nine strains of E. coli were isolated from children with urinary tract infection and six uropathogens were obtained from Korean Colletion for Type Cultures and American Type Culture Collection. Also 135 lactic acid bacteria (LAB) strains were isolated from healthy children, and were identified through physiologic, biochemical methods, 16S rDNA PCR, and data analysis. And with agar disk diffusion assay technique the antimicrobial activities of these LAB strains against those uropathogens were examined. Three strains of separated LAB strains demonstrated major antimicrobial activity against all the uropathogens. In the agar disk diffusion assay technique, antimicrobial activities increased most in the 4th day culture broth with separated Lactobacillus. In summary, some LAB can be used as candidates to develop the probiotic microorganisms that inhibit uropathogens in children, and are expected to be applied to treatment and prevention of pediatric urinary tract infection.

Avaliação da Ação Antimicrobiana da Própolis e de Substâncias Utilizadas em Endodontia sobre o Enterococcus faecalis / Evaluation of the Antimicrobial Action of Propolis and Substances Used

Objetivo: Avaliar a atividade antimicrobiana da própolis e de outras substâncias já utilizadas na proteção pulpar de dentes decíduos, contra cepas de Enterococcus faecalis. Método: Utilizou-se a técnica de difusão em ágar, pelo método do poço, sendo analisadas 11 substâncias: 1) Extrato de própolis verde; 2) Pasta Guedes-Pinto (Iodofórmio + PMCC + Rifocort®); 3)Extrato de própolis verde + Rifocort® + Iodofórmio; 4) Rifocort® +Extrato de própolis verde; 5) Hidróxido de cálcio + Extrato de própolis verde; 6) Hidróxido de cálcio + Soro fisiológico; 7) Iodofórmio +Extrato de própolis verde; 8) Iodofórmio + Soro fisiológico; 9)Rifocort®; 10) Paramonoclorofenol canforado e 11) Soro fisiológico(controle negativo). Resultados: O Rifocort®; o Rifocort® com extrato de própolis verde; a pasta Guedes-Pinto; a pasta de própolis verde com Rifocort® eiodofórmio; e o paramonoclorofenol canforado apresentaram ação antimicrobiana contra o E. faecalis significativamente superior quando comparadas com as demais substâncias testadas. A pasta de hidróxido de cálcio com própolis verde, a solução de extrato de própolis verde, a pasta de iodofórmio com solução de própolis verde apresentaram menor atividade antimicrobiana, não havendo diferença estatisticamente significativa entre estas três formulações. O soro fisiológico, a pasta de hidróxido de cálcio com soro fisiológico e a pasta de iodofórmio com soro fisiológico não apresentaram atividade antimicrobiana. Conclusão: A solução de extrato de própolis verde apresentou baixa atividade antimicrobiana contra o E. faecalis e, dentre as substâncias testadas, o Rifocort® apresentou maior ação antimicrobiana.

Ultrastructural analysis of slime positive & slime negative Staphylococcus epidermidis isolates in infectious keratitis.

BACKGROUND & OBJECTIVE: Slime is a major determinant of Staphylococcus epidermidis adherence.The established methods of laboratory detection of slime production by this organism i.e., Christensen's tube method and congo red agar plate method, can both yield inconclusive and/or intermediate results. We, therefore tried to find out electronmicroscopically the localization of slime in relation to the bacterial cell wall and look for the effect, if any of the slime location on the staphylococcal adherence as well as on the quantum of slime production. METHODS: A total of 132 coagulase negative staphylococci from cases of infectious keratitis were identified as S. epidermidis following the recommended protocol. Slime was detected both by Christensen's tube method and congo red agar plate method. Antibiotic sensitivity testing was performed by standardized disc diffusion method. Adherence of the organisms to artificial surfaces was determined by a quantitative method and transmission electron microscopy was carried out by the conventional techniques. RESULTS: Of the total 132 isolates, 57 (43.2%) were slime positive and 75 (56.8%) were slime negative.Twenty seven (47.4%) of the 57 slime producing organisms were multi drug resistant as compared to only 12 (16%) of 75 nonslime-producing organisms (P<0.001). A majority i.e., 45 (78.9%) of 57 adherent organisms were slime producers as against 12 (16%) of 75 nonadherent organisms. Electron microscopic study revealed a thick viscid layer of slime anchoring to the bacterial cell wall, especially in adherent organisms and those yielding positive slime test. Some of the organisms showed loose nonadherent slime and those were mostly nonadherent to artificial surfaces. INTERPRETATION & CONCLUSION: Slime and multi drug resistance were the important virulence factors of S. epidermidis in bacterial keratitis. It was the adherent slime (i.e., slime in intimate association with the bacterial cell wall as shown by electron microscopy) only, which was responsible for resistance to multiple antibiotics and for the adhesion phenomenon observed in the quantitative slime test.

Comparison among tomato juice agar with other three media for differentiation of Candida dubliniensis from Candida albicans

O presente estudo teve como objetivo comparar o ágar suco de tomate, um tradicional meio utilizado para observação de ascósporos em leveduras, com o ágar semente de niger, ágar caseína e ágar semente de girassol, na diferenciação fenotípica entre C. albicans e C. dubliniensis. Após 48 h de incubação a 30 ºC, os 26 isolados de C. dublinienis (100%) evidenciaram a formação de clamidoconídios igualmente em todos os meios comparados. Entretanto, quando semeados com C. albicans, a formação de clamidoconídios foi raramente observada, resultando nos seguintes percentuais de ausência destas estruturas: ágar suco de tomate (92,47%), ágar niger (96,7%), ágar caseína (91,39%), ágar semente de girassol (96,7%). Estes resultados permitem-nos sugerir a utilização do ágar suco de tomate como mais um meio que, já no primo-isolamento, é capaz de, presuntivamente, diferenciar C. albicans de C. dubliniensis.

Detection of metallo betalactamase producing Pseudomonas aeruginosa in hospitalized patients.

BACKGROUND & OBJECTIVE: Metallo beta-lactamase (MBL)-mediated resistance to carbapenems is an emerging threat in hospital isolates of Pseudomonas aeruginosa. Though there are several screening methods to detect this enzyme production, the National Committee for Clinical Laboratory Standards (NCCLS) does not have performance standards documented so far. There is not enough information from the Indian subcontinent regarding the prevalence and the screening methods for these enzymes. The present study was undertaken to detect MBL in nosocomial isolates of P. aeruginosa by two screening methods. METHODS: Fifty consecutive P. aeruginosa isolates obtained from hospitalized patients were subjected to susceptibility testing to antipseudomonal drugs by disc diffusion, and minimum inhibitory concentration (MIC) of imipenem was determined. The production of MBL was detected by 4-fold reduction in MIC with imipenem-ethylene diamine tetraacetic acid (EDTA) and the zone size enhancement with EDTA impregnated imipenem and ceftazidime discs. RESULTS: Sixteen per cent of the isolates tested were resistant to imipenem by disc diffusion method of which 87.5 per cent exhibited a zone size enhancement with EDTA impregnated imipenem and ceftazidime discs as well as a 4-fold reduction in MIC with imipenem EDTA. The imipenem susceptible isolates (84%) had normal MIC values and exhibited no zone diameter enhancement with EDTA impregnated antibiotic discs. INTERPRETATION & CONCLUSION: MBL-mediated imipenem resistance in P. aeruginosa is a cause for concern in the therapy of critically ill patients. The two confirmatory methods i.e., zone diameter enhancement with EDTA impregnated imipenem and ceftazidime discs and 4-fold reduction in MIC with imipenem EDTA combination are equally effective for their detection.

Can mushrooms fix atmospheric nitrogen?

It is generally reported that fungi like Pleurotus spp. can fix nitrogen (N2). The way they do it is still not clear. The present study hypothesized that only associations of fungi and diazotrophs can fix N2. This was tested in vitro. Pleurotus ostreatus was inoculated with a bradyrhizobial strain nodulating soybean and P. ostreatus with no inoculation was maintained as a control. At maximum mycelial colonization by the bradyrhizobial strain and biofilm formation, the cultures were subjected to acetylene reduction assay (ARA). Another set of the cultures was evaluated for growth and nitrogen accumulation. Nitrogenase activity was present in the biofilm, but not when the fungus or the bradyrhizobial strain was alone. A significant reduction in mycelial dry weight and a significant increase in nitrogen concentration were observed in the inoculated cultures compared to the controls. The mycelial weight reduction could be attributed to C transfer from the fungus to the bradyrhizobial strain, because of high C cost of biological N2 fixation. This needs further investigations using 14C isotopic tracers. It is clear from the present study that mushrooms alone cannot fix atmospheric N2. But when they are in association with diazotrophs, nitrogenase activity is detected because of the diazotrophic N2 fixation. It is not the fungus that fixes N2 as reported earlier. Effective N2 fixing systems, such as the present one, may be used to increase protein content of mushrooms. Our study has implications for future identification of as yet unidentified N2 systems occurring in the environment.

Isolation and characterisation of antibiotic producing microorganisms in composted Nigerian soil.

Compost samples obtained from different locations within the premises of the university of Lagos were analysed to determine the presence and types of antibiotic-producing bacteria, fungi and actinomycetes using nutrient agar, potato Dextrose agar and starch casein nitrate agar respectively as culture media. A variety of bacteria were isolated and these included Staphylococcus aureus, B. subtilis, B. pumilis, B. lactesporus, B. megaterium, B. pulvifaciens, B. licheniformis, Streptococus spp., Corynebacterium spp. and E. coli. The fungal isolates encountered were Aspergillus niger, A. flarus, T. viridae, P. chrysogenum, P. pinofylum and Absida spp., while the following actinomycetes were identified: Norcadia spp., Micromonospora spp., Streptomyces scabies, S. reticuli and S. hygroscopicus. When these organisms were screened for antibiosis, the following species were found to be antibiotic producers: B. licheniformis, B. subtilis, Penicillium chrysogenum, Streptomyces reticuli, S. hygroscopicus and Micromonospora spp. The fungus Penicillium chrysogenum had the highest rate of antibiotic production with an inhibitory zone width of 17mm while Trichoderma viridae produced toxins lytic to other fungal hyphae.

In vitro mass multiplication of Ophiorrhiza mungo Linn.

A protocol for in vitro mass multiplication of plants through seedling (shoot) cultures was established for Ophiorrhiza mungo. Maximum number of adventitious shoots per shoot culture (10.4 +/- 1.72) was initiated on MS solid medium supplemented with BAP (2.22 microM) after 3 weeks. Shoots were further multiplied (12.8 +/- 2.8) through subculture of intact shoots and reculture of nodal segments of aseptic shoots (6.5 +/- 0.94) in MS solid medium containing BAP (0.89 microM). Shoot elongation (1.27 +/- 0.12 cm) was achieved in the medium containing GA3 (1.44 microM) in two weeks. Rooting was favoured in basal agar medium supplemented with IBA (12.3 microM) plus NAA (1.07 microM). The plants were successfully established (100%) in the pots containing sand and top soil (1:1) mixture in a period of two weeks.

Antibacterial and antidiarrhoeal effects of alkaloids of Holarrhena antidysenterica WALL.

The alkaloids from the ethanolic extract of H. antidysenterica seeds were evaluated for their antibacterial activity against clinical isolates of enteropathogenic Escherichia coli (EPEC) in vitro, and their antidiarrhoeal activity on castor oil-induced diarrhoea in rats, in vivo. The plasmid DNA, whole cell lysate and outer membrane protein profile of a clinical isolate of EPEC was determined in presence of alkaloids of H. antidysenterica. The disc diffusion and agar well diffusion methods were used to evaluate the antibacterial efficacy. The alkaloids showed strong antibacterial activity against EPEC strains. In castor oil-induced diarrhoea, alkaloids reduced the diarrhoea with decrease in the number of wet faeces in pretreated rats at a dose of 200-800 mg/kg. The loss of plasmid DNA and suppression of high molecular weight proteins were observed on alkaloids treatment. Taking into account the multiple antibiotic resistance of EPEC, the results suggest usefulness of alkaloids of H. antidysenterica seeds as antibacterial and antidiarrhoeal agents.

Characterization and identification of chitinase producing Streptomyces venezuelae P10.

In an attempt to isolate chitinase producers from soil, a streptomycete strain was found potent using natural chitin as the substrate. Chitinolytic activity was tested directly on agar plates, also with crude enzyme. Chitinase assay showed that the isolate could produce 0.8 U/ml of the enzyme. The morphological, cultural, physiological and biochemical characters of the isolate P10 were studied, and identified as Streptomyces venezuelae P10.

Evaluation of phytochemical and antimicrobial properties of leaf extract of Tapinanthus sessilifolius (P. Beauv) van Tiegh.

Leaf extracts of T. sessilifolius growing on five different host plants (Psidium guajava, Citrus lemon, Vernonia amygdalina, Persea americana and Jatropa curcas) were evaluated for antimicrobial activity of the plant. Powdered leaves of T. sessilifolius collected from each host plant was divided into two portions. One portion was used for aqueous infusion and the other portion was successively extracted with hexane, ethylacetate and methanol. Infusion of aqueous extract of powdered leaves did not show antimicrobial effect even at the concentration of 1000 and 2000 microg/ml on test microorganisms (Staph. aureus, E. coli, Bacillus subtilis, Pseudomonas aeruginosa and Candida albicans). However in broth culture, methanolic and hexane extract had MIC range of 62.5-500 microg/ml and ethylacetate extract had 250-500 microg/ml. Phytochemical screening of leaf samples of T. sessilifolius collected from different host plants showed positive test for hydrolysable tannins, saponins, flavonoids, terpenes, cardiac glycoside, reducing sugars and proteins. LD50 concentration was found to be > 1.500 mg/kg for samples from P. guajava; 489.89 mg/kg for J. curcas and C. lemon; and 692 mg/kg for V. amydalina in mice.

Propiedades físicas del agar de Gracilariopsis tenuifrons (Gracilariaceae) en Sucre, Venezuela / Physical properties of the agar of Gracilariopsis tenuifrons (Gracilariacea) in Sucre, Venezuela

The yield, gel strength, gelling and melting temperatures of Gracilariopsis tenuifrons agar from Guayacán, Araya Peninsula, Sucre State, Venezuela were determined. Yield values with and without alkali treatment ranged from 23.22 to 39.57% and from 16.29 to 22.42% respectively, while gel strength with alkali treatment fluctuated betwen 699.31 and 1231.69 g/cm2 and without treatment varied from 278.0 to 691.06 g/cm2. Gelling and melting temperatures were in the range reported for other agarophytes. Considering gel strength, the agar quality of G. tenuifrons was higher than in other species and its exploitation in economically feasible.

Comparación de los métodos de cultivo para Streptococcus del grupo mutans / Comparison of the culture methods for Streptococcus mutans group

Varios tests microbiológicos han sido propuestos para detectar niveles de Streptococcus grupo mutans (S. mutans) a partir de saliva. En un trabajo previo, se analizó el sistema Dentocult SM (Orion Diagnóstica, Vivadent) y la adaptación local de ese método, registrándose correlación entre ambos. El objetivo de este trabajo fue evaluar 2 tests preparados en el laboratorio para identificar los niveles de S. mutans a partir de saliva en 65 niños entre 6 y 13 años de edad (x 9.76 (2). En ambos tests fue utilizado caldo Mitis Salivarius con bacitracina y sacarosa al 20 por ciento (Matsukubo, 1984). Para la toma de saliva, un método utilizó una espátula asperizada, colocada y rotada 10 veces sobre la lengua, la cual se suspendió en un tubo a rosca con 5 ml. de medio y se incubó en posición vertical, adaptación local del método Dentocult SM. El segundo método utilizó 0.1 ml. de saliva estimulada, la que se sembró en frascos retangulares conteniendo 3 ml. de medi e incubado en posición horizontal. La lectura e interpretación se realizó según tabla patrón de los sistemas Dentocult SM (DC) y Cariescreen (CS) respectivamente. Los resultados mostraron un CPOD + ceod 6.77 +- 9.76 con un componente C+c de 5.21+-0.51. Se registró correlación entre ambos métodos r=0,55 (p menor 0.0001). La mayor coincidencia se observó entre clase 2 de DC y 100.000 UFC dle sistema CS y entre clase 3 de DC y 1.000.000 UFC del sistema CS, con 100 por ciento de concordancia. Los resultados sugieren que ambos métodos son simples y económicos para detectar niveles de S. mutans en saliva, aunque presentan algunas discordancias en las clases intermedias, por lo que se recomienda realizar las lecturas con ambas tablas patrón