Lucio Vera-Cabrera

Comparative genomic and phylogeographic analysis of Mycobacterium leprae.

Reductive evolution and massive pseudogene formation have shaped the 3.31-Mb genome of Mycobacterium leprae, an unculturable obligate pathogen that causes leprosy in humans. The complete genome sequence of M. leprae strain Br4923 from Brazil was obtained by conventional methods (6× coverage), and Illumina resequencing technology was used to obtain the sequences of strains Thai53 (38× coverage) and NHDP63 (46× coverage) from Thailand and the United States, respectively. Whole-genome comparisons with the previously sequenced TN strain from India revealed that the four strains share 99.995% sequence identity and differ only in 215 polymorphic sites, mainly SNPs, and by 5 pseudogenes. Sixteen interrelated SNP subtypes were defined by genotyping both extant and extinct strains of M. leprae from around the world. The 16 SNP subtypes showed a strong geographical association that reflects the migration patterns of early humans and trade routes, with the Silk Road linking Europe to China having contributed to the spread of leprosy.

In Vitro Activities of DA-7157 and DA-7218 against Mycobacterium tuberculosis and Nocardia brasiliensis

ABSTRACT The in vitro activities of DA-7157, a novel oxazolidinone, against clinical isolates of Nocardia brasiliensis and Mycobacterium tuberculosis were determined. Equal MIC50s and MIC90s (0.25 and 0.5 μg/ml, respectively) were found for susceptible and multidrug-resistant isolates of M. tuberculosis. The N. brasiliensis isolates showed an MIC90 of 1 μg/ml and an MIC50 of 1 μg/ml. The DA-7157 prodrug, DA-7218, exhibited similar MICs for M. tuberculosis but fivefold-higher MICs for N. brasiliensis.

In Vitro and In Vivo Activities of Antimicrobials against Nocardia brasiliensis

ABSTRACT In Mexico mycetomas are mostly produced by Nocardia brasiliensis, which can be isolated from about 86% of cases. In the present work, we determined the sensitivities of 30 N. brasiliensis strains isolated from patients with mycetoma to several groups of antimicrobials. As a first screening step we carried out disk diffusion assays with 44 antimicrobials, including aminoglycosides, cephalosporins, penicillins, quinolones, macrolides, and some others. In these assays we observed that some antimicrobials have an effect on more than 66% of the strains: linezolid, amikacin, gentamicin, isepamicin, netilmicin, tobramycin, minocycline, amoxicillin-clavulanic acid, piperacillin-tazobactam, nitroxolin, and spiramycin. Drug activity was confirmed quantitatively by the broth microdilution method. Amoxicillin-clavulanic acid, linezolid, and amikacin, which have been used to treat patients, were tested in an experimental model of mycetoma in BALB/c mice in order to validate the in vitro results. Linezolid showed the highest activity in vivo, followed by the combination amoxicillin-clavulanic acid and amikacin.

Insight into the evolution and origin of leprosy bacilli from the genome sequence of Mycobacterium lepromatosis

Significance Leprosy was thought to be exclusively caused by infection of humans by Mycobacterium leprae. In 2008, Han et al. proposed that Mycobacterium lepromatosis, a separate unculturable species, might be responsible for a rare yet severe form of the disease called diffuse lepromatous leprosy. Here, by using comparative genomics, we show that the two species are very closely related and derived from a common ancestor that underwent genome downsizing and gene decay. Since their separation 13.9 Mya, the two species have continued to lose genes, but from different regions of the genome, and M. leprae appears to be more recent. In a phylogeographic survey, by using differential PCR, we found that M. lepromatosis was scarce and restricted to patients from Mexico. Mycobacterium lepromatosis is an uncultured human pathogen associated with diffuse lepromatous leprosy and a reactional state known as Lucios phenomenon. By using deep sequencing with and without DNA enrichment, we obtained the near-complete genome sequence of M. lepromatosis present in a skin biopsy from a Mexican patient, and compared it with that of Mycobacterium leprae, which has undergone extensive reductive evolution. The genomes display extensive synteny and are similar in size (∼3.27 Mb). Protein-coding genes share 93% nucleotide sequence identity, whereas pseudogenes are only 82% identical. The events that led to pseudogenization of 50% of the genome likely occurred before divergence from their most recent common ancestor (MRCA), and both M. lepromatosis and M. leprae have since accumulated new pseudogenes or acquired specific deletions. Functional comparisons suggest that M. lepromatosis has lost several enzymes required for amino acid synthesis whereas M. leprae has a defective heme pathway. M. lepromatosis has retained all functions required to infect the Schwann cells of the peripheral nervous system and therefore may also be neuropathogenic. A phylogeographic survey of 227 leprosy biopsies by differential PCR revealed that 221 contained M. leprae whereas only six, all from Mexico, harbored M. lepromatosis. Phylogenetic comparisons indicate that M. lepromatosis is closer than M. leprae to the MRCA, and a Bayesian dating analysis suggests that they diverged from their MRCA approximately 13.9 Mya. Thus, despite their ancient separation, the two leprosy bacilli are remarkably conserved and still cause similar pathologic conditions.

Dermatophytoses in Monterrey, México

In the present report we reviewed a total of 2397 cases of dermatophytosis from superficial cutaneous lesions between the years 1978 and 1990. The cases included were from the Department of Dermatology at the University Hospital located in the city of Monterrey, México. A total of 726 tinea pedis, 613 tinea unguium, 441 tinea capitis, 395 tinea corporis and 222 tinea cruris cases were observed. The most commonly isolated dermatophyte species was Trichophyton rubrum (45%), followed by Trichophyton mentagrophytes (23.7%), Trichophyton tonsurans (21%), Microsporum canis (7.1%) and Epidermophyton floccosum (2.5%). Less frequently we isolated Microsporum audouinii, Microsporum gypseum, Trichophyton violaceum and Trichophyton verrucosum. Most of the cases were observed in the warmest months of the year (from March to September), and were equally distributed in both genders, except for tinea cruris which was more prevalent in men (3.5 : 1 ratio).

Actinomycetoma and advances in its treatment

Actinomycetoma is a chronic subcutaneous infection caused by aerobic branching actinomycetes. Its clinical features are firm tumefaction of the affected site and the presence of abscesses, nodules, and sinuses that drain a seropurulent exudate containing filamenting granules. The disease is caused by inoculation of the infectious agent through minor trauma in susceptible individuals. Nocardia brasiliensis, Actinomadura madurae, and Streptomyces somaliensis are among the most frequent agents in the Americas. Cellular and humoral immunity have been studied in animal models. Standard therapy for uncomplicated cases is sulfamethoxazole-trimethoprim given for many months. Bone involvement, disseminated cases, and special locations require combined treatment with amikacin and sulfamethoxazole-trimethoprim. Isolated reports include the addition of other antibiotics such as rifampicin, imipenem, or meropenem. When needed, other aminoglycosides can be used. Amoxicillin-clavulanic acid is indicated in specific cases as alternative treatment. Oxazolidinone antibiotics, such as linezolid and other similar compounds (DA-7218 and DA-7157), have been studied in experimental infections in animal models as well as in vitro and ex vivo, with encouraging results.

In Vitro Activity of PNU-100766 (Linezolid), a New Oxazolidinone Antimicrobial, against Nocardia brasiliensis

ABSTRACT The in vitro activity of a novel oxazolidinone, linezolid, was studied by comparing the activity of linezolid with those of amikacin, trimethoprim-sulfamethoxazole, and amoxicillin-clavulanic acid against 25 strains of Nocardia brasiliensis isolated from patients with mycetoma. All N. brasiliensis strains tested were sensitive to linezolid (MIC at which 90% of strains are inhibited [MIC90], 2 μg/ml; MIC50, 1 μg/ml). This antimicrobial might constitute a good alternative for treatment of actinomycetoma.

Mycobacterium tuberculosis Spoligotypes in Monterrey, Mexico

ABSTRACT Although tuberculosis is still a public health problem in Mexico, there is little information about the genetic characteristics of the isolates. In the present study, we analyzed by spoligotyping 180 Mycobacterium tuberculosis clinical isolates from the urban area of Monterrey, Mexico, including drug-susceptible and drug-resistant isolates. The spoligotype patterns were compared with those in the international SITVIT2 spoligotyping database. Four isolates presented spoligotype patterns not found in the database (orphan types); the rest were distributed among 44 spoligo international types (SITs). SIT53 (clade T1) and SIT119 (clade X1) were predominant and included 43 (23.8%) and 28 (15.5%) of the isolates, respectively. In order to determine if there was a dominant spoligotype in the group of multidrug-resistant isolates, 37 of them were analyzed by IS6110-based restriction fragment length polymorphism assays, and scarce clustering of strains with more than five bands was observed. Fourteen isolates of this multidrug-resistant group presented four bands or less and were distributed in four SITs: SIT53 (n = 8), SIT92 (n = 3), SIT70 (n = 2), and SIT3038 (n = 1). When the molecular detection of mutations in the katG and rpoB genes were analyzed in these isolates with low copy numbers of IS6110, only two isolates shared the same IS6110, spoligotyping, and mutations patterns. When the distribution of the spoligotypes was analyzed by age cohort, SIT119 was predominantly found in patients 0 to 20 years old, especially in males, accounting for up to 40% of the isolates. In contrast, SIT53 was more prevalent in older females. This analysis demonstrates the variability of M. tuberculosis isolates in Monterrey and the partial dominance of SIT53 and SIT119 in that area of Mexico.

Phospholipase Region of Mycobacterium tuberculosis Is a Preferential Locus for IS6110 Transposition

ABSTRACT Enzymes with phospholipase C activity in Mycobacterium tuberculosis have been recently described. The three genes encoding these proteins, plcA, plcB, andplcC, are located at position 2351 of the genomic map ofM. tuberculosis H37Rv and are arranged in tandem. We have previously described the presence of variations in the restriction fragment length polymorphism patterns of the plcA andplcB genes in M. tuberculosis clinical isolates. In the present work we investigated the origin of this polymorphism by sequence analysis of the phospholipase-encoding regions of 11 polymorphic M. tuberculosisclinical isolates. To do so, a long-PCR assay was used to amplify a 5,131-bp fragment that contains the plcA andplcB genes and part of the plcC gene. In the M. tuberculosis strains studied the production of an amplicon ∼1,400 bp larger than anticipated was observed. Sequence analysis of the PCR products indicated the presence of a foreign sequence that corresponded to an IS6110 element. We observed insertion elements in the plcA,plcB, and plcC genes. One site inplcB had the highest incidence of transposition (5 out of 11 strains). In two strains the insertion element was found inplcA in the same nucleotide position. In all the cases, IS6110 was transposed in the same direction. The high level of transposition in the phospholipase region can lead to the excision of fragments of genomic DNA by recombination of neighboring IS6110 elements, as demonstrated by finding the deletion, in two strains, of a 2,837-bp fragment that includedplcA and most of plcB. This can explain the negative results obtained by some authors when detecting themtp40 sequence (plcA) by PCR. Given the high polymorphism in this region, the use of the mtp40sequence as a genetic marker for M. tuberculosis sensu stricto is very restricted.

In vitro antimicrobial susceptibility of Propionibacterium acnes isolated from acne patients in northern Mexico.

Background  Antimicrobials are essential in acne therapy. In the last decades, Propionibacterium acnes has become resistant to different antibiotics.