Use of mueller-iinton broth and agar in the germ tue test / Utilização de ágar e caldo Mueller-Hinton na prova do tubo germinativo

Candida albicans is often isolated from clinical samples, thus its presumptive differentiation from other species of the same genus can be based on its ability to form the germ tube in human serum. Nevertheless, there are two other species that share this characteristic: C. dubliniensis and C. africana. The aim of this study was to compare four different substrates to perform the germ tube (GT) test. The Candida spp. isolates were identified using a manual system (135 C. albicans, 24 C. tropicalis and one C. dubliniensis). The germ tube test was performed with fresh, previously frozen serum and Mueller-Hinton (MH) broth and agar. GT was observed in 96% (130/136) of the isolates through the fresh serum technique, 94% (128/136) through previously frozen serum, 92% (125/136) in MH agar, and 90% (122/136) in MH broth. The sensitivity of each test was higher than 90%, with 100% specificity. Both the MH agar and broth were able to identify the true positives, and false positives were not found. However, some C. albicans isolates were not identified. MH agar and broth may be used in laboratory for the rapid presumptive identification of C. albicans, as an alternative method for germ tube test.

Candida albicans é frequentemente isolada em amostras clínicas, assim a sua diferenciação presuntiva de outras espécies do gênero pode ser baseada na habilidade em formar o tubo germinativo em soro humano. Entretanto, existem outras duas espécies que também possuem essa característica, C. dubliniensis e C. africana. O objetivo foi comparar quatro diferentes substratos para a realização da prova do tubo germinativo (TG). Utilizou-se isolados de Candida spp. identificados através de meio manual (135 C. albicans, 24 C. tropicalis e um C. dubliniensis). A prova do tubo germinativo foi realizada utilizando soro previamente congelado e fresco, caldo e ágar Mueller-Hinton (MH). O TG através da técnica do soro a fresco foi observado em 96% (130/136), 94% (128/136) através do soro previamente congelado, 92% (125/136) no ágar e 90% (122/136) no caldo MH. A sensibilidade de cada teste foi maior que 90% e especificidade de 100%. Tanto o caldo quanto o ágar MH foram capazes de identificar apenas os verdadeiros positivos e não ocorrendo falsos positivos, porém deixaram de identificar alguns isolados de C. albicans. O ágar e o caldo MH podem ser utilizados na rápida e presuntiva identificação laboratorial de C. albicans, como uma alternativa para o teste do tubo germinativo.

Susceptibility Pattern of Aerobic Bacterial Isolates from Wound Swab.

Wound swab culture is the most frequently employed method of confirming wound infection. A regular bacteriological review of infected wounds is necessary to provide qualitative health care particularly when blind treatment is a necessity as in underdeveloped and developing nations. Materials and Methods: A total of 614 Wound swabs sample were received in the department during the study period. Direct Gram staining of the specimens were done after which they were inoculated in Blood agar and MacConkey agar plates and antibiotic sensitivity was done according to CLSI guideline. Result: A total of 496 strains were isolated out of which 232 (46.77%) were Gramnegative bacilli and 264(53.23%) were Gram-positive cocci. Out of the 466 culture positive samples, 29 samples showed polymicrobial growth. E coli was the most common pathogen isolated. Of the 156 isolates of Staphylococcus aureus 68 was from ward and 88 from Out Patient Department (OPD) of which 31(45.58%) and 30(34.09%) were determined to be methicillin resistant (MRSA) respectively. Out of 95 isolates of Coagulase Negative Staphylococcus(CoNS ), 56 was from ward and 39 from OPD. Methicillin Resistant Staphylococcus (MRCoNS) prevalence rate was 46 (82.14%) and 28(71.79%) for ward and OPD respectively. The gram negative isolates were most sensitive to imipenem and it was least sensitive to cephalosporin groups of antibiotics. Conclusion: The most commonly isolated pathogen from wound swab specimens was Gram positive bacteria but 46.77% of the isolates were Gram negative bacteria so antimicrobial coverage for Gram negative bacteria should be included in treatment of wound infection.

Validación del agar Mueller Hinton modificado para la determinación de las concentraciones mínimas inhibitorias a los antifúngicos por etest en candida spp / Validation of the modified Mueller Hinton agar for the determination of the minimum inhibitory concentrations to antifungals by E-test in Candida spp

En este trabajo de investigación se determinaron las Concentraciones Mínimas Inhibitorias (CMI) de los antifúngicos fluconazol, voriconazol, posaconazol, caspofungina, anidulafungina y anfotericina B, por los métodos de microdilución en caldo y difusión en agar con Etest, empleando RPMI 1640 agar y Mueller Hinton modificado (MHm), en 102 cepas de Candida spp., aisladas de sangre, provenientes de la Red de Candidemia del INHRR. El objetivo fue validar el empleo del MHm como medio de cultivo confiable en el método de Etest para la obtención de las CMI. Se utilizaron las cepas C. parapsilosis ATCC 22019 y C. krusei ATCC 6258 como control de calidad. Los resultados demostraron un 94% de frecuencia para C. no albicans y un 6% para C. albicans. Se obtuvo un 100% de sensibilidad para las equinocandinas y anfotericina B, en todas las especies. El MHm demostró una elevada reproducibilidad con respecto al RPMI agar y el método de referencia microdilución en caldo. En base a los resultados obtenidos en esta investigación, se logró la validación del medio MHm para la determinación de las CMI por Etest, así como la detección de resistencia, recomendándolo como metodología confiable, accesible y de fácil ejecución para uso rutinario en el laboratorio de microbiología.

In this research, the Minimum Inhibitory Concentrations (MICs) of the antifungal agent's fluconazole, voriconazole, posaconazole, caspofungin, anidulafungin and amphotericin B were determined by broth microdilution and agar diffusion Etest methods, using RPMI 1640 agar and modified Mueller Hinton (mHM), in 102 strains of Candida spp., isolated from blood, from the Candidemia Network of the INHRR. The objective was to validate the use of mHM as a reliable culture medium for the Etest method to obtain MICs. As a quality control, the strains C. parapsilosis ATCC 22019 and C. krusei ATCC 6258 were used. The results demonstrated a 94% of frequency for non C. albicans and 6% for C. albicans. A 100% of sensitivity was obtained for echinocandins and amphotericin B in all species. The mHM showed high reproducibility with respect to RPMI agar and the reference method of microdilution broth. Based on the results obtained in this investigation, the validation of mHM medium, for the determination of MICs by Etest, as well as the resistance detection was achieved, recommending it as a reliable, affordable and easy to implement method for the routine use in the microbiology laboratory.

Species Identification And Antifungal Susceptibility Pattern Of Candida Isolates From Oropharyngeal Lesions Of HIV Infected Patients..

Background &Objective: Oropharyngeal candidiasis (OPC) is a common feature associated with HIV infection. Over the past decade, reports have documented a shift away from C. albicans as a major cause of infection to non albicans Candida (NAC) species. Several NAC spp are inherently resistant to commonly used antifungal drugs. The objective of the present study was to investigate the distribution pattern of Candida spp. from HIV infected patients with OPC and evaluate its antifungal susceptibility pattern. Methods: A total of 192 HIV infected patients with oropharyngeal lesions (OPL) suggestive of candidiasis and 60 non HIV infected healthy individuals presenting without any OPL were included in the study.Swabs collected from the site of lesions were used for the demonstration and isolation of Candida. Speciation of Candida isolates was done and antifungal susceptibility testing was performed by the disc diffusion method. Results: Out of 192 HIV-infected patients with OPL, 179(93.2%) showed growth of Candida. Isolation of NAC species was higher than C. albicans. Azole resistance was more in NAC species as compared to C. albicans.Conclusions: NAC species has emerged as an important cause of OPC in HIV infected patients. The increased isolation rates of NAC species and a gradual shift in the antifungal susceptibility profile underlines the need of early and accurate diagnosis of infecting Candida spp along with antifungal susceptibility testing for selecting the most appropriate antifungal agent for therapy.

Comparative in vitro antibacterial analysis of different brands of cefixime against clinical isolates of Staphylococcus aureus and Escherichia coli.

The objective of this study was to compare the antibacterial activity of standard and different brands of Cefixime, against standard samples and clinical isolates of E. coli and S. aureus collected from different hospitals. Standard samples and isolates of E. coli and S. aureus were separately cultured in Mueller Hinton broth. After the bacterial incubation, 5 ml solution each of standard Cefixime and its different brands were added to the test tubes containing bacterial culture. Cefixime samples were added in the concentration of 0.0625, 0.125, 0.25, 0.5, 1, 2, 4, 8, 16, 32, 64 and 128μg/ml to separate test tubes. The cultures were again incubated and then the culture samples were analyzed by UV-spectrophotometer, and minimum inhibitory concentrations of all samples were determined. The analysis and interpretation of results were done by single factor ANOVA. An MIC of 0.75μg/ml and 8μg/ml of standard Cefixime was found for standard E. coli and S. aureous respectively. Standard Cefixime and its six selected brands exhibited a higher MIC range for clinical isolates of S. aureus than the clinical isolates of E. coli. Higher MIC values of standard Cefixime and its brands were observed for clinical isolates of E. coli and S. aureus. Higher MIC values for the clinical isolates of E. coli and S. aureus indicated that both the organisms have developed resistance to Cefixime in comparison to standard microorganisms acquired from ATCC.

Phenotypic and Genotypic Methods for the Detection of Methicillin Resistant Coagulase Negative Staphylococci : A Comparative Study.

CONS are the major cause of nosocomial infection in last decade and methicillin resistant CoNS has emerged as a major clinical problem. The present study was to compare different phenotypic methods with genotypic method PCR, for the detection of methicillin resistance in CoNS. 100 CoNS isolates from different samples were studied for the detection of mecA gene. PCR was considered as “gold standard”. Oxacillin and cefoxitin antibiotics were used for different phenotypic tests (DD, Agar dilution and MHOX). The sensitivities of oxacillin and cefoxitin disks for all CONS were found to be 92.30% and 88.46% respectively and the specificities were 87.5% and 100% respectively. The sensitivities of the agar dilution test for oxacillin and cefoxitin were 86.53% and 80.76%, respectively, where as the specificities were 79.16% and 85.41%, respectively. The sensitivity of MHOX was observed to be 96.16% and specificity 72.91%. Cefoxitin D.D and oxacillin AD methods could be used as initial test for the determination of methicillin resistance in CoNS isolates. The result of MHOX shows that it could be the best single method for the evaluation of oxacillin resistance mediated by the mec A gene for all CoNS species.

Caracterización de un hidrolizado ácido de caseína / Characterization of a casein acid hydrolysate

INTRODUCCIÓN: la mayor disponibilidad de caseína seca en polvo en el mercado y la escasa oferta de caseína húmeda, ocasionaron la factibilidad de modificar el proceso tecnológico de obtención del hidrolizado ácido de caseína. OBJETIVO: caracterización del hidrolizado ácido de caseína obtenido a escala industrial por una nueva tecnología en el Centro Nacional de Biopreparados (Cuba) y su desempeño en los medios de cultivo. MÉTODOS: se evaluaron 10 lotes del producto, a los cuales se les realizó el análisis químico, así como la evaluación del desempeño mediante la determinación de la densidad microbiana en el tiempo y la realización de las pruebas de susceptibilidad antimicrobiana. RESULTADOS: se demostró que el hidrolizado posee valores de los principales componentes químicos característicos de los productos que se comercializan en el mercado internacional: nitrógeno amínico 6,59 ± 0,71 por ciento, nitrógeno total 8,12 ± 0,41 por ciento y composición aminoacídica. Como características distintivas el producto muestra un contenido reducido de cloruro de sodio (32,63 ± 2,46 por ciento), calcio (334 mg/L), magnesio (133 mg/L) y pérdida por desecación (3,34 ± 0,66 por ciento). La capacidad de promoción de crecimiento de Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 25923 y Pseudomonas aeruginosa ATCC 27853 no mostraron diferencia significativa con respecto a hidrolizados proteicos de otros proveedores: Biotécnica International, Merck y Oxoid Ltd. (p< 0,05). Se alcanzó un crecimiento bacteriano en el caldo Mueller Hinton a altas diluciones (10-6). Los valores de susceptibilidad de diferentes microorganismos a los antibióticos en el medio agar Mueller Hinton concordaron en relación con los establecidos en las normas del Clinical and Laboratory Standards Institute (CLSI). CONCLUSIÓN: el producto obtenido puede ser empleado como componente de medios de cultivo con diferentes requerimientos y con adecuada estabilidad.

INTRODUCTION: the great availability of dry powdered casein in the market and low accessibility of wet casein induced the viability of modifying the technological process for obtaining casein acid hydrolysate. OBJECTIVES: to characterize the casein acid hydrolysate obtained at industrial scale by a new technology in the National Center of Biological Products of Cuba and to test its performance in culture media. METHODS: ten product batches were tested by chemical composition analysis, and their performance was evaluated through measuring the microbial density vs time and conducting susceptibility antimicrobial tests. RESULTS: it was demonstrated that the hydrolysate had values of the main chemical components inherent to the products in the international market such as amino nitrogen 6,59 ± 0,71 percent, total nitrogen 8,12 ± 0,41 percent and aminoacid composition. As distinctive characteristics, the product shows reduced contents of some components like: sodium chloride (32,63 ± 2,46 percent), calcium (334 mg/L), magnesium (133 mg/L) and loss on drying (3,34 ± 0,66 percent). The growth encouraging capacity for Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 25923 and Pseudomonas aeruginosa ATCC 27853 did not show significant differences with protein hydrolysates from other commercial sources, namely Biotecnica International, Merck and Oxoid Ltd. (p<0,05). It appeared bacterial growth in Mueller Hinton´s broth at high dilutions (10-6). The antimicrobial susceptibility values in the Mueller Hinton Agar agreed with those established by the Clinical and Laboratory Standards Institute (CLSI). CONCLUSIONS: the product may be used as a component of culture media with different requirements and adequate stability.

Phenotypic and Genotypic Methods for the Detection of Methicillin Resistant Coagulase Negative Staphylococci: A Comparative Study.

CoNS are the major cause of nosocomial infection in last decade and methicillin resistant CoNS has emerged as a major clinical problem. The present study was to compare different phenotypic methods with genotypic method PCR, for the detection of methicillin resistance in CoNS. 100 CoNS isolates from different samples were studied for the detection of mecA gene. PCR was considered as “gold standard”. Oxacillin and cefoxitin antibiotics were used for different phenotypic tests (DD, Agar dilution and MHOX). The sensitivities of oxacillin and cefoxitin disks for all CoNS were found to be 92.30% and 88.46% respectively and the specificities were 87.5% and 100% respectively. The sensitivities of the agar dilution test for oxacillin and cefoxitin were 86.53% and 80.76%, respectively, where as the specificities were 79.16% and 85.41%, respectively. The sensitivity of MHOX was observed to be 96.16% and specificity 72.91%. Coagulase negative staphylococcus(CoNS), antibiotic sensitivity, MIC, polymerase chain reaction (PCR), Mueller Hinton Oxacillin Agar Screening test (MHOX). Cefoxitin D.D and oxacillin AD methods could be used as initial test for the determination of methicillin resistance in CoNS isolates. The result of MHOX shows that it could be the best single method for the evaluation of oxacillin resistance mediated by the mec A gene for all CoNS species.

Evaluation of Disk Diffusion Test with Glucose- and Methylene blue-Enriched Mueller-Hinton Agar for Susceptibility Testing of Fluconazole against Candida Isolates / 대한진단검사의학회지

BACKGROUND: Recently, the disk diffusion testing of fluconazole against Candida spp. has been attempted in order to provide a simple inexpensive method in the routine laboratory. We investigated the possibility and reliability of a fluconazole disk diffusion method using a Mueller-Hinton agar supplemented with glucose and methylene blue (GM-MH). METHODS: One hundred and seven isolates of Candida spp. (54 C. albicans, 21 C. glabrata, 20 C. tropicalis, 6 C. parapsilosis, 4 C. krusei, and 2 C. lusitaniae) were tested with the broth microdilution method of National Committee for Clinical Laboratory Standards (NCCLS) document M27-A2 and a disk diffusion test using GM-MH agar. RESULTS: The overall categorical agreement between the NCCLS method and the disk diffusion method was 89.8% for fluconazole, with 0.9% very major errors and 9.3% minor errors; no major errors were detected. CONCLUSIONS: These data suggest that the fluconazole disk diffusion test on GM-MH agar can be used as a routine screening procedure for susceptibility of Candida spp. in the clinical laboratory.