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M I Romano

Publications and source records attributed to M I Romano.

At least 19 recordsLinked to original sources
PubMed2006

Lpp34, a novel putative lipoprotein from Mycobacterium avium subsp. paratuberculosis.

A Mycobacterium avium subsp. paratuberculosis expression library in lambda ZAP was screened with immunized mice sera. One clone was selected, sequenced and further characterized. The sequence analysis of the hypothetical open-reading frame (ORF) predicts a protein of 20.8 kDa with a probable signal sequence compatible with Cys-acylation at Cys24, characteristic of lipoproteins. In consequence, the protein was termed Lpp34. Recombinant expression of Lpp34 was achieved by cloning the lpp34 gene into the histidine-tag expression vector pRSET-A. Western blot analysis showed a protein band with a molecular weight of 34 kDa. The native protein was localized in the membrane fraction of M. avium subsp. paratuberculosis and extracted in the detergent phase of Triton X-114. Southern blot and polymerase chain reaction showed that the gene is absent from all the non-M. avium complex mycobacterial genomes tested. Humoral reactivity using bovine sera demonstrated that this protein is widely recognized by both the infected and non-infected animals. This could partly be due to the conserved sequence in close-related environmental bacteria such as M. avium subsp. avium and to the presence of a conserved epitope in other bacteria such as Escherichia coli. In conclusion, these findings show that Lpp34 is a membrane protein and a putative lipoprotein present in M. avium complex mycobacteria and absent in the M. tuberculosis complex.

Animals↗
PubMed2005

Further analysis of VNTR and MIRU in the genome of Mycobacterium avium complex, and application to molecular epidemiology of isolates from South America.

All members of Mycobacterium avium complex are serious pathogens for humans and animals. The aim of this study was to look for and analyze VNTR-MIRU loci in the genome of M. avium complex and their preliminary application to test these isolates. In the present study, we identified 22 novel VNTR-MIRU by using Tandem Repeat software: five with a structure similar to MIRU and 17 without MIRU structure; these latter were designated as VNTR. Most VNTR were located within predicted coding regions. Most MIRU were intercistronic with their extremities overlapping the termination and initiation codons of their flanking genes. Some of these VNTR-MIRU exhibited polymorphism among M. avium complex isolates due to insertion or deletion of whole repeats and/or of nucleotide sequence degeneration. We determined the variability of six VNTR-MIRU loci in 21 M. avium subsp. hominissuis and 26 M. avium subsp. paratuberculosis. The analysis identified 15 different alleles with the combination of six VNTR-MIRU in the 21 M. avium subsp. hominissuis with 16 different IS1245 RFLP and four different profiles with PCR-restriction analysis of hsp65 (PRA). However, neither the six VNTR-MIRU loci nor the PRA were able to distinguish M. avium subsp. paratuberculosis isolates with five different IS900 RFLP profiles. In conclusion, some of the VNTR-MIRU loci identified were useful to differentiate M. avium subsp. hominissuis but not M. avium subsp. paratuberculosis isolates here included. However, we observed polymorphism in VNTR-MIRU loci between M. avium subsp. hominissuis and M. avium subsp. paratuberculosis genomes, which could be important in the understanding of the obvious differences in the pathogenic effects of these mycobacteria.

Animals↗
PubMed2005

Mutation in the P36 gene of Mycobacterium bovis provokes attenuation of the bacillus in a mouse model.

P36 is a member of a family of secreted proteins distributed throughout the genus Mycobacterium. The central domain of these proteins contains several amino acid PGLTS repeats, which differ considerably between species. P36, also called exported repetitive protein (Erp) in M. tuberculosis, has been shown to be associated with virulence since the disruption of its gene impaired multiplication of both virulent M. tuberculosis and M. bovis BCG in cultured macrophages and immunocompetent mice. In order to demonstrate that P36 is a putative virulence factor of wild-type Mycobacterium bovis we generated a P36 mutant by gene disruption and we evaluated its replication in spleen and lungs of infected mice. In this study, the mutant strain displays low levels of multiplication in mice, indicating that the P36 gene is important for in vivo growth of M. bovis.

Animals↗
PubMed2004

Non-O157 Shiga toxin-producing Escherichia coli isolated from diarrhoeic calves in Argentina.

Faecal samples from 76 diarrhoeic calves belonging to 36 farms located in the Pampas plain, Argentina, were examined for Shiga toxin-producing Escherichia coli (STEC). A total of 15 STEC strains were isolated from 12 (15.8%) calves which came from six different farms. All stx positive strains assayed by PCR were also positives in the Vero cell cytotoxicity test. The majority (60.0%) of the STEC strains carried the stx(1) gene. Twelve (80.0%) of the STEC isolates which belonged to serotypes O5:H- (n = 4), O26:H11 (n = 4), O26:H- (n = 1), O111:H- (n = 2), and O123:H38 (n = 1) were also enterohaemolysin (EHly) positive and carried the gene encoding for intimin (eae). All the stx positive strains were negative for the bfpA gene. Localized adherence to HEp-2 cells were observed in 83.3% of the eae+ STEC strains. STEC belonging to serotype O5:H- showed atypical biochemical properties, including urease production. Urease was also produced by two strains belonging to serotypes O153:H? and non-typeable, respectively. Resistance to three or more antibiotics was observed in 12 (80.0%) of the STEC isolates. Most of the serotypes of STEC recovered in this survey carried virulence traits that are associated with increased human and bovine pathogenicity. The present study shows that highly virulent STEC strains are being shed by diarrhoeic calves from farms located in a high incidence area of human STEC infections.

Animals↗
PubMed2002

Detection of Shiga toxin-producing Escherichia coli by PCR in cattle in Argentina. Evaluation of two procedures.

Different experimental approaches were evaluated for their ability to detect stx genes by PCR and identify Shiga toxin-producing Escherichia coli (STEC) in bovine fecal samples. One hundred and sixty fecal samples from steers in Argentina were processed by protocols that involved: (1) enrichment of fecal samples and DNA extraction using a commercially available kit (Protocol A); (2) plating on selective media after enrichment of the fecal sample followed by heat-lysis DNA extraction from the confluent growth zone (Protocol B); (3) analysis of individual colonies isolated from direct fecal culture on MacConkey agar and sorbitol MacConkey agar supplemented with cefixime and potassium tellurite (Protocol C), used as Gold Standard. PCR performed on bacteria from the confluent growth zone (Protocol B) proved to be the most sensitive methodology. In addition, enrichment for greater than 6h, enhanced sensitivity. Among eight STEC isolates, four were O8:H19 and four were stx2/eae-negative. An STEC isolate was characterized as O26:H11 with a stx1/eae/EHEC-hlyA genotype, often associated with human disease. Finally, no STEC O157 strains were isolated using these methods.

Animals↗
PubMed2001

Characterization of P55, a multidrug efflux pump in Mycobacterium bovis and Mycobacterium tuberculosis.

The Mycobacterium bovis P55 gene, located downstream from the gene that encodes the immunogenic lipoprotein P27, has been characterized. The gene was identical to the open reading frame of the Rv1410c gene in the genome of Mycobacterium tuberculosis H37Rv, annotated as a probable drug efflux protein. Genes similar to P55 were present in all species of the M. tuberculosis complex and other mycobacteria such as Mycobacterium leprae and Mycobacterium avium. By Western blotting, P55 was located in the membrane fraction of M. bovis. When transformed into Mycobacterium smegmatis after cloning, P55 conferred aminoglycoside and tetracycline resistance. The levels of resistance to streptomycin and tetracycline conferred by P55 were decreased in the presence of the protonophore carbonyl cyanide m-chlorophenylhydrazone and the pump inhibitors verapamil and reserpine. M. smegmatis cells expressing the plasmid-encoded P55 accumulated less tetracycline than the control cells. We conclude that P55 is a membrane protein implicated in aminoglycoside and tetracycline efflux in mycobacteria.

Amino Acid Motifs↗
PubMed2001

Sequence analysis of the direct repeat region in Mycobacterium bovis.

Spoligotyping is a major tool for molecular typing of Mycobacterium bovis. This technique is based on the polymorphism of spacers that separate direct repeats (DRs) in the M. tuberculosis complex DR region. Numerous M. bovis strains show a lack of several spacers which appears as a gap in the spoligotyping pattern. To determine whether these gaps contain alternative spacers not included in the spoligotyping membrane, PCRs using primers that hybridize to the spacers adjacent to the gaps were performed. Comparing the sizes of products obtained by PCR with those deduced from spoligotyping patterns, fragments were selected and sequenced to look for alternative spacers. Upon analysis of the sequences, five alternative spacers were detected, although deletions of spacers are mainly responsible for the observed gaps. The alternative spacers, which are more frequent in M. bovis than in M. tuberculosis, may contribute to increased M. bovis differentiation.

Animals↗
PubMed2001

Mycobacterium bovis: polymerase chain reaction identification in bovine lymphonode biopsies and genotyping in isolates from Southeast Brazil by spolygotyping and restriction fragment length polymorphism.

Diagnosis of the Mycobacterium tuberculosis complex by direct PCR of mediastinal lymphnode DNA and microbiological tests were compared in cattle suspicious of bearing tuberculous-like lesions detected during slaughter. The PCR procedure applied on DNA samples (n=54) obtained by adding alpha -casein into the thiocyanate extraction mix was positive in 70% of the samples. PCR confirmed the identification of 23 samples (100%) that grew in culture, 9 samples (60%) that failed to grow in culture, plus 6 (37.5%) samples that resulted in growth of bacterial contaminants. Genotyping by IS6110-RFLP and DR-spoligotyping analysis of seven samples revealed the presence of several polimorphisms. Seven of the isolates contained multiple copies of IS6110, thus defining the existence of five singular genotypes.

Animals↗
PubMed2001

PCR-restriction fragment length polymorphism analysis (PRA) of Mycobacterium leprae from human lepromas and from a natural case of an armadillo of Corrientes, Argentina.

Polymerase chain reaction (PCR)-restriction fragment length polymorphism analysis (PRA) which relies on the amplification of a 439-bp portion of the hsp65 gene present in all mycobacteria, followed by two distinct digestions (with BstEII and HaeIII) of the PCR product, offers a rapid and easy alternative that allows identification of the species without the need for specialized equipment. Wild leprosy in the nine-banded armadillo (Dasypus novemcinctus) is characterized by the presence of multiple bacilli in internal organs such as lymph nodes, spleen and liver, as well as in nerves and skin. We could observe this in 9 out of 132 animals captured in Corrientes, Argentina, an area endemic for leprosy in humans. Mycobacterium leprae were recognized in those naturally infected animals through different techniques. Three samples of extracted DNA of the mycobacteria present in the spleen, liver and popliteal lymph node of a naturally infected animal during the Experimental Program in Armadillo (PEA) and three samples of human lepromas were processed by PRA. The patterns of the six samples analyzed were identical and were characteristic of M. leprae. These studies, made for the first time in Argentina, corroborate the initial discoveries in South America made by our investigative group on the detection of armadillos naturally infected with the Hansen bacillus.

Animals↗
PubMed2000

The gene encoding P27 lipoprotein and a putative antibiotic-resistance gene form an operon in Mycobacterium tuberculosis and Mycobacterium bovis.

P27 is an antigenic membrane lipoprotein synthesized by members of the Mycobacterium tuberculosis complex. Northern blotting and RT-PCR experiments indicated that the genes encoding P27 and a putative antibiotic transporter (P55) form an operon. A promoter region was identified and characterized by deletion analysis in Mycobacterium smegmatis. Two transcription initiation points were mapped in Mycobacterium bovis BCG by primer extension analysis to 76 bp and 87 bp upstream of the ATG initiation codon. Putative -10 and -35 promoter consensus sequences associated with these showed 66% similarity to previously identified mycobacterial promoters. These results suggest that the P27/P55 operon is transcribed from two promoters in M. bovis BCG.

Drug Resistance, Microbial↗
PubMed2000

Evaluation of the polymerase chain reaction for the detection of Mycobacterium tuberculosis.

The development of nucleic acid-based technologies has improved the sensitivity, specificity and speed of detection of Mycobacterium tuberculosis in clinical samples. Both commercially available and 'in-house' polymerase chain reaction (PCR) systems are in use, and a significant number of reports compare such systems with more traditional diagnostic tools for tuberculosis. Few studies, however, have focused on the reproducibility of the results when submitting a sample batch to PCR in different laboratories, especially in developing countries. Consequently, PCR results obtained from six laboratories in six different Latin American countries for samples reconstituted with defined amounts of M. tuberculosis cells were evaluated. Each laboratory used specific conditions of sample processing, nucleic acid amplification and amplicon detection. Analysis of results allowed large differences in sensitivity and specificity to be observed. We conclude that in its present setting, in-house PCR cannot be used as a single diagnostic tool for tuberculosis, and that special care needs to be taken upon interpretation of results by inclusion of a proper number of positive and negative controls.

Evaluation Studies as Topic↗
PubMed1999

Antigenic characterization of mycobacteria from South American wild seals.

Tuberculosis-producing mycobacteria have been previously described in marine mammals (Cousins et al., 1990, 1993; Romano et al., 1995; Bernardelli et al., 1996). The strains belonged to the M. tuberculosis complex (M. tuberculosis, M. bovis, M. microti and M. africanum), but showed genetic and biochemical differences. The antigenic composition of mycobacteria isolated from wild seals was analyzed by Western blots, using antibodies against some selected antigens. The antigenic content was compared with that of M. bovis, M. tuberculosis and M. microti isolates. The lack of Hsp65 protein in supernatants suggested a low degree of cell lysis in the three-week cultures used. SOD, P27 lipoprotein, MPB64 and antigen 85 were observed in all the strains studied. The wild seal strains, as well as M. tuberculosis, did not produce MPB70 and MPB83. Only very weak bands of P36 antigen were observed in culture supernatants from wild seal mycobacteria. Summarizing, the antigenic composition of mycobacterial strains from wild seals is different from M. bovis strains.

Animals↗
PubMed1999

Molecular characterization of mycobacteria isolated from seals.

Tuberculosis (TB) was diagnosed in 10 seals from three species (Arctocephalus australis, Arctocephalus tropicalis and Otaria flavescens) found in South America. The mycobacteria isolated from these cases belonged to the Mycobacterium tuberculosis complex, as determined by RFLP using an IS6110 probe, spoligotyping, analysis of the 16S rRNA gene sequence and by PCR-restriction analysis of hsp65. Polymorphisms in gyrA, katG, oxyR and pncA were investigated in some of the isolates, as well as the presence of the MPB70 antigen. The insertion sequence IS6110 was present in three to seven copies in the genome of the mycobacteria isolated from seals. Using the IS6110 probe, six patterns (designated A, B, C, D, E and F) were identified from 10 different isolates. Patterns A and B were found for the mycobacteria isolated from two and four seals, respectively, indicating an epidemiological relationship between isolates grouped according to their IS6110 RFLP. The mycobacteria isolated from seals shared the majority of their IS6110 DNA-containing restriction fragments, and nine isolates had an identical spoligotype; only one isolate showed a minor difference in its spoligotype. In addition, none of these spoligotypes were found in other M. tuberculosis complex strains. These results suggest that the isolates from seals constitute a unique group of closely related strains. The mycobacteria isolated from seals showed polymorphisms at gyrA codon 95 and katG codon 463, as do group 1 M. tuberculosis, and M. bovis. Group 1 mycobacteria are associated with cluster cases. The spoligotypes found in the mycobacteria isolated from seals lack spacers 39-43, as does M. bovis, but the MPB70 antigen, which is highly expressed in M. bovis and minimally expressed in M. tuberculosis, was not detected in these mycobacteria. The mycobacteria isolated from seals also showed oxyR and pncA polymorphisms specific to M. tuberculosis. In conclusion, the mycobacteria that cause TB in seals in the South-Western Atlantic are a related group, and based on the combination of genetic characteristics, belong to a unique genotypic group within the M. tuberculosis complex.

Animals↗
PubMed1999

Usefulness of spoligotyping in molecular epidemiology of Mycobacterium bovis-related infections in South America.

Two hundred twenty-four Mycobacterium bovis isolates, mainly from South American countries, were typed by spoligotyping, and 41 different spoligotypes were identified. A total of 202 M. bovis isolates (90%) were grouped into 19 different clusters. The largest cluster contained 96 isolates (42.8%) on the basis of the most frequently observed spoligotype, spoligotype 34. Nineteen M. bovis isolates from humans in Argentina had spoligotypes and polymorphic GC-rich repetitive sequence (PGRS) types that represented the most common types found among isolates from cattle. All five isolates from Uruguay and three of the six isolates from Paraguay had spoligotypes that were also detected for isolates from Argentina. The spoligotypes of isolates from Brazil, Costa Rica, and Mexico and of some of the isolates from Paraguay could not be found in Argentina. A total of 154 M. bovis isolates were selected in order to compare the discriminative power of spoligotyping and restriction fragment length polymorphism (RFLP) analysis with direct repeat (DR) and PGRS probes. By spoligotyping, 31 different types were found, while AluI-digested DR probe-associated RFLP analysis identified 42 types, and RFLP analysis with the PGRS probe also detected 42 types; these were partly independent of the DR types. By combining the results obtained by spoligotyping and by RFLP analysis with the DR and PGRS probes, 88 different types were obtained. Although the differentiation of M. bovis by spoligotyping was less discriminatory than differentiation by RFLP analysis with the DR and PGRS probes, spoligotyping is easier to perform and its results are easier to interpret. Therefore, for the purpose of typing of M. bovis isolates, spoligotyping could be performed first and the isolates could be grouped into clusters and then analyzed by RFLP analysis with the DR and PGRS probes.

Animals↗
PubMed1999

The IS6110 restriction fragment length polymorphism in particular multidrug-resistant Mycobacterium tuberculosis strains may evolve too fast for reliable use in outbreak investigation.

To study possible nosocomial transmission of multidrug-resistant (MDR) Mycobacterium tuberculosis, strain types and other information on 24, mostly human immunodeficiency virus-positive patients, were collected. Isolates from 11 patients had identical IS6110 restriction fragment length polymorphism (RFLP) patterns as well as spoligotype patterns and resistance profiles. Noticeably, nine other isolates from related cases also exhibited identical spoligotypes but slightly different RFLP patterns. These results indicate that for some MDR strains, the evolutionary clock of IS6110 RFLP may run too fast for reliable interpretation of strain typing results over a period of a few years.

AIDS-Related Opportunistic Infections↗
PubMed1999

Amplification of a 500-base-pair fragment from cultured isolates of Mycobacterium bovis.

The presence of a 500-bp fragment which amplifies a region from the genome of Mycobacterium bovis (J. G. Rodriguez, G. A. Meija, P. Del Portillo, M. E. Patarroyo, and L. A. Murillo, Microbiology 141:2131-2138, 1995) was evaluated by carrying out PCR on 121 M. bovis isolates. The M. bovis strains, previously characterized by culture and biochemical tests, were isolated from cattle in different regions of Argentina, Mexico, and Colombia. Four additional strains isolated from sea lions that belong to the M. tuberculosis complex were also included in the study. All of the isolates tested were PCR positive, rendering the expected 500-bp band and giving a correlation of 100% with previous microbiological characterization. Southern blot analysis revealed a common band of 1, 800 bp and a polymorphic high-molecular-mass hybridization pattern. The results show that this assay may be useful for diagnosis and identification of M. bovis in cattle.

Animals↗
PubMed1999

Expression of the Mycobacterium bovis P36 gene in Mycobacterium smegmatis and the baculovirus/insect cell system.

In the present study we evaluated different systems for the expression of mycobacterial antigen P36 secreted by Mycobacterium bovis. P36 was detected by Western blot using a specific antiserum. The P36 gene was initially expressed in E. coli, under the control of the T7 promoter, but severe proteolysis prevented its purification. We then tried to express P36 in M. smegmatis and insect cells. For M. smegmatis, we used three different plasmid vectors differing in copy number and in the presence of a promoter for expression of heterologous proteins. P36 was detected in the cell extract and culture supernatant in both expression systems and was recognized by sera from M. bovis-infected cattle. To compare the expression level and compartmentalization, the MPB70 antigen was also expressed. The highest production was reached in insect cell supernatants. In conclusion, M. smegmatis and especially the baculovirus expression system are good choices for the production of proteins from pathogenic mycobacteria for the development of mycobacterial vaccines and diagnostic reagents.

Animals↗