Extended safety studies of the attenuated live tuberculosis vaccine SO2 based on phoP mutant.

Safety is one of the main concerns for attenuated live vaccine candidates. Here we extend the stability and attenuation studies of the promising tuberculosis vaccine candidate based on Mycobacterium tuberculosis phoP mutant strain, SO2. Stability of the phoP mutation was tested after sub-culturing SO2 strain for 6 months in laboratory media and also after 3 months of infection in SCID mice. Results showed no reversion of the phoP mutation either in vitro or in vivo. In addition, SO2 was fully sensitive to four major first-line antituberculous drugs against tuberculosis. Safety and toxicity studies were performed in guinea pigs. Animals were infected with a quantity of SO2 equivalent to 50 vaccination doses (2.5x10(6) CFUs) and weight was monitored for 6 months. All animals survived and no histological lesions were found, showing full attenuation of SO2. Studies in a post-exposure model of guinea pigs and mice, previously infected with M. tuberculosis, were performed and no toxicity effects were found after inoculation of SO2. All these results together confirm that SO2 has a secure safety profile that encourages its use in clinical trials.

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.

Molecular cloning and characterization of Tap, a putative multidrug efflux pump present in Mycobacterium fortuitum and Mycobacterium tuberculosis.

A recombinant plasmid isolated from a Mycobacterium fortuitum genomic library by selection for gentamicin and 2-N'-ethylnetilmicin resistance conferred low-level aminoglycoside and tetracycline resistance when introduced into M. smegmatis. Further characterization of this plasmid allowed the identification of the M. fortuitum tap gene. A homologous gene in the M. tuberculosis H37Rv genome has been identified. The M. tuberculosis tap gene (Rv1258 in the annotated sequence of the M. tuberculosis genome) was cloned and conferred low-level resistance to tetracycline when introduced into M. smegmatis. The sequences of the putative Tap proteins showed 20 to 30% amino acid identity to membrane efflux pumps of the major facilitator superfamily (MFS), mainly tetracycline and macrolide efflux pumps, and to other proteins of unknown function but with similar antibiotic resistance patterns. Approximately 12 transmembrane regions and different sequence motifs characteristic of the MFS proteins also were detected. In the presence of the protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP), the levels of resistance to antibiotics conferred by plasmids containing the tap genes were decreased. When tetracycline accumulation experiments were carried out with the M. fortuitum tap gene, the level of tetracycline accumulation was lower than that in control cells but was independent of the presence of CCCP. We conclude that the Tap proteins of the opportunistic organism M. fortuitum and the important pathogen M. tuberculosis are probably proton-dependent efflux pumps, although we cannot exclude the possibility that they act as regulatory proteins.

The molecular epidemiology of tuberculosis in Zaragoza, Spain: a retrospective epidemiological study in 1993.

SETTING: The incidence of tuberculosis (TB) in Spain is one of the highest in Europe. In Zaragoza region the incidence rate of tuberculosis and the acquired immune deficiency syndrome (AIDS) are close to the national average. OBJECTIVE: To better define the molecular epidemiology of tuberculosis in an area of Europe where this has not been previously studied. DESIGN: A retrospective epidemiological study on tuberculosis was conducted in Zaragoza, a region of Spain, in 1993. The study population consisted of 226 patients from whom positive culture and complete clinical and demographic data were available. Mycobacterium tuberculosis strains were typed by standard restriction fragment length polymorphism (RFLP). A cluster was defined as two or more isolates with identical RFLP patterns when five or more copies of IS6110 are present. The 137 non-clustered patients were compared with the 89 clustered patients and studied by using univariate analysis. RESULTS: Thirty-nine percent of the patients were clustered, suggesting possible recent transmission. Infection with drug-resistant M. tuberculosis was associated with a decreased risk of being in a cluster. The strains isolated from human immunodeficiency virus (HIV)-positive patients were not associated with clustering. We found that immigration was not a major determinant in the total number of TB cases. CONCLUSION: Immigration, HIV and drug resistance were not associated with recent transmission. More than 50% of the clusters contained two or three patients, indicating that small outbreaks were responsible for most of the tuberculosis cases. Our RFLP typing results indicate that a TB control programme should be implemented in Spain in order to lower transmission of TB.

Isolation by genetic labeling of a new mycobacterial plasmid, pJAZ38, from Mycobacterium fortuitum.

In a two-step mating experiment with recipient strains of Mycobacterium smegmatis, the Mycobacterium fortuitum cryptic plasmid pJAZ38 was isolated. Plasmid pJAZ38 was genetically labeled by cointegration formation mediated by the kanamycin-resistant mycobacterial transposon Tn611. The region responsible for replication of pJAZ38 was located and sequenced. This region showed homology with the Mycobacterium avium plasmid pLR7 and the Mycobacterium scrofulaceum plasmid pMSC262, a family of plasmids which have been found to be widespread throughout the mycobacteria. Further experiments showed pJAZ38 to be stably inherited in the absence of selection pressure and compatible with the most commonly used mycobacterial replicon, pAL5000. In contrast to pLR7 and pMSC262, pJAZ38 was able to replicate in M. smegmatis mc(2)155, making it a useful tool for mycobacterial genetics.

Use of a PCR method based on IS6110 polymorphism for typing Mycobacterium tuberculosis strains from BACTEC cultures.

Two PCR typing methods, based on polymorphism of the insertion sequence IS6110, were compared with Mycobacterium tuberculosis strains by using a single primer complementary to the inverted repeats of IS6110. Total M. tuberculosis DNA either was amplified directly (IS6110-PCR) or was amplified following digestion and ligation (IS6110-inverse-PCR). Both PCR techniques showed a similar degree of discrimination. Because of its simplicity, IS6110-PCR was chosen to confirm that a single M. tuberculosis strain was responsible for an outbreak of tuberculosis in a secondary school. IS6110-PCR was used to study the degree of differentiation in 85 clinical M. tuberculosis isolates from BACTEC 12B broth cultures. Results were consistent with those of the standardized IS6110 restriction fragment length polymorphism (RFLP) analysis method, showing identical PCR types for identical RFLPs, although the degree of discrimination was greater by RFLP analysis. The study concludes that due to its simplicity, IS6110-PCR is a good screening method when quick differentiation between M. tuberculosis strains is needed because BACTEC cultures may be used directly.

The Mycobacterium tuberculosis purine biosynthetic pathway: isolation and characterization of the purC and purL genes.

Genes from the Mycobacterium tuberculosis purine biosynthetic pathway were identified using purine auxotrophic mutants of Mycobacterium smegmatis obtained by Tn611 transposon mutagenesis. Two approaches were followed in parallel. The first consisted of the complementation of the M. smegmatis purine auxotrophs using a M. tuberculosis H37Rv shuttle cosmid library. In the second approach, specific probes corresponding to the regions adjacent to the insertion sites of Tn611 in the M. smegmatis genome were used to screen a M. tuberculosis plasmid library by colony hybridization for inserts carrying homologous DNA fragments. Nucleotide sequence analysis of two M. tuberculosis genes isolated by these methods revealed high similarities with purC and purL genes from other bacterial and fungal sources. Transcriptional start sites were mapped for both genes, which revealed similar-10 boxes but with a higher GC content than the Escherichia coli sigma 70 consensus.

Efficient transposition in mycobacteria: construction of Mycobacterium smegmatis insertional mutant libraries.

The Tn611 transposon was inserted into pCG63, a temperature-sensitive plasmid isolated from an Escherichia coli-mycobacterial shuttle vector which contains the pAL5000 and pUC18 replicons. The resulting plasmid, pCG79, was used to generate a large number of insertional mutations in Mycobacterium smegmatis. These are the first mycobacterial insertional mutant libraries to be constructed by transposition directly into a mycobacterium. No highly preferential insertion sites were detected by Southern blot analysis of the chromosomal DNAs isolated from the insertion mutants. Auxotrophic mutants with various phenotypes were isolated at a frequency ranging from 0.1 to 0.4%, suggesting that the libraries are representative. The pCG79 system thus seems to be a useful tool for the study of M. smegmatis genetics and may be applicable to other mycobacteria, such as the M. tuberculosis complex.

[Application of RFLP to typing strains of Mycobacterium tuberculosis]. / Aplicación del RFLP a la tipificación de cepas de Mycobacterium tuberculosis.

BACKGROUND: Typing of Mycobacterium tuberculosis strains using RFLP (Restriction Fragments Length Polymorphism) had been shown to be useful for the detection of tuberculosis outbreaks in different countries. METHODS: A non-radioactive hybridization technique with the insertion sequence IS6110 has been used for typing M. tuberculosis strains. This technique detects polymorphism in the genome of M. tuberculosis following digestion with the restriction enzyme PvuII, and hybridisation with IS6110. RESULTS: One hundred and fourteen strains of M. tuberculosis isolated from Hospital Clínico Universitario (HCU) of Zaragoza, have been typed. Identical banding patterns were obtained in successive isolates from the same patient (at different times or from different culture sites) as was the case with some strains isolated in the same geographical location. The latter may be interpreted as a possible outbreak or perhaps a higher prevalence of a particular strain type in certain areas. CONCLUSIONS: The high level of polymorphism observed in the strains of M. tuberculosis isolated in HCU of Zaragoza, allows their typing using RFLP. The standardised use of this technique in different Spanish hospitals may allow the detection of outbreaks, which is of special interest for multiresistant strains of M. tuberculosis.

Analysis of the regions responsible for IS6110 RFLP in a single Mycobacterium tuberculosis strain.

A high degree of IS6110 restriction fragment length polymorphism (RFLP) is observed amongst the different strains of the Mycobacterium tuberculosis complex. The sequences of the IS6110 flanking regions from a M. tuberculosis strain harbouring four IS6110 copies were determined. Duplication of 3-4 nucleotides was found at the extremities of the four IS6110 copies, suggesting that IS6110 RFLP is due to transposition of the IS element. One of the copies of IS6110 analysed in the study was shown to be located at the same site in the genome of M. tuberculosis as the single copy present in an M. bovis BCG strain.

Restriction fragment length polymorphism analysis using IS6110 as an epidemiological marker in tuberculosis.

The mycobacterial insertion sequence IS6110 has been shown to be present in multiple copies in the chromosome of Mycobacterium tuberculosis. IS6110 restriction fragment length polymorphism analysis of strains isolated from patients who developed tuberculosis showed identical patterns over a 2- to 3-year period. In contrast, a high degree of polymorphism was observed between strains of the M. tuberculosis complex isolated from different patients. This study demonstrates that the presence of IS6110 does not induce in vivo major genomic rearrangements over a 2- to 3-year period and confirms its use as a valuable epidemiological marker in tuberculosis.

Aminoglycoside acetyltransferase 3-IV (aacC4) and hygromycin B 4-I phosphotransferase (hphB) in bacteria isolated from human and animal sources.

Members of the family Enterobacteriaceae harboring an enzyme of the aminoglycoside acetyltransferase 3 class (AAC-3-IV) (apramycin and gentamicin resistance) and hygromycin B phosphotransferase 4 (HPH-4-I) (hygromycin B resistance) have been isolated from human clinical sources in Europe. A cluster of genes containing IS140, aacC4, and hphB was found in these strains. We demonstrate by Southern hybridization that this cluster is identical to the operon found in animals that also contains insertion sequences belonging to the ISO family. This provides another example of presumptive transfer of antibiotic resistance genes between bacteria of animal and human origin.

The susceptibility to amoxycillin/clavulanate of Enterobacteriaceae with plasmid-mediated ampicillin resistance: a twelve-year study of strains in one Spanish hospital.

We studied the antibacterial activity of amoxycillin, clavulanic acid and the combination, against 1210 clinical isolates with plasmid-mediated ampicillin resistance, isolated at the Zaragoza Clinical University Hospital during 12 years. MICs were determined by an agar dilution technique with an inoculum of 10(4) cfu per spot. MIC50 values of amoxycillin/clavulanate ranged from 8 to 16 mg/l. The antibacterial activity of amoxycillin/clavulanate did not show any statistically significant variation during the 12-year period.

Comparative activity of cefotetan against plasmid-encoded aminoglycoside-resistant enterobacteria strains.

304 strains of R-plasmid harbouring enterobacteria resistant to aminoglycosides were studied for their susceptibilities to a range of antibiotics, including cefotetan. Cefotetan and latamoxef were the most active of the four cephamycins tested and all were stable to the beta-lactamases produced by these strains. No new beta-lactamases (SHV-2, CTX-1, TEM-4, CAZ-1) were found in these strains capable of hydrolysing third generation cephalosporins. The activity of cefotetan against these multi-resistant, beta-lactamase producing strains may be of clinical value.

Characterization of an hospital disseminated plasmid encoding resistance to gentamicin and other antimicrobial agents.

A preliminary report has shown the existence of an endemic R plasmid in the University Hospital of Zaragoza. The results presented in this paper demonstrate the dissemination of a new 73 kilobases plasmid into multiple strains and species of gram-negative bacilli. This transferable plasmid belongs to Incompatibility group P and mediates resistance to ampicillin, tetracycline, gentamicin, kanamycin, streptomycin, chloramphenicol and sulfamethoxazole, synthesizing the aminoglycoside-modifying enzymes 3-acetyltransferase, 3'phosphotransferase, and 3"nucleotidyltransferase, and a TEM-1 beta-lactamase. These results and the previous findings show that a family of gentamicin-resistance plasmids exists among the gram-negative bacteria in the University Hospital. Resistance to gentamicin in all these plasmids is associated with the formation of 3-N-acetyltransferases.

Enzymatic modification of aminoglycoside antibiotics by Branhamella catarrhalis carrying an R factor.

Fifteen out of 89 clinical strains of Branhamella catarrhalis isolated from patients at the University Hospital of Zaragoza were resistant to aminoglycosides and other antimicrobials. In two strains, B. catarrhalis 220 and B. catarrhalis 115, the resistance to aminoglycosides was associated with synthesis of aminoglycoside-modifying enzymes, namely 3"-O-phosphotransferase [APH(3")] and 3'-O-phosphotransferase [APH(3')]. B. catarrhalis 115 was resistant to ampicillin, streptomycin, kanamycin, neomycin, butirosin, lividomycin, ribostamycin, paromomycin and trimethoprim-sulfamethoxazole and harboured a 32 megadalton (Md) plasmid. The resistance determinants of the latter were transferred to Neisseria subflava by conjugation and to Escherichia coli by transformation. The transconjugant strain presented an antibiotic resistance pattern similar to the donor strain and carried the same plasmid. The transformant strain acquired the 32 Md plasmid but presented, besides the resistance pattern already mentioned, resistance to tetracycline, gentamicin and tobramycin. Resistance to gentamicin and tobramycin was mediated by the synthesis of a 3-N-acetyltransferase. This resistance and the related enzyme were expressed neither in the donor B. catarrhalis strain nor in the transconjugant N. subflava strain.