Microbiological Water Evaluation from Biofilm Adhered to Dental Unit Waterlines / Evaluación Microbiológica del Agua de Biofilm Adherido a Líneas de Agua de la Unidad Dental

ABSTRACT: The contamination of the dental units' waterlines is a reality, which can develop individual and collective disorders. The aim of this study was to evaluate the prevalence and resistance profile of bacteria on the internal surfaces of waterlines in a dental clinic from a Dentistry school of a Brazilian university. The design was an exploratory, descriptive study with quantitative and qualitative approach. Samples (n=4) were collected for analysis at different points: the portion closest to the water reservoir of the chair, and the portion closest to the triple syringe. After collection the samples were cultured in BHI medium in an oven for 24-48 h at 37 °C. For the quantitative analysis 1 ml of each sample was used for serial dilution up to the dilution value seven. The colonies were counted after pour plate and the results expressed in UFC/cm2. The qualitative analysis was initiated with the cultivation of Agar Blood, EMB-Levine and Cetrimide Agar for 24 h, and the identification of bacteria was performed by microscopic analyses. The resistance profile was verified by classical antibiogram. The internal surfaces of unit waterlines units exhibited a mean of 2.44 x 109 CFU / cm2. Bacillus subtilis and Pseudomonas aeruginosa were identified. The resistance profile of Pseudomonas aeruginosa indicated sensitivity to all tested antibiotics. A large number of microorganisms was quantified from biofilm accumulated in the dental units' waterlines. However, they were not resistant to classic antibiogram. Better management and application of decontamination protocols for waterlines need to be applied since opportunistic infections may be associated with Pseudomonas aeruginosa.

RESUMEN: La contaminación de líneas de agua en las unidades dentarias es una realidad, generando enfermedades individuales y colectivas. El objetivo de este estudio fue evaluar la prevalencia y resistencia de las bacterias en las zonas internas de las líneas de agua de la Facultad de Odontología de una universidad brasileña. El diseño del estudio fue exploratorio, descriptivo con enfoques cuantitativos y cualitativos. Las muestras para análisis (n=4) fueron selecionadas de distintos lugares: el punto más cercano al sistema de agua del sillón odontológico y el punto más cercano a la jeringa tríplice. Las muestras obtenidas fueron cultivadas en un médio BHI por 24-48 h, en un horno a 37 ºC. Para el análisis cuantitativo, se utilizó 1 ml de cada muestra para dilución hasta el valor siete. Las colonias fueran contadas y los resultados fueron expresados en UFC/cm2. El análisis cualitativo fue iniciado con el cultivo de Agar Sangre, EBM-Levine y Agar Cetrimide por 24 h y la identificación de la bacteria fue realizada por análisis microscópicos. El perfil de resistencia fue verificado por el antibiograma clásico. Las zonas internas de las unidades de líneas de agua mostraron un promédio de 2,44 x 109 UFC/cm2. Bacillus subtilis y Pseudomonas aeruginosa fueron encontrados. El perfil de resistencia de Pseudomonas aeruginosa indicó sensibilidad a todos los antibióticos testados. Un gran número de microorganismos fue cuantificado desde la biopelícula acumulada en las líneas de agua de unidades dentales. Sin embargo, no resistieron al antibiograma clásico. Se requiere una mejor gestión y aplicación de protocolos de decontaminación en las líneas de agua debido a que las infecciones oportunistas puedan estar asociadas a Pseudomonas aeruginosa.

Comparison between Ogawa-Kudoh and modified Petroff techniques for mycobacteria cultivation in the diagnosis of pulmonary tuberculosis / Comparação entre os métodos de Ogawa-Kudoh e Petroff modificado para o cultivo de micobactérias no diagnóstico da tuberculose pulmonar

ABSTRACT Objective To compare the performance of the Ogawa-Kudoh method with the modified Petroff technique in diagnosis of pulmonary tuberculosis. Methods A total of 205 sputum samples from 166 patients with clinical suspicion or under pulmonary tuberculosis follow-up, seen at a public tertiary care hospital, from July 2014 to July 2016 were used. All samples were simultaneously processed using the Ogawa-Kudoh and modified Petroff decontamination methods, according to the recommendations of the Ministry of Health. In the statistical analysis, the McNemar test and the Kappa index were used, respectively, to compare proportions and verify agreement between data. Results The Ogawa-Kudoh and modified Petroff methods were efficient in mycobacteria detection, with no significant differences in results (p=0.549) and contamination rate of the cultures (p=0.065). The agreement between techniques was considered excellent (Kappa index of 0.877) and Ogawa-Kudoh, as compared to the modified Petroff technique, showed sensitivity of 90.4%, specificity of 96.6%, positive predictive value of 94.3% and negative predictive value of 94.2%. Conclusion The Ogawa-Kudoh technique proved to be sufficiently sensitive and specific for diagnosis of pulmonary tuberculosis, and, therefore, suitable for routine laboratory application. Since it is simple, low-cost and has less technical requirements for biosafety and professional training, Ogawa-Kudoh is an alternative for managers and healthcare professionals to promote the expansion of bacteriological diagnostic coverage of pulmonary tuberculosis.

RESUMO Objetivo Comparar o desempenho do método de Ogawa-Kudoh ao de Petroff modificado no diagnóstico da tuberculose pulmonar. Métodos Utilizaram-se 205 amostras de escarro de 166 pacientes com suspeita clínica ou controle de tuberculose pulmonar atendidos em um hospital público terciário, entre os meses de julho de 2014 a julho de 2016. Todas as amostras foram processadas simultaneamente pelos métodos de descontaminação Ogawa-Kudoh e Petroff modificado, seguindo as recomendações do Ministério da Saúde. Na análise estatística, foi empregado o teste de McNemar, para comparação de proporções, e o índice Kappa, para verificar o grau de concordância entre os dados. Resultados Os métodos Ogawa-Kudoh e Petroff modificado mostraram-se eficientes na detecção de micobactérias, não sendo verificadas discordâncias significativas tanto nas comparações de pares de resultados (p=0,549), como na taxa de contaminação das culturas (p=0,065). O grau de concordância das técnicas foi considerado excelente (índice Kappa de 0,877), e o Ogawa-Kudoh, em relação ao Petroff modificado, apresentou 90,4% de sensibilidade, 96,6% de especificidade, 94,3% de valor preditivo positivo e 94,2% de valor preditivo negativo. Conclusão O método de Ogawa-Kudoh revelou-se suficientemente sensível e específico para o diagnóstico da tuberculose pulmonar e, portanto, adequado para a aplicação na rotina laboratorial. Por ser mais simples, de baixo custo e com menores exigências técnicas de biossegurança e capacitação profissional, o Ogawa-Kudoh apresenta-se como alternativa para gestores e profissionais da área promoverem a ampliação da cobertura diagnóstica bacteriológica da tuberculose pulmonar.

Incidence of variant hemoglobins in newborns attended by a public health laboratory / Incidência de hemoglobinas variantes em neonatos assistidos por um laboratório de saúde pública

ABSTRACT Objective To evaluate the incidence of variant hemoglobins in different health regions. Methods A descriptive, observational, and cross-sectional study with a quantitative approach based on secondary data in the internal records of the neonatal screening service - Laboratório Central de Saúde Pública do Estado do Piauí (PI, Brazil). The variables related to sex, ethnicity and positive diagnosis for variant hemoglobins were analyzed, with further population distribution of hemoglobinopathies among the macroregions of the state. Results A total of 69,180 samples of newborns were analyzed, and 3,747 were diagnosed as hemoglobinopathies, from February 1st, 2014 and December 31st, 2015. Sickle cell trait was the most frequent (4.1%), followed by hemoglobinopathy C in 0.9%; homozygous hemoglobin S cases 0.1% stood out and there were no cases of hemoglobinopathy D in the state. It is also worth noting that the highest frequencies of hemoglobin alterations in Piauí were in males (49.8%) and of parda skin color (38.5%). The region of Piauí presenting the highest incidence of heteroygous variant hemoglobins was Tabuleiros do Alto Parnaíba and Vale do Sambito, due to importance of the region's population Entre Rios. Conclusion Neonatal screening programs are important for screening, orientations regarding health actions and monitoring of families with hemoglobinopathies, in order to reduce morbidity and mortality rates.

RESUMO Objetivo Avaliar a incidência de hemoglobinas variantes em diferentes regiões de saúde. Métodos Estudo descritivo, observacional e transversal com abordagem quantitativa baseada em dados secundários presentes nos registros internos do serviço de triagem neonatal do Laboratório Central de Saúde Pública do Estado do Piauí (PI, Brasil). Foram analisadas as variáveis referentes a sexo, etnia e diagnóstico positivo para hemoglobinas variantes, com posterior distribuição populacional das hemoglobinopatias entre as macrorregiões do Estado. Resultados Foram analisadas 69.180 amostras de recém-nascidos, e 3.747 diagnosticados com hemoglobinopatias, entre 1° de fevereiro de 2014 e 31 de dezembro de 2015. Constatou-se maior ocorrência do traço falciforme em 4,1% da amostra e da hemoglobinopatia C em 0,9%, com destaque para os casos de hemoglobina S em homozigose (0,1%) e ausência de hemoglobinopatia D no Estado. Destacou-se, ainda, o fato de as maiores frequências de alteração hemoglobínica do Piauí estarem presentes em recém-nascidos do sexo masculino (49,8%) e de etnia parda (38,5%). As regiões do Piauí que apresentaram as maiores incidências de hemoglobinas variantes em heterozigose foram Tabuleiros do Alto Parnaíba e Vale do Sambito, devendo-se ressaltar a relevância populacional da região Entre Rios. Conclusão Os programas de triagem neonatal são importantes para rastreamento, orientação de ações de saúde e acompanhamento de famílias acometidas com hemoglobinopatias, a fim de diminuir a morbimortalidade provocada por estas patologias.

Directriz para reducir el riesgo de transmisión de mycobacterium abscessus durante la práctica clínica odontológica / Guideline to reduce the risk of transmission of mycobacterium abscessusin clinical dental practice

Las medidas de bioseguridad están predestinadas a reducir el riesgo de transmisión de microorganismos a partir de fuentes de infección reconocidas o no reconocidas en clínicas dentales vinculadas con lacontaminación de los materiales, aparatos y/o instrumentos. Un microorganismo reemergente es el Mycobacterium abscessus, que es unabacteria ambiental que puede ocasionar problemas de salud muy serios, por lo que debe ser controlada y prevenida su transmisión.

Biosafety measures are designed to reduce the risk of transmission ofmicroorganisms from recognized or unrecognized sources of infectionin dental procedures associated with the contamination of materials,apparatus, and/or instruments. One reemerging microorganism isMycobacterium abscessus, which is an environmental bacterium thatcan cause serious health problems and therefore needs to be controlledand prevented.

Rapid culture-based detection of living mycobacteria using microchannel electrical impedance spectroscopy (m-EIS)

BACKGROUND: Multiple techniques exist for detecting Mycobacteria, each having its own advantages and drawbacks. Among them, automated culture-based systems like the BACTEC-MGIT™ are popular because they are inexpensive, reliable and highly accurate. However, they have a relatively long "time-to-detection" (TTD). Hence, a method that retains the reliability and low-cost of the MGIT system, while reducing TTD would be highly desirable. METHODS: Living bacterial cells possess a membrane potential, on account of which they store charge when subjected to an AC-field. This charge storage (bulk capacitance) can be estimated using impedance measurements at multiple frequencies. An increase in the number of living cells during culture is reflected in an increase in bulk capacitance, and this forms the basis of our detection. M. bovis BCG and M. smegmatis suspensions with differing initial loads are cultured in MGIT media supplemented with OADC and Middlebrook 7H9 media respectively, electrical "scans" taken at regular intervals and the bulk capacitance estimated from the scans. Bulk capacitance estimates at later time-points are statistically compared to the suspension's baseline value. A statistically significant increase is assumed to indicate the presence of proliferating mycobacteria. RESULTS: Our TTDs were 60 and 36 h for M. bovis BCG and 20 and 9 h for M. smegmatis with initial loads of 1000 CFU/ml and 100,000 CFU/ml respectively. The corresponding TTDs for the commercial BACTEC MGIT 960 system were 131 and 84.6 h for M. bovis BCG and 41.7 and 12 h for M smegmatis, respectively. CONCLUSION: Our culture-based detection method using multi-frequency impedance measurements is capable of detecting mycobacteria faster than current commercial systems.

Mycobacteriosis in patients with breast implants: a case review from the Ivo Pitanguy Institute / Micobacteriose em implantes mamários: revisão da casuística do Instituto Ivo Pitanguy

INTRODUCTION: Reports of infections caused by rapidly growing mycobacteria during plastic surgery have increased in recent years despite improvements in techniques of asepsis/antisepsis and antibiotic prophylaxis. Infections occurring after the insertion of breast implants are a cause of patient morbidity and a significant problem for the surgeon. METHODS: Breast implant surgery cases complicated by mycobacterial infections at the Infirmary ward 38th of the Santa Casa da Misericórdia, Rio de Janeiro were retrospectively reviewed. A description of the current guidelines for the prevention and treatment of mycobacteriosis is included. Laboratory confirmed and clinically suspected cases were included in this study. RESULTS: Of 483 augmentation mammaplasty cases, 3 patients developed mycobacterial infections in the last 3 years. In 2 patients, there was a suspicion of infection that was not confirmed by laboratory data. CONCLUSIONS: Prophylaxis is fundamental for reducing the incidence of mycobacteriosis during plastic surgery procedures. However, the identification, diagnosis, and treatment of mycobacterial diseases are important to minimize the morbidity of this type of infection.

INTRODUÇÃO: Nos últimos anos, foram crescentes os registros de infecções por micobactéria de crescimento rápido em cirurgia plástica, mesmo com a melhoria dos métodos de assepsia/ antissepsia e da antibioticoprofilaxia. A infecção após inclusão de implantes mamários causa grande morbidade às pacientes e transtorno ao cirurgião. MÉTODO: Estudo retrospectivo dos casos de infecção por micobactéria de crescimento rápido da 38ª Enfermaria da Santa Casa da Misericórdia do Rio de Janeiro, após inclusão de implantes mamários, em que são apresentadas propostas de prevenção e tratamento da micobacteriose. Foram incluídos os casos confirmados laboratorialmente e os clinicamente suspeitos. RESULTADOS: Até o presente momento foram confirmados 3 casos de infecção por micobactéria, num total de 483 mamaplastia de aumento no decorrer de 3 anos. Em 2 pacientes, houve suspeita de infecção, porém sem confirmação laboratorial. CONCLUSÕES: A profilaxia é o pilar fundamental para a redução do impacto da micobacteriose em procedimentos de cirurgia plástica. Entretanto, saber identificar, diagnosticar e tratar corretamente a micobacteriose é de suma importância para minimizar a morbidade da paciente.

Infecções por micobactérias de crescimento rápido resistentes a desinfetantes: uma problemática nacional? / Infections by rapidly growing mycobacteria resistant to disinfectants: a national matter?

Rapidly growing mycobacteria (RGM) are opportunistic microorganisms and widely distributed into aqueous environment and soil. Human RGM infections are usually associated with contaminated solutions or medical instruments used during invasive procedures. RGM postsurgical infections have recently emerged in Brazil and have caused national alert, considering the risk factors and epidemiological aspects. This study aimed at analysing the main factors linked to the recent RGM outbreaks, with focus on the national epidemic of Mycobacterium massiliense infections related to the BRA100 strains resistant to 2 percent glutaraldehyde commercial solutions commonly used for preoperative high-level disinfection. Based on previous studies and laboratorial results of assays and colaborations, it has been observed that the cases have been associated with videolaparoscopy for different applications and elective esthetic procedures, such as lipoaspiration and mammary prosthesis implant. Furthermore, outbreaks between 2004 and 2008 and the epidemic in Rio de Janeiro state may be considered particular Brazilian events. Although there are a few epidemiological published studies, some hypotheses based on common aspects related to most national nosocomial occurrences are possible, such as lack of protocols for cleaning and high-level disinfection, use of 2 percent glutaraldehyde as high-level disinfectant for surgical instruments, and dissemination of M. massiliense BRA100 by unknown mechanisms.

Infecção por micobactérias de crescimento rápido após procedimentos videocirúrgicos: a hipótese do glutaraldeído / Rapidly growing mycobacteria infection after videosurgical procedures: the glutaraldehyde hypothesis

Between August 2006 and February 2007, in the state of Rio de Janeiro, Brazil, a massive outbreak of RGM infections after video laparoscopy was mainly associated to the recently described Mycobacterium massiliense species. All confirmed and probable cases reports described the use of high-level disinfection of medical devices by using 2 percent glutaraldehyde (2 percent GA) for 30 min before the surgical procedures. We investigated the susceptibility of the M. massiliense isolates recovered during the outbreak to high-level disinfection after 30 min, 1h, 6h and 10h of exposure to the commercial disinfectants. Reference strains for official mycobactericidal tests such as Mycobacterium abscessus, Mycobacterium bovis, Mycobacterium chelonae, Mycobacterium neoaurum and Mycobacterium smegmatis were included as controls. Although all the reference strains were eliminated in 30 min of exposure to 2 percent GA, we observed the recovery of all M. massiliense clinical isolates even after 10h of exposure. This study suggests that failures in high-level disinfection and the high tolerance of these M. massiliense clinical strains to the 2 percent GA were strongly associated to the magnitude of the outbreak.

In vitro synergic effect of beta-lapachone and isoniazid on the growth of Mycobacterium fortuitum and Mycobacterium smegmatis

Nontuberculous mycobacteria are ubiquitous and saprophytic organisms that have been implicated in a wide spectrum of diseases due to an increasing number of immunocompromised patients. The natural resistance of atypical mycobacteria to classical antituberculous drugs has encouraged research into new chemotherapeutic agents and drug combinations. The aim of this study was to determine the in vitro antimycobacterial activities of ²-lapachone alone and in combination with isoniazid against Mycobacterium fortuitum and Mycobacterium smegmatis via the Time-Kill Curve method. A 2 log10 CFU/mL reduction in the M. smegmatis culture was observed 72 h after adding ²-lapachone at its minimum inhibitory concentration. This drug sterilised the culture in 120 h. For M. fortuitum, a reduction of 1.55 log10 CFU/mL occurred in 24 h, but regrowth was seen in contact with ²-lapachone. Both microorganisms were resistant to isoniazid. Regrowth of M. fortuitum and M. smegmatis was observed at 48 h and 72 h, respectively. In combination, these two drugs had a bactericidal effect and sterilised both cultures in 96 h. These results are valuable because antibiotic-resistant bacteria are a major public health problem.

Estudo fenotípico e molecular de micobactérias de crescimento rápido de interesse em Saúde Pública / Phenotypic and molecular study of mycobacteria fast-growing interest in Public Health

Os complexos Mycobacterium chelonae – M. abscessus e Mycobacterium fortuitum – M. peregrinum são compostos por espécies bacterianas de crescimento rápido e potencialmente patogênicas. Sua distribuição é ubíqua no ambiente, são resistentes a cloração da água e a sua replicação ocorre mesmo em condições de escassez de nutrientes. Estão envolvidos em casos de infecção pulmonar e extrapulmonar, e causam infecções em pacientes imunocomprometidos e submetidos a procedimentos cirúrgicos invasivos. Os objetivos do presente trabalho foram: confirmar através de testes fenotípicos e com as técnicas de PRA hsp65 e sequenciamento do fragmento do rpoB, a identificação de micobactérias de crescimento rápido, incluídas dos complexos M. chelonae-M.abcessus e M. fortuitum-M.peregrinum. Foram incluídos no estudo os isolados provenientes de pacientes com dois ou mais isolamentos provenientes de sítio não estéril ou um isolamento de sítio estéril. O estudo de 38isolados demonstrou que as provas fenotípicas disponíveis atualmente não permitem a identificação de todas as espécies de micobactérias de crescimento rápido já descritas na literatura. O PRA hsp65 possibilitou a identificação rápida e precisa de 63 por cento das espécies de micobactérias e demonstrou um perfil compartilhado pelas espécies M. abcessus 2; M. bolletti 1 e M. massiliense 1. O sequenciamento do gene rpoB confirmou a identificação das espécies citadas. Nossos resultados demonstram que o PRA-hsp65 e o sequenciamento do gene rpoB são ferramentas úteis para fornecer a identificação das espécies de micobactérias com mais acurácia. O uso dessas técnicas poderiam ser consideradas em laboratório de referência para identificar Micobactérias de crescimento rápido uma vez que elas são patógenos emergentes implicados em surtos e isolados de pacientes em centros de referência para tratamento de tuberculose multirresistente.

Evaluation of Combination of BACTEC Mycobacteria Growth Indicator Tube 960 System and Ogawa Media for Mycobacterial Culture / 대한진단검사의학회지

BACKGROUND: The combined use of liquid media and solid media is recommended for mycobacterial culture. We evaluated diagnostic performance of combination of BACTEC Mycobacteria Growth Indicator Tube (MGIT; Becton Dickinson, USA) and 2% Ogawa media (Korean Institute of Tuberculosis, Korea) for recovery of mycobacteria. METHODS: In September 2007, 1,764 specimens from 1,059 patients were cultured with MGIT and Ogawa. Acid fast bacilli (AFB) smear was fluorochrome-stained. The isolates were identified into Mycobacterium tuberculosis (MTB) and nontuberculous mycobacteria (NTM) with PCR using Seeplex TB Detection Kit (Seegene, Korea). Recovery rate, time to detection (TTD), contamination rate, mixed growth rate and species distribution were analyzed. RESULTS: Two hundred thirty-five specimens (13.3%) from 165 patients (15.6%) were positive for mycobacterial culture. Recovery rates of mycobacteria from the group using both media, MGIT only, and Ogawa only were 13.3%, 12.1%, and 7.8%, respectively. While MGIT recovered 98.9% of MTB and 79.7% of NTM, Ogawa recovered 65.9% of MTB and 54.1% of NTM. TTDs of total mycobacteria/MTB/NTM in MGIT and Ogawa were 10.6/11.4/9.7 days and 31/29/33 days, respectively. MGIT TTDs of total mycobacteria/MTB/NTM from AFB-positive specimens were significantly shorter than those of AFB-negative specimens; 8.2/9.5/4.4 days vs 11.6/12.7/10.7 days. Contamination and mixed growth rate of MGIT were 9.6% and 3.7%. Primary culture of Ogawa recovered 1 MTB and 1 NTM among the 170 MGIT-contaminated specimens and 38 mycobacteria among 66 specimens that showed mixed cultures of MGIT. CONCLUSIONS: MGIT warrants sensitive and rapid isolation of mycobacteria. However, the combination of MGIT and Ogawa is more desirable to recover mycobacteria in the case of contaminations or mixed cultures.

Protection by live Mycobacterium habana vaccine against Mycobacterium tuberculosis H37Rv challenge in mice.

BACKGROUND & OBJECTIVES: In recent years the efficacy of BCG vaccine against tuberculosis has been questioned and there is no alternative vaccine available. Several strategies are being applied to get a satisfactory vaccine. Two approaches are generally considered: the subunit vaccines and the whole cell vaccines. The objective of this investigation was to evaluate an avirulent mycobacteria, Mycobacterium habana, as a whole cell vaccine to protect mice from infection of M. tuberculosis H37Rv. METHODS: AKR and immunocompromised SJL/J mice were immunized with live M. habana vaccine. These mice were challenged with M. tuberculosis H37Rv eight weeks later along with unimmunized control mice. Protection by M. habana vaccine was measured through several parameters, which included survival of challenged mice, dissemination of challenge strain and histopathology of lung tissues. RESULTS: M. habana vaccinated animals were healthier than the unvaccinated mice after challenge with M. tuberculosis and survived with significant increase in mean survival time. The viable count of challenge strain was at least 100-fold less in vaccinated mice than the control mice. The lung tissues in unvaccinated mice showed marked bronchopneumonia with clusters of acid fast bacilli, whereas vaccinated mice showed small areas of damage and evidence of protection subsequently. INTERPRETATION & CONCLUSION: It may be concluded from the evidence presented here that mice vaccinated with M. habana were protected from challenge with M. tuberculosis in both normal and immunocompromised states.

Water soluble palmitic acid in media for cultivation of leprosy derived psychrophilic mycobacteria from Mycobacterium leprae infected tissues

Cultivation trials for Mycobacterium leprae resulted in growth of Mycobacterium psychrophilum (L). Media were inoculated with host grown Mycobacterium leprae cells from armadillo tissues, Nu mice foot pads or human lepromata. Cultures were obtained in liquid and on semisolid multifactoria 1 media containing water soluble palmitic acid or its salts. Ammonium thioglycolate and Napalmitate served as carbon and energy sources. The water soluble palmitic acid remained in perfect solution following sterilization in the autoclave, thus easily accessible to the cells. The cyclodextrin-Fe complex served as a siderophore to grow the obtained leprosy derived psychrophilic cells. The leprosy derived cultures and subcultures grew opimally at+10 degrees Celssius but deteriorated rapidly at + 32 degrees Celsius, in the multifactorial media. No growth occurred in 7H9 media. Cultures were not identified for classification.

Presence of mycobactin-like substance in Mycobacterium leprae.

Chloroform extracts of M. leprae suspensions--crude, partially purified and purified--were prepared by standard methods. Similar extracts were also prepared from the livers of normal armadillos using the same methods that were used to prepare crude and partially purified M. leprae suspensions. The only chloroform extract that supported the growth of M. paratuberculosis was the one prepared from Percoll gradient-purified M. leprae. Other four extracts not only did not support the growth of mycobactin-dependent M. paratuberculosis, but also inhibited the growth of mycobactin-independent strain of M. paratuberculosis. These results suggest the presence of mycobactin-like substance in M. leprae, and also, the presence of other unknown substance(s) in the crude suspensions of armadillo livers that inhibits the growth of M. paratuberculosis.

Metabolic studies on mycobacteria. V. A preliminary report on the ATP synthesis by mycobacteria including M. leprae by using different substrates.

By deletion and addition of various substrates in Sauton's and Dubos media, an experimental system has been standardised in which the role of various nutrients in the energy synthesis of mycobacteria can be determined. By using this system with cultivable mycobacteria it was observed that glycerol and asparagine are the important ingredients for ATP synthesis by mycobacteria. Glucose further enhanced the ATP synthesis and growth of these mycobacteria. In the media containing asparagine or glycerol, there was marginal increase in the ATP in the M. leprae suspensions initially but this was not sustained and there was no progressive increase in biomass or multiplication. When M. leprae was incubated in the media from which both these substrates were deleted, there was progressive decline in ATP levels right from the beginning. From these preliminary results, it appears that asparagine and glycerol may be useful as substrates for ATP synthesis by M. leprae and need to be investigated further. In depth studies are necessary to find out the factors which results in the inability of M. leprae to utilise these and other substrates in a substrained manner for its multiplication and growth in artificial media.

Estudio comparativo de diferentes medios para micobacterias, que incluye la nueva modificación UIT-A / Comparative study of different media for mycobacteria, including new UIT-A modification

Se estudia la eficiencia de la producción de biomasa micobacteriana entre diferentes medios de cultivos, éstos son: Sauton agar, Middlebrook, UIT y la modificación UIT-A. Los resultados obtenidos después del análisis estadístico son satisfactorios con respecto a la modificación UIT-A

Absence of mycobactin in Mycobacterium leprae; probably a microbe dependent microorganism implications.

Ferric mycobactins were prepared from Mycobacterium phlei. Mycobacterium avium--intracellulare A and H, isolated respectively from armadillo and human leprosy specimens. Attempts were made to extract mycobactin from host grown M. leprae cells. The crude ferric mycobactin extracts were tested for growth supporting effect on the mycobactin dependent M. paratuberculosis strain ATCC 19698. Mycobactins prepared from M. phlei and the two M. avium--intracellulare strains had growth promoting effect on M. paratuberculosis. The same test organism did not grow in media supplemented with the extract prepared from M. leprae. Results indicate the absence of mycobactin from host grown M. leprae. Since M. leprae cells contain cytochrome c and since mycobactin is essential to growth of all mycobacteria, M. leprae might be considered as a microbe dependent microbe. It is proposed that secondary mycobacteria present in M. leprae infected humans and armadillos might provide mycobactin for in vivo multiplication of M. leprae.

Comparison of Mycobacterial Growth in Dubos Medium, Hyaluronate Supplemented Medium and Umbilical Cord Extract Based Medium

This is a report of attempts to compare the growth yields of various species of fastidious mycobacterium inch1ding human pathogens and non-pathogens in the conventional Dubos liquid medium and two simple media formulated recently; one is a medium containing 0.1% hyaluronic acid and 6.0% bovine serum albumin and the other is a semisyntheic medium made of umbilical cord extract supplemented with 10% sheep serum as a final concentration. All mycobacterial strains employed in experiments gave the heaviest growth yields in the hyaluronic acid-bovine serum albumin medium (HAS medium), among the three media. Dubos liquid medium seemed to be inferior to a medium made of umbilical cord extract (UCE medium) in supporting mycobacterial growth. There were three-to seven-fold increases in dry weight of the bacteria grown in the HAS medium as compared with those in the Dubos liquid medium. We also looked for the possible effect of bovine serum albumin (BSA)in the HAS medium on mycobacterial growth. As a result, we found that the amount of BSA in the HAS medium, ranging from zero to 6.0% in the medium, showed no substantial effect on the mycobacteria1 growth.