Selective Liquid Media Research Articles

Экспрессионная система Pichia pastoris рекомбинантного химозина Camelus dromedarius с подбором оптимальных условий индукции белка

The paper introduces optimal conditions for chymosin protein induction. The research featured the correlation between the cell mass gain of transformed P. pastoris/pPICZ(alpha)B/proCYM_camel_pp_IDT and the composition of nutrient media with different carbon sources and zeocin resistance producer strains. P. pastoris GS115/his4 was transformed to obtain P. pastoris/pPICZ(alpha)B/proCYM_camel_pp_IDT as a strain producer. The cell mass growth rate correlated with the zeocin concentrations in the YPD nutrient medium without glycerol and biotin and the YPD nutrient medium fortified with 0.0000% biotin and 1% glycerol. The biomass density increased faster when cultured on the fortified medium, regardless of zeocin concentration, especially during cultivation day 1. The cell mass growth on solid and liquid selective media was multidirectional. The two experimental samples on solid selective media with 200 μg/ml zeocin started growing on cultivation day 3 if the YPD medium was not fortified. The samples on fortified medium, on the contrary, showed no growth on day 3. The optical density of the cell mass grown on liquid selective medium demonstrated a particular pattern: the rate of biomass growth in the fortified medium samples decreased gradually whereas the unfortified medium samples demonstrated signs of growth, which intensified on cultivation day 2. To increase the cell density of P. pastoris strain GS115/his4, a preliminary application of 0.5 % methanol is recommended as a carbon source to activate the MUT metabolic pathway, i.e., methanol utilization. Adding the alternative oxidase enzyme is also recommended since strain GS115/his4 belongs to the MUT+ phenotype with a high growth rate and productivity, hence the need for additional methanol. The obtained data may help to optimize the commercial yield of P. pastoris as a producer of recombinant protein.

Droplet microfluidic system for high throughput and passive selection of bacteria producing biosurfactants.

Traditional methods for the enrichment of microorganisms rely on growth in a selective liquid medium or on an agar plate, followed by tedious characterization. Droplet microfluidic techniques have been recently used to cultivate microorganisms and preserve enriched bacterial taxonomic diversity. However, new methods are needed to select droplets comprising not only growing microorganisms but also those exhibiting specific properties, such as the production of value-added compounds. We describe here a droplet microfluidic screening technique for the functional selection of biosurfactant-producing microorganisms, which are of great interest in the bioremediation and biotechnology industries. Single bacterial cells are first encapsulated into picoliter droplets for clonal cultivation and then passively sorted at high throughput based on changes in interfacial tension in individual droplets. Our method expands droplet-based microbial enrichment with a novel approach that reduces the time and resources needed for the selection of surfactant-producing bacteria.

ATP Bioluminescence for Rapid and Selective Detection of Bacteria and Yeasts in Wine

Microbial contamination may represent a loss of money for wine producers as several defects can arise due to a microorganism’s growth during storage. The aim of this study was to implement a bioluminescence assay protocol to rapidly and simultaneously detect bacteria and yeasts in wines. Different wines samples were deliberately contaminated with bacteria and yeasts at different concentrations and filtered through two serial filters with decreasing mesh to separate bacteria and yeasts. These were resuscitated over 24 h on selective liquid media and analyzed by bioluminescence assay. ATP measurements discriminated the presence of yeasts and bacteria in artificially contaminated wine samples down to 50 CFU/L of yeasts and 1000 CFU/L of bacteria. The developed protocol allowed to detect, rapidly (24 h) and simultaneously, bacteria and yeasts in different types of wines. This would be of great interest for industries, for which an early detection and discrimination of microbial contaminants would help in the decision-making process.

Inhibitory Effects of Iso-α and β Hop Acids Against Pediococcus pentosaceus

The goal of the research was to assess the inhibitory effects of hop extracts, iso-α and β acids, against Pediococcus pentosaceus bacteria, during a short incubation period, both in liquid selective media (high pH values) and beer wort fermentation (low pH values) and testing if the identified iso-α acid stress changes the activity of S. cerevisiae boulardii yeast and ethanol production. Flow cytometry analysis was used for bacterial and yeast cell viability. In relation to the antibacterial activity of β-acids, a lower viability of Pediococcus pentosaceus cells was observed after a short incubation period in selective media, under iso-α acid stress. In beer wort, for a mixed culture with P. pentosaceus bacteria and S. cerevisiae boulardii yeast, under iso-α acid stress conditions at pH 4.0-5.0, Pediococcus pentosaceus exhibited lower cell viability (20.7%) than in selective media (61.4%). Regarding iso-α hop acid on S. cerevisiae boulardii yeast, the results showed that iso-α does not change the S. cerevisiae activity but prevents the culture from being contaminated by Pediococcus pentosaceus. The results highlighted reliable inhibitory effects of iso-α and β-acids against P. pentosaceus, both at pH 6.0-7.0 and pH 4.0-5.0, which open the possibility of hops being used as a supplement to prevent beverage contamination with spoilage microorganisms.
 
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 In press - Online First. Article has been peer reviewed, accepted for publication and published online without pagination. It will receive pagination when the issue will be ready for publishing as a complete number (Volume 47, Issue 4, 2019). The article is searchable and citable by Digital Object Identifier (DOI). DOI link will become active after the article will be included in the complete issue.
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New Alternative of Enhancing Hospital Hygiene Facing Pseudomonas aeruginosa Drug Resistance Impact of Hypertonic Saline Solutions on the Behavior of P. aeruginosa

The objective of this study was to elucidate the antibacterial effect of hypertonic saline of KH2PO4 and NaCl solutions for enhancing hospital hygiene facing drug resistance of the biofilm of Pseudomonas aeruginosa isolated from a hospital environment on an agar cetrimide medium. The variations of P. aeruginosa biofilm density were measured following optical surface density by a correlated imaging method. Inhibition tests of 90% of the biofilm in a selective liquid medium monitored by spectrophotometry showed inhibition rates of 91.2 and 91.1% for minimal biofilm inhibition concentrations of 5.4 and 3.6% for NaCl and KH2PO4, respectively. The results of the eradication tests of 50% of the biofilm showed minimal biofilm eradication concentrations of 6.3 and 3.6% and eradication ratios of 50.6 and 50.9% for NaCl and KH2PO4, respectively. In the case of a selective solid medium, the results exhibited a minimal biofilm inhibition concentration of 4.2 and 3.9%, a biofilm inhibition rate of 90.8 and 90.5%, and ...

Agrobacterium-mediated transformation of Vitis Cv. Monastrell suspension-cultured cells: Determination of critical parameters.

Although some works have explored the transformation of differentiated, embryogenic suspension-cultured cells (SCC) to produce transgenic grapevine plants, to our knowledge this is one of the first reports on the efficient transformation of dedifferentiated Vitis vinifera cv Monastrell SCC. This protocol has been developed using the sonication-assisted Agrobacterium-mediated transformation (SAAT) method. A construct harboring the selectable nptII and the eyfp/IV2 marker genes was used in the study and transformation efficiencies reached over 50 independent transformed SCC per gram of infected cells. Best results were obtained when cells were infected at the exponential phase. A high density plating (500 mg/dish) gave significantly better results. As selective agent, kanamycin was inefficient for the selection of Monastrell transformed SCC since wild type cells were almost insensitive to this antibiotic whereas application of paromomycin resulted in very effective selection. Selected eyfp-expressing microcalli were grown until enough tissue was available to scale up a new transgenic SCC. These transgenic SCC lines were evaluated molecularly and phenotypically demonstrating the presence and integration of both transgenes, the absence of Agrobacterium contamination and the ability of the transformed SCC to grow in highly selective liquid medium. The methodology described here opens the possibility of improving the production of valuable metabolites. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:725-734, 2016.

Effective degradation of organic pollutants in aqueous media by microbial strains isolated from soil of a contaminated industrial site

Bioremediation is a low-cost technology, whose efficacy is often enhanced with preliminary mild physical–chemical remediation methods. A further advantage of bioremediation resides in its eco-compatibility and, thus, sustainability. Two autochthonous microbial strains, Methylobacterium populi VP2 and Aspergillus sydowii VP4, were isolated from a soil of a highly contaminated industrial site and used to degrade the aqueous extract of contaminants (AEC) obtained from the same polluted soil. The AEC incubation with both strains produced a significant removal of most organic pollutants, although the degradation capacity decreased with increasing AEC concentration in the minimal selective liquid medium (MSML) of the experiments. At 30 % of AEC, M. populi VP2 determined the removal of most pollutants and the appearance of new products due to oxidation and enzymatic degradation. Incubation of A. sydowii VP4 at the same AEC concentration in MSML removed the same pollutants but also the derived degradation products. Our results showed that the strains isolated from a highly contaminated soil maintained the capacity to use organic contaminants as metabolic carbon in aqueous extracts from the same soil. The greater biodegradation efficiency of the fungal strain in comparison to M. populi VP2 may be caused by a modification of the A. sydowii VP4 cell surface that increases cell permeability to hydrophobic compounds and thus enhances the extent of pollutants degradation. This work indicates that two specific strains, M. populi VP2 and A. sydowii VP4, isolated from the soil of a highly contaminated site are not only useful in the treatment of leaching polluted waters but may also be used in bioaugmentation practices during remediation of contaminated soils.

Environmental Prevalence of Pathogens in Different Drinking Water Resources in Makkah City (Kingdom of Saudi Arabia)

Water is the most important substance in our daily life. Without it, life would not have been possible. Potable water is essential to humans and other life forms, as water is important to the mechanics of biological metabolisms in the body. Drinking water should be pure and free of contaminants to ensure proper health and wellness. Drinking water from different water resources such as wells and tankers should be free from contamination with waterborne pathogens including bacteria, fungi, viruses and parasites.Treatment of water using many ways is generally done in order to purify it.However, some water treatment techniques may not properly handled. In addition, water transferring techniques may contaminate the drinking water. Therefore, this study was aim toinvestigate drinking water in wells and tankers to observe any microbial pathogen presence as a source of health hazard.One hundred and eightwater samples from different sources were examined for microbial pathogens using filtration method on solid and liquid selective media. Four sources include sea desalinated water(SDW) from governmental water desalination factories, drinkable wells water (DWW), non-drinkable wells water (NDWW) and commercial desalinated water(CDW)from small commercial water desalination factories.Seven DWW samples (58.3%) and five NDWW samples (41.7%) were contaminated with E. coli. Eleven DWW samples (91.7%) and all NDWW samples (100%) were contaminated with P. aeruginosa. One DWW sample (8.3%) and twoNDWW samples (16.7%) were contaminated with E. faecalis. Four DWW samples (33.3%) and one NDWW sample (8.3%) were found contaminated with aspergillus spp. Four SDW samples (100%) and four CDW samples (50%) were contaminated with Penicillium spp. Conclusion:CDWwas found to be the more suitable than other sources for drinking if a biological hazard is the main target. However, contamination at transferring process should be addressed. Yet, water tanker which is a common transferring technique in many areas in Saudi Arabia and should be tested for safety level from point of contamination hazard during the transferring process.

Identification of select fumonisin forming Fusarium species using PCR applications of the polyketide synthase gene and its relationship to fumonisin production in vitro.

A polymerase chain reaction (PCR) based diagnostic assay was used to develop markers for detection of Fusarium verticillioides (=F. moniliforme), a fumonisin producing fungus in maize tissues. Species-specific primers were designed based on sequence data from the polyketide synthase (PKS) gene (FUM1- previously FUM5) responsible for fumonisin production in fungi. Four sets of oligonucleotide primers were tested for their specificity using 24 strains of F. verticillioides, 10 F. proliferatum, and 12 of other Fusarium species. In addition, 13 species of other fungal genera, from four phyla, were tested as negative controls. Among the four sets, primer set B consistently amplified a 419-bp fragment from the DNA 96% of all F. verticillioides strains and 83% of F. proliferatum. All other fungi tested were negative using primer set B. A total of 38% of the F. verticillioides strains grown on a selective liquid medium produced fumonisin and 92% formed the toxin on standard rice medium. When fumonisin formed in culture, PCR assay using primer set B detected every strain of F. verticillioides, but only amplified 80% of F. proliferatum strains that produced the toxin. PCR detection was consistent at 100 pg/microl concentration of genomic DNA from 4 F. verticillioides strains, but varied at 10 pg/microl. Two duplicate greenhouse tests using artificially inoculated maize plants, had greater levels of F. verticillioides detected after re-evaluting using primer set B than from culturing of the tissues. The molecular protocols described in this study requires only 1 day for completion compared to approximately 10 days for cultural work and morphological determination. In conclusion, conventional PCR assay using primer set B provides a sensitive and accurate detection assay that can be used as a primary or secondary confirmation method for identification and occurrence of F. verticillioides within the maize tissues. However, studies using primer set B for fumonisin production determined by strains of F. verticillioides and F. proliferatum will require further verification.

Cloning and characterization of three genes encoding Qb-SNARE proteins in rice

Qb-SNARE proteins belong to the superfamily of SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) and function as important components of the vesicle trafficking machinery in eukaryotic cells. Here, we report three novel plant SNARE (NPSN) genes isolated from rice and named OsNPSN11, OsNPSN12 and OsNPSN13. They have about 70% nucleotide identity over their entire coding regions and similar genomic organization with ten exons and nine introns in each gene. Multiple alignment of deduced amino acid sequences indicate that the OsNPSNs proteins are homologous to AtNPSNs from Arabidopsis, containing a Qb-SNARE domain and a membrane-spanning domain in the C-terminal region. Semi-quantitative RT-PCR assays showed that the OsNPSNs were ubiquitously and differentially expressed in roots, culms, leaves, immature spikes and flowering spikes. The expression of OsNPSNs was significantly activated in rice seedlings treated with H(2)O(2), but down-regulated under NaCl and PEG6000 stresses. Transient expression method in onion epidermal cells revealed that OsNPSNs were located in the plasma membrane. Transformed yeast cells with OsNPSNs had better growth rates than empty-vector transformants when cultured on either solid or liquid selective media containing various concentrations of H(2)O(2), but more sensitive to NaCl and mannitol stresses. The 35S:OsNPSN11 transgenic tobacco also showed more tolerance to H(2)O(2) and sensitivity to NaCl and mannitol than non-transgenic tobacco. These results indicate that OsNPSNs may be involved in different aspects of the signal transduction in plant and yeast responses to abiotic stresses.

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Isolation of Leptospirillum ferriphilum by single-layered solid medium

According to physiological and biochemical characteristics of Leptospirillum ferriphilum, a strain of object bacteria was isolated successfully. Bacteria were enriched by selective liquid medium and plated on designed single-layered agar solid medium. Colony was cultured and bacteria were collected. The morphologies of the object bacteria were observed using crystal violet staining, scanning electron microscope(SEM) and transmission electron microscope (TEM). The result of 16S rDNA identification shows that this bacterium belongs to Leptospirillum ferriphilum and it is named as Leptospirillum ferriphilum strain D1. These results indicate that this new single-layered agar solid medium is efficient and simple for isolation of Leptospirillum ferriphilum. Additionally, physiological-biochemical characteristics show that the optimum initial pH value and its growth temperature are 1.68 and 40 °C, respectively. The culture of it is used to leach a complex concentrate chalocpyrite, the leaching efficiencies of copper and iron are 1.93% and 13.74%, respectively, and it is more effective than the A. ferrooxidans culture in the leaching of the complex concentrate chalcopyrite.

FUNCTIONAL COMPLEMENTATION OF AN ARGININE AUXOTROPHIC YEAST MUTANT BY AN ARGININOSUCCINATE SYNTHETASE FROM PORPHYRA YEZOENSIS (RHODOPHYTA)1

The objective of this study was to develop a tool for studying Porphyra yezoensis Ueda gene function using the yeast transformation system. The gene encoding argininosuccinate synthetase (EC 6345) of P. yezoensis (PyARG1) was obtained by PCR using an expressed sequence tag clone as a template, and subcloned into the yeast expression vector pYES2. The gene was expressed when the vector harboring PyARG1 was introduced into an ARG1‐deficient strain of Saccharomyces cerevisiae, which resulted in successful complementation of the mutant phenotype. The transformed cells could survive on a selective medium lacking arginine, and transcripts of PyARG1 were detected by reverse transcription‐PCR. A quantitative comparison showed that the rescued mutant cells grew in the selective liquid medium with a minor reduction in growth rate relative to wild‐type cells. This is the first report in which the function of a P. yezoensis gene has been directly demonstrated. This technique will provide new opportunities for further investigations into the functions of various genes in P. yezoensis and other macroalgal species.

DNA based detection of blackleg and soft rot disease causing Erwinia strains in seed potatoes

Erwinia carotovora subsp. atroseptica (Eca), Erwinia carotovora subsp. carotovora (Ecc) and Erwinia chrysanthemi (Ech) are the different sub species of Erwinia that cause the diseases commonly known as blackleg, aerial stem rot and soft rot on potato. Blackleg and aerial stem rot affect vines during the growing season, whereas soft rot affects tubers in the field and during transit and storage. The three species can cause soft rot under cool and moist conditions. E. carotovora subsp. atroseptica is the major cause of blackleg, a blackening of the stem base of potato plants, which originates from the mother tuber (Pérombelon and Kelman, 1987). Erwinia carotovora subsp. carotovora mainly causes aerial stem rot (aerial blackleg), but under high temperatures it has been reported to cause blackleg like symptoms. E. chrysanthemi also induces blackleg-like symptoms. Until recently E. chrysanthemi had been mainly confined to warmer climates of Europe, Australia and the tropics. To date the species has been known to occur in cool temperate regions including Finland. In Finland E. chrysanthemi the strain has been reported for the first time in 2004 and it appears to spread fast in certain parts of country (Degefu, unpublished, Pirhonen, personal communication). Although infested crop residues and rotting tubers are among the important sources of inoculum, latent infections in seed tuber provide the major source of infection in potato production (Hannukkala and Segertedt, 2004). At the seed potato laboratory MTT, Ruukki we are carrying out research and services on PCR (DNA) based detection of latent infection of blackleg and soft rot Erwinia strains. The enrichment of the bacteria in semi selective liquid medium prior to PCR ( BIO-PCR) is an important initial step for the success in PCR detection extremely low number of the target bacteria from tubers. The different strains appear to differ in their ability to compete with other saprophytes and reach the target detection limit of bacterial population during the enrichment culture of the potato peel extract. Results of prior PCR enrichment of the bacteria, detection limits of the different strains and preliminary data, from the analysis of some seed lots from the high grade area of north Ostrobothnia and Åland regions, on the occurrence of the strains and the new trends of introduction and spread of E. chrysanthemi in Finland are presented and discussed. Evaluation of the current status of Erwinia diagnostics and areas of future research are highlighted

A simple cultural method for the presumptive detection of the yeasts Brettanomyces/Dekkera in wines

The development of a simple and reliable procedure, compatible with routine use in wineries, for the presumptive detection of Brettanomyces/Dekkera from wine and wine-environment samples. The method of detection of these yeasts employs a selective enrichment medium. The medium contains glucose (10 g l(-1)) as carbon and energy source, cycloheximide (20 mg l(-1)) to prevent growth of Saccharomyces, chloramphenicol (200 mg l(-1)) to prevent growth of bacteria and p-coumaric acid (20 mg l(-1)) as the precursor for the production of 4-ethyl-phenol. After the inoculation with wine, the medium is monitored by visual inspection of turbidity and by periodic olfactive analysis. Contaminated wines will develop visible turbidity in the medium and will produce the 4-ethyl-phenol off-odour, which can be easily detected by smelling. A selective enrichment liquid medium was developed to differentially promote the growth and activity of Brettanomyces/Dekkera. The method is simple to execute, employing a simple-to-prepare medium and a periodic olfactive detection. The characteristics of the procedure make it particularly applicable in a wine-making environment thus presenting important advantages to the wine industry.

Yeast as a Tractable Genetic System for Functional Studies of the Insulin-degrading Enzyme

We have developed yeast as an expression and genetic system for functional studies of the insulin-degrading enzyme (IDE), which cleaves and inactivates certain small peptide molecules, including insulin and the neurotoxic A beta peptide. We show that heterologously expressed rat IDE is enzymatically active, as judged by the ability of IDE-containing yeast extracts to cleave insulin in vitro. We also show that IDE can promote the in vivo production of the yeast a-factor mating pheromone, a function normally attributed to the yeast enzymes Axl1p and Ste23p. However, IDE cannot substitute for the function of Axl1p in promoting haploid axial budding and repressing haploid invasive growth, activities that require an uncharacterized activity of Axl1p. Particulate fractions enriched for Axl1p or Ste23p are incapable of cleaving insulin, suggesting that the functional conservation of these enzymes may not be bidirectionally conserved. We have made practical use of our genetic system to confirm that residues composing the extended zinc metalloprotease motif of M16A family enzymes are required for the enzymatic activity of IDE, Ste23p, and Axl1p. We have determined that IDE and Axl1p both require an intact C terminus for optimal activity. We expect that the tractable genetic system that we have developed will be useful for investigating the enzymatic and structure/function properties of IDE and possibly for the identification of novel IDE alleles having altered substrate specificity.

The Diagnosis and Therapy of Tuberculosis During the Past 100 Years

Methods for the radiographic diagnosis of tuberculosis have improved from simple fluoroscopy to computerized tomography. Although direct smear examination is still the most widely used bacteriological method of diagnosis, cultural methods with selective liquid media are sensitive and rapid. The use of antituberculosis drugs has changed tuberculosis from a disease with about a 50% mortality, treated by measures to collapse the affected lung lesions and by rest for the patient, to a condition successfully curable by chemotherapy. Key steps in the development of modern chemotherapy regimens were the demonstrations in clinical trials that (1) streptomycin was effective; (2) combination of drugs prevented the emergence of drug-resistant Mycobacterium tuberculosis; (3) chemotherapy under domiciliary conditions was effective and did not put family members at risk of infection; (4) patient compliance could be assisted by fully supervised intermittent regimens, or more effectively, by (5) shortening treatment by the introduction of rifampin and pyrazinamide, the two most potent sterilizing drugs, into the regimens. Regimens were divided into an initial intensive phase, while bacterial populations were high, and a longer continuation phase to complete sterilization. Pyrazinamide was shown to sterilize only in the intensive phase. The treatment of nonpulmonary tuberculosis followed the same plan, but when bacterial populations are low, fewer drugs are required in combination.

Population dynamics of Pythium aphanidermatum and response of tomato plants as affected by root-zone temperature

Tomato plants were cultivated in a climate chamber in 12-L-containers with aerated nutrient solution at root-zone temperatures of 20, 25 and 30 °C. Half of the containers were inoculated with oospores of Pythium aphanidermatum. During cultivation, the density of oospores in a sample of the nutrient solution was estimated one, three and five weeks after inoculation, using a haemocytometer, and the numbers of propagules in the nutrient solution and in the roots were also measured, by incubating serial dilutions of samples in a selective liquid medium. Six weeks after inoculation, plants were harvested, and their root and shoot characteristics recorded. With increasing root-zone temperature, the population densities of P. aphanidermatum in the roots and in the nutrient solution increased, while the growth of inoculated plants was reduced. Five weeks after inoculation, the pathogen density at 30 °C was 3.0 · 107 propagules per g dry mass in the roots and 6.6 · 104 propagules l−1 in the nutrient solution. At 20 °C, these quantities were 1.1 · 106 and 1.9 · 104, respectively. A high pathogen density at 30 °C resulted in significantly reduced photosynthesis, transpiration and dry mass of all plant components. Nitrogen and potassium concentration in the leaves decreased, which indicated limitations in the nutrient uptake of severely infected roots. At 20 °C, however, plant characteristics other than transpiration were not affected. It was concluded that the tomato tolerates infections of P. aphani- dermatum at low root-zone temperatures.

Comparison of Oxford Agar, PALCAM and Listeria monocytogenes Blood Agar for the recovery of L. monocytogenes from foods and environmental samples

This work had as the main objective a comparison between Listeria monocytogenes Blood Agar (LMBA) and the conventional selective agar media, Oxford and PALCAM, relative to its efficacy in the detection of L. monocytogenes in naturally contaminated food and environmental samples. 173 environmental samples and 272 samples of foods were analysed. A higher sensitivity for detection of L. monocytogenes was verified for LMBA than for PALCAM and Oxford. In LMBA L. monocytogenes could be distinguished from other Listeria spp. by detection of hemolysis. In Oxford and PALCAM this distinction was not possible. The higher growth rate of L. innocua cf. L. monocytogenes in selective liquid media could result in a high number of false negatives (non-detection of the target organism on plates, although its presence was observed by other tests, eg. mini-VIDAS LMO). The need for specific media for the detection of L. monocytogenes in food was confirmed. LMBA could be an alternative medium to use together with PALCAM or Oxford.

Nontuberculous mycobacteria in patients with cystic fibrosis.

The prevalence and clinical implications of colonization with nontuberculous mycobacteria were prospectively studied in 37 patients who had cystic fibrosis. Sputum samples were cultured on Coletsos and Löwenstein-Jensen selective media after decontamination with sodium hydroxide and oxalic acid. Oxalic acid-decontaminated fractions were also cultured in selective liquid medium. Nontuberculous mycobacteria were isolated from 6 patients (16.1%). Mycobacterium chelonae and Mycobacterium avium-intracellulare complex were the most common species. Three patients with positive results of culture had at least 1 positive result by acid-fast smear. Oxalic acid decontamination and culture in liquid medium had the lowest contamination rate (6.7%). Colonization with nontuberculous mycobacteria was associated with humoral response to mycobacteria (immunoglobulin G titers against antigen A60) in patients with samples that tested positive by acid-fast smear. An improvement in pulmonary function was observed in 2 patients after they received a course of antimycobacterial therapy. Screening for nontuberculous mycobacteria in patients with cystic fibrosis will contribute to understanding the relevance of these pathogens with regard to deterioration of pulmonary function in patients with cystic fibrosis.