Multiplex PCR using 16S rRNA gene-targeted primers for the identification of bifidobacteria from human origin

Three multiplex polymerase chain reactions (PCRs) targeted on Bifidobacterium and related species were designed to identify human species. The selected primers yielded amplified products of various sizes, each specific for a species. Three to four pairs were gathered in one PCR reaction and their specificity under multiplex conditions was confirmed using DNA from 26 reference strains. Using this technique on unidentified faecal strains, B. bifidum, B. longum and B. breve species were commonly recovered in infants while B. adolescentis, B. catenulatum/B. pseudocatenulatum continuum and B. longum species were predominant in adults. Thus, a single PCR can provide the assignment of a strain to one these species, reducing the number of PCR reactions and hands-on time for the identification of human isolates of bifidobacteria. Moreover, this technique is also applicable for the in situ detection of bifidobacteria in DNA extracts from human stools.     

Differentiation of velogenic, mesogenic and lentogenic strains of Newcastle disease virus by multiplex RT-PCR

A multiplex RT‐PCR technique has been developed for differentiation of velogenic, mesogenic and lentogenic pathotypes of Newcastle disease virus (NDV), using a set of three oligonucleotide primers designed from NDV genomic RNA (P1, P2 and P3). The primer pair P1 and P2 generated a RT‐PCR product of 204 bp, only with RNA from velogenic and mesogenic strains, whereas the P1 and P3 generated a 364 bp product only with RNA from mesogenic and lentogenic strains. Thirty four NDV strains, including some reference strains (known pathotypes), NDV field isolates and NDV vaccine strains, as well as other avian virus strains, were tested with multiplex RT‐PCR. All reference strains tested were differentiated in agreement with their intracerebral pathogenicity index (ICPI) values or with the pathotypes known in previous reports. The nucleotide sequence analysis of RT‐PCR products for four NDV strains was fully in agreement with the RT‐PCR characterisations of these strains. The RT‐PCR results of other avian RNA viruses further confirmed the reliability and specificity of this technique. However, the RT‐PCR failed to detect some other avian NDV, which may not originate from chicken. This multiplex RT‐PCR technique is simple and easy to perform. It could be applied not only to determine the origin of NDV, but also may be used diagnostically in molecular epidemiological analysis of ND and for prediction of pathotypes of NDV isolates.     

Use of molecular methods in identification of Candida species and evaluation of fluconazole resistance

The aim of this study was to evaluate the use of one of the molecular typing methods such as PCR (polymerase chain reaction) following by RFLP (restriction fragment length polymorphism) analysis in the identification of Candida species and then to differentiate the identified azole susceptible and resistant Candida albicans strains by using AP-PCR (arbitrarily primed-polymerase chain reaction). The identification of Candida species by PCR and RFLP analysis was based on the size and primary structural variation of rDNA intergenic spacer regions (ITS). Forty-four clinical Candida isolates comprising 5 species were included to the study. The amplification products were digested individually with 3 different restriction enzymes: HaeIII, DdeI, and BfaI. All the isolates tested yielded the expected band patterns by PCR and RFLP analysis. The results obtained from this study demonstrate that Candida species can be differentiated as C. albicans and non-C. albicans strains only by using HaeIII restriction enzyme and BfaI maintains the differentiation of these non-C. albicans species. After identification Candida species with RFLP analysis, C. albicans strains were included to the AP-PCR test. By using AP-PCR, fluconazole susceptible and resistant strains were differentiated. Nine fluconazole susceptible and 24 fluconazole resistant C. albicans were included to the study. Fluconazole resistant strains had more bands when evaluating with the agarose gel electrophoresis but there were no specific discriminatory band patterns to warrant the differentiation of the resistance. The identification of Candida species with the amplification of intergenic spacer region and RFLP analysis is a practical, short, and a reliable method when comparing to the conventional time-consuming Candida species identification methods. The fluconazole susceptibility testing with AP-PCR seems to be a promising method but further studies must be performed for more specific results.     

Identification and differentiation of Lactobacillus, Streptococcus and Bifidobacterium species in fermented milk products with bifidobacteria

The aim of this study was to identify and discriminate bacteria contained in commercial fermented milks with bifidobacteria by the use of amplified ribosomal DNA restriction analysis (ARDRA) and randomly amplified polymorphic DNA (RAPD) techniques. ARDRA of the 16S rDNA gene and RAPD were performed on 13 Lactobacillus strains, 13 Streptococcus and 13 Bifidobacterium strains isolated from commercial fermented milk. Lactobacillus delbrueckii, Streptococcus thermophilus and Bifidobacterium animalis isolates were identified by genus- and species-PCR and also, they were differentiated at genus and species level by ARDRA using MwoI restriction enzyme. The ARDRA technique allowed for the discrimination among these three related genus with the use of only one restriction enzyme, since distinctive profiles were obtained for each genus. Therefore it can be a simple, rapid and useful method for routine identification. Also, RAPD technique allowed the discrimination of all bacteria contained in dairy products, at genus- and strain-level by the performance of one PCR reaction.     

Development of 16S rRNA-gene-targeted group-specific primers for the detection and identification of predominant bacteria in human feces

For the detection and identification of predominant bacteria in human feces, 16S rRNA-gene-targeted group-specific primers for the Bacteroides fragilis group, Bifidobacterium, the Clostridium coccoides group, and Prevotella were designed and evaluated. The specificity of these primers was confirmed by using DNA extracted from 90 species that are commonly found in the human intestinal microflora. The group-specific primers were then used for identification of 300 isolates from feces of six healthy volunteers. The isolates were clearly identified as 117 isolates of the B. fragilis group, 22 isolates of Bifidobacterium, 65 isolates of the C. coccoides group, and 17 isolates of Prevotella, indicating that 74% of the isolates were identified with the four pairs of primers. The remaining 79 isolates were identified by 16S ribosomal DNA sequence analysis and consisted of 40 isolates of Collinsella, 24 isolates of the Clostridium leptum subgroup, and 15 isolates of disparate clusters. In addition, qualitative detection of these bacterial groups was accomplished without cultivation by using DNA extracted from the fecal samples. The goal for this specific PCR technique is to develop a procedure for quantitative detection of these bacterial groups, and a real-time quantitative PCR for detection of Bifidobacterium is now being investigated (T. Requena, J. Burton, T. Matsuki, K. Munro, M. A. Simon, R. Tanaka, K. Watanabe, and G. W. Tannock, Appl. Environ. Microbiol. 68:2420-2427, 2002). Therefore, the approaches used to detect and identify predominant bacteria with the group-specific primers described here should contribute to future studies of the composition and dynamics of the intestinal microflora.     

PCR Analysis of the Tri13 gene to Determine the Genetic Potential of Fusarium graminearum Isolates from Iran to Produce Nivalenol and Deoxynivalenol

Fusarium graminearum trichothecene producing isolates can be broadly divided into two chemotypes based on the production of the 8- ketotrichothecenes deoxynivalenol (DON) and nivalenol (NIV). Functional Tri13 gene required for the production of NIV and 4- acetyl NIV, whereas in the isolates producing DON and its acetylated derivates, this gene is nonfunctional. In this study, a total of 57 isolates from different fields of Mazandaran province, Iran were identified as F. graminearum using classical methods and species specific primers. In order to assess the potential of isolates to produce NIV or DON, we used PCR to determine whether isolates carried a functional or nonfunctional Tri13 gene. Out of the 57 tested F. graminearum isolates with Tri13 PCR assays, 46 yielded an amplicon similar to the size predicted for nivalenol production, while 11 yielded an amplicon similar to the size predicted for deoxynivalenol production. From regions where more than one F. graminearum isolate was obtained, isolates were not exclusively of a single chemotype. It seems that genetic diversity among the isolates has relation with geographical region and wheat cultivar. The assay can provide information about the distribution of Tri13 haplotype that can be used in tracing of trichothecene contaminated samples.     

RAPD Analysis of Pathogenic Variability in Ascochyta rabiei

Thirty Italian isolates of the phytopathogenic fungus Ascochyta rabiei (Pass.) Labr., the causal organism of Ascochyta blight on chickpea (Cicer arietinum L.), were analysed by a random oligonucleotide primer dependent polymerase chain, reaction (PCR) technique called random amplified polymorphic DNA analysis (RAPD) using three decamer primers. In previous investigations these isolates had been differentiated in six pathogenic groups. RAPD results were summarized in an analysis using the program PAUP. With each of the primers several amplification products were observed which were common to all isolates. The results of the RAPD analyses also showed that all isolates could be identified by a unique RAPD pattern. No correlation between RAPD patterns and the division of the isolates in pathogenic groups could be established. The application of the RAPD technique for cataloguing isolates and to obtain specific genetic markers for all isolates of the species Ascochyta rabiei is discussed.     

Phenotypic evaluation to differentiate Candida albicans from Candida dubliniensis

This study evaluated the phenotypic tests used to differentiate Candida albicans from Candida dubliniensis. A total of 55 isolates from vaginal secretions, oral cavity and hemoculture were studied. They were originally identified as C. albicans, based on their morphological and physiological characteristics. These isolates were tested for colony color development on CHROMagar Candida medium, growth at 45 degrees C on Sabouraud Dextrose agar, lipolytic activity on Tween 80 Agar medium and colony morphology and chlamydoconidia formation on Staib agar medium. Of the 55 isolates studied, seven yielded one or more phenotypic characteristics suggestive of Candida dubliniensis. These isolates were tested by PCR with specific primers for Candida dubliniensis and API ID 32. The seven isolates were confirmed as Candida albicans. All of these finding indicate that DNA based tests should be used for definitive identification of Candida dubliniensis.     

Polymorphic restriction patterns of ribosomal internal transcribed spacers in the biocontrol fungus Puccinia carduorum correlate with weed host origin.

The internal transcribed spacer (ITS) regions of ribosomal DNA were amplified by PCR and used to develop genetic markers for isolates of Puccinia carduorum being evaluated for biological control of Carduus thoermeri (musk thistle). Unique patterns were produced upon restriction of ITS DNA amplified from four separate Puccinia spp. Restriction patterns of ITS DNA of isolates of P. carduorum from Carduus acanthoides and C. thoermeri were distinct from those of P. carduorum from Carduus tenuiflorus and Carduus pycnocephalus. By this technique, isolates of P. carduorum from four different weed hosts can be differentiated from other Puccinia spp. and separated into two host groups.     

Use of Random Amplified Polymorphic DNA (RAPD) Analysis and Genetic Fingerprinting to Differentiate Isolates of Race O, C and T of Bipolaris maydis

Isolates of three races of Bipolaris maydis from China (races O, C and T) were compared using two techniques. Random Amplified Polymorphic DNA (RAPD) analysis using 24 primers indicated that race O and C isolates were more similar to one another than to the race T isolate. Twenty of the primers produced RAPD profiles that were similar for the race O and C isolates but differed for the race T isolate (four primers did not amplify products in any of the isolates). Four primers produced profiles which differed for all three races and two of these (A-09 and B-18) clearly differentiated the race O and race C isolates. Genetic fingerprinting of B. maydis using M13 DNA as a probe differentiated race O and C isolates from the race T with all four restriction enzymes used. Furthermore, when DNA was digested with Hind III, the hybridization profiles of the race O and C isolates differed from one another.     

Typing Primus necrotic ringspot virus isolates by serology and restriction endonuclease analysis of PCR products

Primus necrotic ringspot virus (PNRSV) isolates were characterised by bioassays, serotyping and restriction fragment length polymorphism (RFLP) analysis of PCR products. Based on symptoms in host trees and bioassays it was concluded that only one of the 16 tested isolates is severe. The serotyping results demonstrated that by using four different MAbs in TAS-ELISA the tested isolates could be divided into four subgroups, however, the severe isolate could not be singled out. RFLP analysis of PCR products supported the serotyping data but did not differentiate between isolates of the two main serological subgroups. A restriction map, derived from sequence analysis of the PCR products obtained from selected isolates, allowed exact location of the restriction sites within the PCR products of each isolate. A mild isolate with a unique genome structure was identified by both serological and RFLP assays. As far as we are aware, this is the first report on sub-grouping of PNRSV isolates by bioassays, serotyping with MAbs and RFLP analysis.     

Identification procedure for Pasteurella pneumotropica in microbiologic monitoring of laboratory animals.

Discrepancies have been recognized in the identification of Pasteurella pneumotropica between testing laboratories. To determine the causes of the differences and to propose a reliable identification procedure for P. pneumotropica, a working group was organized and 69 isolates identified or suspected as P. pneumotropica were collected from 8 laboratories in Japan. These isolates were examined by colony morphology, Gram-staining, the slide agglutination test using two antisera (ATCC35149 and MaR), two commercially available biochemical test kits (ID test, API20NE) and two primer sets of PCR tests (Wang PCR, CIEA PCR). The 69 isolates and two reference strains were divided into 10 groups by test results. No single procedure for P. pneumotropica identification was found. Among tested isolates, large differences were not observed by colony morphology and Gram-straining except for colony colors that depended on their biotypes. Sixty-eight out of 69 isolates were positive by the slide agglutination test using two antisera except for one isolate that tested with one antiserum. The ID test identified 61 out of 69 isolates as P. pneumotropica and there was no large difference from the results of CIEA PCR. From these results, we recommend the combination of colony observation, Gram-straining, the slide agglutination tests with two antisera and biochemical test using the ID test for practical and reliable identification of this organism.     

Validation of a polymerase chain reaction/restriction enzyme analysis method for species identification of thermophilic campylobacters isolated from domestic and wild animals

AIMS: To compare and evaluate a polymerase chain reaction/restriction enzyme analysis (PCR/REA) method with standard phenotypic tests for the identification and differentiation of the thermophilic campylobacters Campylobacter jejuni, C. coli, C. lari and C. upsaliensis. METHODS AND RESULTS: One hundred and eighty-two presumptive thermophilic campylobacters from 12 different animal species were tested by a recently published PCR/REA and standard phenotypic tests. By PCR/REA, 95% of the isolates were clearly identified as either one of the four thermophilic Campylobacter species or as not belonging to this group of organisms at all. By standard phenotyping, 174 of the 182 isolates were initially identified as either C. jejuni, C. coli, C. lari or C. upsaliensis. Additional genotypic tests and phenotyping showed that 52 of these identifications were either incorrect or unreliable. Of the C. jejuni isolates, 19% were identified as C. coli by initial phenotyping and 27 sheep isolates phenotyped as C. coli or C. lari were, in fact, arcobacters. CONCLUSIONS: The PCR/REA was more reliable than standard phenotyping for the identification of thermophilic campylobacters from different animals. SIGNIFICANCE AND IMPACT OF THE STUDY: Routinely used phenotypic tests often resulted in unreliable identifications, requiring additional testing. The PCR/REA, however, gave unequivocal results and was considered useful for the routine identification of thermophilic campylobacters from different animals.     

Culture-based and non-growth-dependent detection of the Burkholderia cepacia complex in soil environments

Burkholderia cepacia complex (Bcc) bacteria reside in soil, plant rhizospheres, and water, but their prevalence and distribution in outdoor environments is not clear. We sampled a variety of soil and rhizosphere environments with which people may have contact: playgrounds, athletic fields, parks, hiking trails, residential yards, and gardens. A total of 91 sites was sampled in three large U.S. cities. In the first phase of the study, putative Bcc isolates were recovered on Burkholderia cepacia selective agar and trypan blue tetracycline medium and subsequently examined for biochemical reactivity and growth at 32 and 22 degrees C. Isolates were further examined by PCR assays targeting Bcc-specific ribosomal DNA and recA gene sequences. Among the 1,013 bacterial isolates examined, 68 were identified as Bcc; 14 (15%) of 91 sampled sites yielded Bcc isolates. In the second phase, DNA was extracted directly from soil samples and examined with PCR assays targeting Bcc 16S rRNA gene sequences. Either 82 or 93% of the soil samples were positive for at least one Bcc genomovar, depending on the PCR assay system used. Cloning and sequencing were performed to check the specificity of the PCR assays. Sequence analysis of the 463-bp 16S rRNA inserts from eight clones indicated that all were from members of the Bcc. The four soil samples from which these clones were generated did not yield isolates identified as Bcc. Based on PCR detection, Bcc appears to be prevalent in soil from urban and suburban environments. Culture-based recovery of Bcc may underestimate environmental populations.     

Speciating Campylobacter jejuni and Campylobacter coli isolates from poultry and humans using six PCR-based assays

Six previously published polymerase chain reaction (PCR) assays each targeting different genes were used to speciate 116 isolates previously identified as Campylobacter jejuni using routine microbiological techniques. Of the 116 isolates, 84 were of poultry origin and 32 of human origin. The six PCR assays confirmed the species identities of 31 of 32 (97%) human isolates and 56 of 84 (67%) poultry isolates as C. jejuni. Twenty eight of 84 (33%) poultry isolates were identified as Campylobacter coli and the remaining human isolate was tentatively identified as Campylobacter upsaliensis based on the degree of similarity of 16S rRNA gene sequences. Four of six published PCR assays showed 100% concordance in their ability to speciate 113 of the 116 (97.4%) isolates; two assays failed to generate a PCR product with four to 10 isolates. A C. coli-specific PCR identified all 28 hippuricase gene (hipO)-negative poultry isolates as C. coli although three isolates confirmed to be C. jejuni by the remaining five assays were also positive in this assay. A PCR-restriction fragment length polymorphism assay based on the 16S rRNA gene was developed, which contrary to the results of the six PCR-based assays, identified 28 of 29 hipO-negative isolates as C. jejuni. DNA sequence analysis of 16S rRNA genes from four hipO-negative poultry isolates showed they were almost identical to the C. jejuni type strain 16S rRNA sequences ATCC43431 and ATCC33560 indicating that assays reliant on 16S rRNA sequence may not be suitable for the differentiation of these two species.     

Identification and genetic fingerprinting of Brachyspira species

Six Brachyspira type and reference strains, and 14 well characterized porcine field isolates representing all recognised porcine Brachyspira spp. were compared by different molecular methods. Sequence analysis of the 16S rRNA and the nox genes, pulsed-field gel electrophoresis (PFGE) and randomly amplified polymorphic DNA (RAPD) were used in the study. In addition the isolates were analysed by five species-specific PCR systems. The topologies of the dendrograms obtained from each of the four typing systems were different. The B. pilosicoli isolates formed monophyletic clusters in all dendrograms, but with different sister lines. All five porcine Brachyspira species formed monophyletic clusters in the nox gene-based dendrogram only. All five PCR systems accurately identified their targets, except for the nox gene-based B. intermedia-specific system, by which it was not possible to identify one of the presumed B. intermedia isolates, and the other B. intermedia-specific system, based on the 23S rRNA gene, gave a positive reaction for one B. innocens isolate. In an extended study, 46 additional isolates and the original eight isolates with the phenotypes of B. hyodysenteriae or B. intermedia were compared by PFGE and PCR. The PFGE results indicated a high genetic diversity of isolates with the phenotype of B. intermedia. Thirty-three of 34 tested isolates could be identified by one or both of the two B. intermedia-specific PCR systems used, however, only 19 of the 34 isolates were positive in both systems.     

Molecular characterization of Leptospira sp. strains isolated from human subjects in São Paulo, Brazil using a polymerase chain reaction-based assay: a public health tool

A polymerase chain reaction (PCR)-based assay which amplifies repetitive DNA elements present within bacterial genomes was used to characterize and differentiate Leptospira sp. Thirty-five strains from a reference culture collection and 18 clinical isolates which had been previously analyzed by cross agglutinin absorption test (CAAT) were evaluated by this technique. PCR results from analysis of the reference culture collection showed no bands corresponding to serogroups Australis, Autumnalis, Bataviae, Celledoni, Cynopteri, Djasiman, Panama, Pomona, Pyrogenes, and Tarassovi. However, the PCR method was able to clearly discriminate the serogroups Andamana, Ballum, Canicola, Grippotyphosa, Hebdomadis, Icterohaemorrhagiae, Javanica, Sejroe, Semaranga, and Shermani. Clinical isolates previously characterized by CAAT as serovar Copenhageni, serovar Castellonis, and as serovar Canicola were in agreement with PCR results. The clinical isolate previously characterized as serovar Pomona was not differentiated by PCR. Forty additional clinical isolates from patients with leptospirosis obtained in S?o Paulo, Brazil were also evaluated by this PCR method. Thirty-nine of these were determined to belong to serogroup Icterohaemorrhagiae (97.5%) and one to serogroup Sejroe (2.5%). These results demonstrate that the PCR method described in this study has utility for rapid typing of Leptospira sp. at the serogroup level and can be used in epidemiological survey.     

Evaluation of molecular techniques to biotype Giardia duodenalis collected during an outbreak

Twenty-seven Giardia duodenalis cyst-positive specimens (human, animal, or drinking water) were obtained from a waterborne outbreak in a community in British Columbia, western Canada. Parasite isolates were characterized using molecular techniques at 4 different steps of organism retrieval. None of the drinking water samples (n = 20) infected gerbils and none was successfully amplified using polymerase chain reaction (PCR). We were able to genotype 4 of 7 (human and animal) isolates by amplification of DNA from original specimens at the triosephosphate isomerase (tpi) gene locus using PCR followed by restriction fragment length polymorphism (RFLP) analysis. Five of the original specimens inoculated into Mongolian gerbils (Meriones unguiculatus) were infective and genotyped at the tpi locus using parasite material collected from the gerbil (cysts and trophozoites). Pulsed field gel electrophoresis (PFGE) was used to biotype trophozoites collected from the gerbils as well as trophozoites from the 4 isolates that adapted to culture. Four of these 5 isolates displayed the same (designated outbreak) biotype at all parasite retrieval steps with all molecular techniques including the originally amplified isolates. PCR-RFLP identified an additional biotype group. The 4 isolates that adapted to in vitro culture were also characterized by isoenzyme electrophoresis (IE). Biotype groups identified in these axenized isolates were all the same with each molecular technique (PCR-RFLP, PFGE, IE) tested. Results of this study demonstrate a need for more sensitive molecular methods to detect and characterize Giardia in original host and environmental samples. Results are also consistent with evidence of biotype changes that occur during the presently used process of isolate retrieval.     

Specific and sensitive detection of Ralstonia solanacearum in soil on the basis of PCR amplification of fliC fragments

Ralstonia solanacearum is the causative agent of bacterial wilt in many important crops. A specific and sensitive PCR detection method that uses primers targeting the gene coding for the flagella subunit, fliC, was established. Based on the first fliC gene sequence of R. solanacearum strain K60 available at GenBank, the Ral_fliC PCR primer system was designed; this system yielded a single 724-bp product with the DNAs of all of the R. solanacearum strains tested. However, R. pickettii and four environmental Ralstonia isolates also yielded amplicons. The Ral_fliC PCR products obtained with 12 strains (R. solanacearum, R. pickettii, and environmental isolates) were sequenced. By sequence alignment, Rsol_fliC primers specific for R. solanacearum were designed. With this primer system, a specific 400-bp PCR product was obtained from all 82 strains of R. solanacearum tested. Six strains of R. pickettii and several closely related environmental isolates yielded no PCR product; however, a product was obtained with one Pseudomonas syzygii strain. A GC-clamped 400-bp fliC product could be separated in denaturing gradient gels and allowed us to distinguish P. syzygii from R. solanacearum. The Rsol_fliC PCR system was applied to detect R. solanacearum in soil. PCR amplification, followed by Southern blot hybridization, allowed us to detect about one target DNA molecule per PCR, which is equivalent to 10(3) CFU g of bulk soil(-1). The system was applied to survey soils from different geographic origins for the presence of R. solanacearum.     

Non-tuberculous mycobacteria I: one year clinical isolates identification in Tertiary Hospital Aids Reference Center,Rio de Janeiro,Brazil, in pre highly active antiretroviral therapy era

The aim of this study was to determine the prevalence of non-tuberculous mycobacteria (NTM) isolates at University Hospital, Reference Center for Aids in Rio de Janeiro, Brazil, during one year. We used standard biochemical tests for species identification and IS1245 PCR amplification was applied as a Mycobacterium avium specific identification marker. Four hundred and four specimens from 233 patients yielded acid-fast bacilli growth. M. tuberculosis was identified in 85% of the patients and NTM in 15%. NTM disseminated infection was a common event correlated with human immunodeficiency virus (HIV) infected patients and only in HIV negative patients the source of NTM was non sterile site. M. avium complex (MAC) was biochemically identified in 57.8% (49/83) of NTM isolates, most of them from sterile sites (75.5%), and in 94% (46/49) the IS 1245 marker specific for M. avium was present. Twenty NTM strains showed a MAC biochemical pattern with the exception of a urease-positive (99% of MAC are urease-negative), however IS1245 was detected in 96% of the strains leading to their identification as M. avium. In this group differences in NTM source was not significant. The second most frequently isolated NTM was identified as M. scrofulaceum (7.2%), followed by M. terrae (3.6%), M. gordonae (2.4%), M. chelonae (1.2%), M. fortuitum (1.2%) and one strain which could not be identified. All were IS1245 negative except for one strain identified as M. scrofulaceum. It is interesting to note that non-sterile sites were the major source of these isolates (92.8%). Our finding indicated that M. avium is still the major atypical species among in the MAC isolates recovered from Brazilian Aids patients without highty active antiretroviral therapy schema. Some discrepancies were seen between the identification methods and further investigations must be done to better characterize NTM isolates using other phenotypic and genotypic methods.