Study of growth, metabolism, and morphology of Akkermansia muciniphila with an in vitro advanced bionic intestinal reactor.

BACKGROUND: As a kind of potential probiotic, Akkermansia muciniphila abundance in human body is directly causally related to obesity, diabetes, inflammation and abnormal metabolism. In this study, A. muciniphila dynamic cultures using five different media were implemented in an in vitro bionic intestinal reactor for the first time instead of the traditional static culture using brain heart infusion broth (BHI) or BHI + porcine mucin (BPM). RESULTS: The biomass under dynamic culture using BPM reached 1.92 g/L, which improved 44.36% compared with the value under static culture using BPM. The biomass under dynamic culture using human mucin (HM) further increased to the highest level of 2.89 g/L. Under dynamic culture using porcine mucin (PM) and HM, the main metabolites were short-chain fatty acids (acetic acid and butyric acid), while using other media, a considerable amount of branched-chain fatty acids (isobutyric and isovaleric acids) were produced. Under dynamic culture Using HM, the cell diameters reached 999 nm, and the outer membrane protein concentration reached the highest level of 26.26 µg/mg. CONCLUSIONS: This study provided a preliminary theoretical basis for the development of A. muciniphila as the next generation probiotic.

A descriptive study of culture media in Brazilian assisted reproduction clinics.

OBJECTIVE: The present study aimed to draw a profile of the most commonly used media and protocol characteristics from assisted reproduction technology (ART) facilities in Brazil. METHODS: To obtain an overview of ART methods and culture media, a questionnaire was given to embryologists from ART clinics in Brazil. Further research in scientific papers and journals was carried out for describing the processes around Brazil, USA and Europe. RESULTS: From the questionnaire, we found that the embryo medium mostly used is CSCMTM from Irvine Scientific, represented 37.04% in Brazilian ART clinics; interestingly, 70.37% of clinics exchange the embryo media bath; however, 70.37% do not change the media type. Transfers in Brazilian clinics were variable, but day 3 transfer was a procedure seen in 37.04%. The remaining embryos are habitually maintained in prolonged cultivation in 51.85% of the clinics interviewed. CONCLUSION: Although there are numerous studies trying to better understand embryo culture media influences, there is a lack of evidence for choosing one as the most appropriate. In short, it is a random decision for such an essential stage of In Vitro Fertilization.

Growth of MRSA and Pseudomonas aeruginosa in a fine-celled foam model containing sessile commensal skin bacteria.

Sessile cultures of the skin bacteria Staphylococcus saprophyticus and Corynebacterium xerosis were grown using novel fine-celled foam substrata to test the outcome of challenge by methicillin-resistant Staphylococcus aureus or Pseudomonas aeruginosa under three growth medium regimens (simulated sweat, simulated serum or simulated sweat substituted with simulated serum during the microbial challenge). S. saprophyticus and C. xerosis significantly limited MRSA and P. aeruginosa immigration respectively, under the simulated sweat and serum medium regimes. Under the substitution medium regime however, MRSA and P. aeruginosa integrated into pre-established biofilms to a significantly greater extent, attaining cell densities similar to the axenic controls. The outcome of challenge was influenced by the medium composition and test organism but could not be predicted based on planktonic competition assays or growth dynamics. Interactions between skin and wound isolates could be modelled using the fine-celled foam-based system. This model could be used to further investigate interactions and also in preclinical studies of antimicrobial wound care regimens.

Impact of Implementation of an Automated Liquid Culture System on Diagnosis of Tuberculous Pleurisy.

This study was conducted to evaluate the impact of implementation of an automated liquid culture system on the diagnosis of tuberculous pleurisy in an HIV-uninfected patient population. We retrospectively compared the culture yield, time to positivity, and contamination rate of pleural effusion samples in the BACTEC Mycobacteria Growth Indicator Tube 960 (MGIT) and Ogawa media among patients with tuberculous pleurisy. Out of 104 effusion samples, 43 (41.3%) were culture positive on either the MGIT or the Ogawa media. The culture yield of MGIT was higher (40.4%, 42/104) than that of Ogawa media (18.3%, 19/104) (P<0.001). One of the samples was positive only on the Ogawa medium. The median time to positivity was faster in the MGIT (18 days, range 8-32 days) than in the Ogawa media (37 days, range 20-59 days) (P<0.001). No contamination or growth of nontuberculous mycobacterium was observed on either of the culture media. In conclusion, the automated liquid culture system could provide approximately twice as high yields and fast results in effusion culture, compared to solid media. Supplemental solid media may have a limited impact on maximizing sensitivity in effusion culture; however, further studies are required.

[An alternative biphasic nutrient medium for the diagnosis of cutaneous leishmaniasis]. / Kutanöz leysmanyazis tanisinda alternatif bifazik nutrient besiyeri.

Cutaneous leishmaniasis (CL) caused by the Leishmania spp. parasites, is a disease characterized by nodulo-ulcerative lesions in the skin. CL is transmitted to humans by infected sandflies during blood sucking, and is endemic in about 98 countries over the world. The demonstration of amastigotes via microscopic examination, and the growth of promastigotes in NNN (Novy-MacNeal-Nicolle) medium are gold standard methods for laboratory diagnosis. The aim of this study was to compare the biphasic NNN medium that is frequently used in routine laboratories with the biphasic nutrient medium that can be prepared easily in microbiology laboratories, for the growth of promastigotes. In the study, the aspiration fluid sample was used as clinical sample which was obtained from the skin lesion of a 47-year-old female patient admitted to Izmir Katip Celebi Ataturk Education and Research Hospital dermatology outpatient clinic and pre-diagnosed as CL. The aspirate sample taken from the lesion was evaluated with microscopy, cultivation in two different media and real-time polymerase chain reaction (Rt-PCR) methods. In microscopic examination Leishmania amastigotes were observed in Giemsa-stained smears prepared from the aspiration fluid. In Rt-PCR performed by using specific primers and probes targeting ITS1 region of Leishmania parasite, a melting-curve compatible with L.tropica was detected. For cultivation, triple inoculations of the aspirate sample into NNN (NNN + RPMI 1640 + 10% fetal calf serum) and nutrient media (nutrient agar + nutrient broth + 10% fetal calf serum) were used. The cultures were incubated at 27°C for 10 days, and the number of propagated promastigotes were counted on the third, seventh and tenth days. The growth of Leishmania promastigotes was detected in both media on the third day. The number of promastigotes grown in NNN medium on the third, seventh and tenth days were 105/ml, 106/ml and 108/ml, respectively. Those values in nutrient medium were 106/ml, 107/ml and 108/ml on the third, seventh and tenth days, respectively. Although the number of promastigotes on the third and seventh days were higher in nutrient medium than NNN medium, the number of cultivated promastigotes were equal on the tenth day. As a result, nutrient medium is considered to have an impact in the diagnosis of CL, by providing an alternative to the routine medium used and can readily be available in microbiology and parasitology laboratories with long shelf-life. It was concluded that biphasic nutrient medium could be used as a supplementary medium for diagnosis in laboratories in the absence of NNN medium or can not be provided.

[Evaluation of second-line antituberculosis drug susceptibilities of multidrug-resistant Mycobacterium tuberculosis complex isolates by E-test method]. / Çok ilaca dirençli Mycobacterium tuberculosis kompleks izolatlarinda ikinci basamak antitüberküloz ilaç duyarliliklarinin E-test yöntemi ile degerlendirilmesi.

Multidrug-resistant (MDR) tuberculosis (TB) constitutes a restricting factor for the effective treatment of TB worldwide. Early diagnosis and appropriate treatment of patients are the most effective strategy in the control of MDR-TB. Therefore, knowledge of drug resistance patterns of the MDR-TB clinical isolates are necessary in planning of an appropriate treatment regimen for the patient. The aims of this study were to detect the susceptibilities of MDR-TB isolates to second-line anti-TB drugs by E-test method, and to compare their results with Löwenstein-Jensen (LJ) proportion method. A total of 122 MDR (resistant to isoniazid and rifampicin) Mycobacterium tuberculosis complex (MTC) strains isolated from samples of patients with pulmonary TB were included in the study. The isolates were identified by conventional methods and first-line anti-TB drug susceptibility testing was performed by the proportion method using LJ medium. The susceptibilities of the isolates to second-line anti-TB drugs [kanamycin (KN), ofloxacin (OFL), ethionamid (ETN), linezolid (LIN)] were tested by proportion method on LJ medium and E-test method on Middlebrook 7H11 medium. For this purpose, E-test strips (bioMerieux, Fransa) of KN (0.016-256 mg/ml), OFL (0.02-32 mg/ml), ETN (0.016-256 mg/ml), and LIN (0.016-256 mg/ml) were used. The susceptibility tests were evaluated in 5., 7., and 10. days after application of the E-test strips, and proportion method on LJ medium was evaluated 28 days later. Second line-anti-TB drug susceptibility results were obtained in 5 to 10 days by E-test. Of the MDR MTC strains 98% (119/122) were susceptible to KN, OFL and LIN, while 2% (3/122) of the strains were resistant to KN and ETN. The correlation between E-test and LJ proportion method was estimated as 96% for KN and ETN, 98% for OFL, and 100% for LIN. When compared with LJ proportion method, the specificity of E-test in the detection of susceptibility to KN, OFL, ETN and LIN were 60%, 38%, 60%, and 100%, respectively, while the sensitivity was 100% for all drugs. Our results indicated that E-test method exhibited high sensitivity and specificity (100%) for LIN, so it may be used alone in susceptibility testing for this drug, however since the specificity is low (38%) for OFL it should be used together with the proportion method. In conclusion, E-test method might contribute for initiation of an early and effective anti-TB drug treatment and control of infection by rapid diagnosis in MDR-TB cases.

Impact of Implementation of an Automated Liquid Culture System on Diagnosis of Tuberculous Pleurisy

This study was conducted to evaluate the impact of implementation of an automated liquid culture system on the diagnosis of tuberculous pleurisy in an HIV-uninfected patient population. We retrospectively compared the culture yield, time to positivity, and contamination rate of pleural effusion samples in the BACTEC Mycobacteria Growth Indicator Tube 960 (MGIT) and Ogawa media among patients with tuberculous pleurisy. Out of 104 effusion samples, 43 (41.3%) were culture positive on either the MGIT or the Ogawa media. The culture yield of MGIT was higher (40.4%, 42/104) than that of Ogawa media (18.3%, 19/104) (P<0.001). One of the samples was positive only on the Ogawa medium. The median time to positivity was faster in the MGIT (18 days, range 8-32 days) than in the Ogawa media (37 days, range 20-59 days) (P<0.001). No contamination or growth of nontuberculous mycobacterium was observed on either of the culture media. In conclusion, the automated liquid culture system could provide approximately twice as high yields and fast results in effusion culture, compared to solid media. Supplemental solid media may have a limited impact on maximizing sensitivity in effusion culture; however, further studies are required.

Letter to the editor: Respective Contribution of Liquid and Solid Media to Mycobacterial Yields from Pleural Fluid in Tuberculous Pleural Effusion

No abstract available.

Evaluation of different culture media for improvement in bioinsecticides production by indigenous Bacillus thuringiensis and their application against larvae of Aedes aegypti.

Production of indigenous isolate Bacillus thuringiensis sv2 (Bt sv2) was checked on conventional and nonconventional carbon and nitrogen sources in shake flasks. The effects on the production of biomass, toxin production, and spore formation capability of mosquito toxic strain were determined. Toxicity differs within the same strain depending on the growth medium. Bt sv2 produced with pigeon pea and soya bean flour were found highly effective with LC50 < 4 ppm against larvae of Aedes aegypti. These results were comparable with bacteria produced from Luria broth as a reference medium. Cost-effective analyses have revealed that production of biopesticide from test media is highly economical. The cost of production of Bt sv2 with soya bean flour was significantly reduced by 23-fold. The use of nonconventional sources has yielded a new knowledge in this area as the process development aspects of biomass production have been neglected as an area of research. These studies are very important from the point of media optimization for economic production of Bacillus thuringiensis based insecticides in mosquito control programmes.

Modern principles of classification and development of nutrient media for culturing of human and animal cells.

Analysis of the main principles of classification and development of nutrient media used for culturing of human and animal cells in biology and medicine is presented. The key moments of induction and regulation of mitogenic cascades and their differences in cells of continuous lines, diploid cells, and primary cultures are discussed. Some variants of classification of nutrient media for various cell culture types are described. Peculiarities of composition and the prospects of using serum-free media are discussed. Practical results obtained by the authors in the development of diverse purpose nutrient media are presented.

Evaluation of three selective media for isolation of Aggregatibacter actinomycetemcomitans.

BACKGROUND AND OBJECTIVE: Aggregatibacter actinomycetemcomitans is a pathogen in oral and nonoral infections. Detection and quantification of this pathogen can be performed using selective culture techniques. The aim of this study was to establish the efficacy of two known selective media in their ability to select and support the growth of A. actinomycetemcomitans. MATERIAL AND METHODS: Trypticase soy bacitracin vancomycin (TSBV) medium and brain-heart infusion agar with vancomycin (Dentaid-1), as well as a modified Dentaid-1 medium (in which the brain-heart infusion agar was substituted with brain-heart infusion broth), were compared. Two-hundred and eighteen clinical samples were used to establish the recovery rate, the number of colony-forming units (CFUs) of A. actinomycetemcomitans as well as the total number of CFUs on the three different types of medium. In addition, the numbers of gram-negative aerobic rods and yeasts were determined. RESULTS: Both types of Dentaid-1 medium showed a higher recovery of A. actinomycetemcomitans compared with TSBV. However, these differences did not reach statistical significance. The total number of CFUs of A. actinomycetemcomitans recovered was significantly higher on Dentaid-1 compared with TSBV (p = 0.029). The mean number of gram-negative aerobic rods recovered was statistically higher on both types of Dentaid-1 medium in comparison with TSBV. Low numbers of yeasts were recovered occasionally on all test plates. CONCLUSION: Dentaid-1 is a low-cost effective alternative to TSBV for the isolation and growth of A. actinomycetemcomitans from clinical samples, such as dental plaque, which contain a complex microflora.

Recovery of mutans streptococci on MSB, SB-20 and SB-20M agar media.

OBJECTIVE: The recovery of mutans streptococci in saliva and dental biofilm samples depends, in part, on the culture medium used. In this study, we compared (i) the culture media Sucrose-Bacitracin agar (SB-20), Modified SB-20 (SB-20M) and Mitis Salivarius Bacitracin agar (MSB) in the count of colony forming units (cfu) of mutans streptococci and (ii) in the morphological and biochemical differentiation between Streptococcus mutans and Streptococcus sobrinus. DESIGN: Samples of non-stimulated saliva from 20 children were plated on SB-20, SB-20M and MSB, and incubated in microaerophilia at 37°C for 72h. Identification of microorganisms was based on analysis of colony morphology under stereomicroscopy. The biochemical identification of colonies was done by biochemical tests using sugar fermentation, resistance to bacitracin and hydrogen peroxide production. RESULTS: There was no significant difference (p>0.05) in the number of cfu of mutans streptococci recovered on SB-20 and SB-20M agar. Comparing the media, SB-20 and SB-20M yielded a larger number of mutans streptococci colonies (p<0.05) and were more effective than MSB in the identification of S. sobrinus (p<0.05), but not of S. mutans (p>0.05). CONCLUSION: There was no significant difference between SB-20 and SB-20M culture media in the count of mutans streptococci, demonstrating that the replacement of sucrose by coarse granular cane sugar did not alter the efficacy of the medium. Compared with MSB, SB-20 and SB-20M allowed counting a larger number of mutans streptococci colonies and a more effective morphological identification of S. sobrinus.

Susceptibilidad antibiótica in vitro a fluoroquinolonas / In vitro antibiotic susceptibility to fluoroquinolones

Objetivo: Determinar la susceptibilidad antibiótica de la bacterias obtenidas en cultivos de infecciones oculares en la Fundación Oftalmológica de Santander - Clínica Carlos Ardila Lulle (FOSCAL). Materiales y métodos: Estudio descriptivo retrospectivo de una serie de registros de cultivos de muestras de superficie ocular y líquidos intraoculares del laboratorio OCULAB-FOSCAL en Floridablanca (Colombia) realizados entre enero y diciembre de 2007. Se realizó antibiograma por el método de Kirby-Bauer con sensi-discos impregnados de concentraciones determinadas de antibiótico. Resultados: Se recogieron un total de 352 muestras de los cuales 160 fueron de conjuntiva, 150 fueron de córnea y 42 de líquidos intraoculares. Se recuperó más de un microorganismo en el 45,65% del total de las muestras. El 78,7 y el 18,4% de las bacterias identificadas correspondieron a Gram positivos y a Gram negativos, respectivamente. El 6,3, 8,9, 33,2 y 35,6% de las bacterias Gram positivas fueron resistentes a gatifloxacino, moxifloxacino, ciprofloxacino y levofloxacino, respectivamente. El 7,4, 16,7, 16,7 y 25,9% de las bacterias Gram negativas fueron resistentes a gatifloxacino, moxifloxacino, ciprofloxacino y levofloxacino, respectivamente. La resistencia bacteriana global (tanto Gram positivos como Gram negativos) a moxifloxacino fue del 10,15% y a gatifloxacino del 6,46%, siendo esta diferencia estadísticamente significativa (p<0,05)(AU)

Conclusiones: En nuestro estudio, se evidenció el desarrollo de resistencia bacteriana en muestras oculares incluso con las fluoroquinolonas de cuarta generación. Sin embargo se encontraron menores niveles de resistencia para las fluoroquinolonas de cuarta generación que para las de tercera y segunda generación, especialmente entre Gram positivos. Gatifloxacino mostró menores niveles de resistencia que la moxifloxacino. La interpretación de esta superioridad debe, sin embargo, hacerse con cuidado en el campo clínico, ya que se deben tener en cuenta otros factores como la penetración tisular y la actividad in vivo(AU)

Objective: To determine the antibiotic susceptibility of bacteria recovered from cultures of ocular infections in the Fundación Oftalmológica de Santander - Clínica Carlos Ardila Lulle (FOSCAL). Materials and methods: Retrospective descriptive study of a series of registries of cultures of samples from ocular surfaces and intraocular fluids from the OCULAB-FOSCAL laboratory in Floridablanca (Colombia) made between January and December of 2007. Antibiotic sensitivity screening by the method of Kirby-Bauer with impregnated Sensi-Discs™ of determined antibiotic concentrations was performed. Results: A total of 352 samples were studied: 160 from conjunctiva, 150 from cornea and 42 from intraocular fluids. Of the total of the samples more than one microorganism was recovered 45.65% of the samples. Gram positive and Gram negative bacteria were identified in 78.7 and 18.4%, respectively. Resistance to gatifloxacin, moxifloxacin, ciprofloxacin and levofloxacin was observed in 6.3, 8.9, 33.2 and 35.6%, respectively, of Gram positive bacteria. Resistance to gatifloxacin, moxifloxacin, ciprofloxacin and levofloxacin was also observed in 7.4, 16.7, 16.7%and 25.9%, respectively, of Gram negative bacteria. The overall bacterial resistance (Gram positive and Gram negative) to moxifloxacin was 10.15%, and to gatifloxacin it was 6.46%, being which showed a statistically significant difference (P<.05). Conclusions: In our study the development of bacterial resistance to fourth generation fluoroquinolones was demonstrated in ocular samples. However, lower levels of resistance to fourth generation fluoroquinolones compared with that of third and second generation were found, particularly to Gram positive. Gatifloxacin showed lower resistance levels than moxifloxacin. Nevertheless, interpretation of this superiority must be made with caution in the clinical field, since other factors, like tissue penetration and in vivo activity, must be taken into account(AU)

[Evaluation of biofilm formation by Staphylococcus aureus isolated from sputum of cystic fibrosis patients]. / Ocena tworzenia biofilmu przez szczepy Staphylococcus aureus wyizolowane z plwociny pacjentów z mukowiscydoza.

The purpose of this study was to evaluate two screening methods for detection of biofilm formation by eighty clinical Staphylococcus aureus isolates from patients with cystic fibrosis, and evaluation of biofilm production on the polystyrene 96-well tissue culture plates, depending on media applied. All clinical strains were incubated in three different media: Luria-Bertani broth (LB), tryptic soy broth supplemented with 2% glucose (TSBglu) and brain heart infusion (BHI). Biofilm production was screened by staining with crystal violet (CV) or with 2,3,5-triphenyltetrazolium chloride (TTC). Both CV and TTC assays showed, that all analyzed isolates created biofilm, in all tested media, however with different intensity. In conclusion, the CV method was found to be more sensitive than the TTC method, when we need information about whole mass of biofilm. The most optimal medium for the biofilm culture was LB medium.

Study of the lactic acid bacteria throughout the manufacture of dry-cured lacón (a Spanish traditional meat product). Effect of some additives.

Total aerobic mesophilic microflora (on SPC agar), lactic acid bacteria (on MRS agar) and lactobacilli (on Rogosa agar) were enumerated in samples from the surface and the interior of the pieces throughout the manufacture of six batches of lacón. Three of the batches were made without additives and three with additives (glucose (2 g/kg), sodium nitrite (E(250)) (125 mg/kg), sodium nitrate (E(251)) (175 mg/kg), sodium ascorbate (E(301)) (500 mg/kg), and sodium citrate (E(331)) (100 mg/kg)). The counts decreased throughout the manufacturing process, particularly after the salting stage. The use of additives did not affect the counts or the evolution of the microbial groups, except for the lactobacilli, which were present in higher numbers in the batches with additives. In four batches (two without and two with additives), from MRS agar and from Rogosa agar plates, 10 colonies were randomly taken from each sampling point of each batch (five from the surface sample and five from the interior sample) and from each culture medium; a total of 224 strains from MRS agar, and 176 strains from Rogosa agar that were identified by classical methods. The MRS agar displayed moderate selectivity for the isolation of lactic acid bacteria, and only 59% of the isolated strains belonged to this microbial group. Homofermentative and facultative heterofermentative lactobacilli (particularly Lactobacillus curvatus and Lactobacillus sakei) were the most abundant species isolated on this medium. The selectivity of the Rogosa agar for lactobacilli was extremely high. The species of lactobacilli isolated on this medium at different stages of manufacture of the four batches of lacón were consistent with those isolated from MRS agar. The use of additives in the lacón did not appreciably affect the kinds and proportions of species isolated on either MRS agar or Rogosa agar.