Howard Takiff - Academia.edu (original) (raw)

Papers by Howard Takiff

Emerging Microbes & Infections, 2020

Mycobacterial persistence mechanisms remain to be fully characterized. Screening a transposon ins... more Mycobacterial persistence mechanisms remain to be fully characterized. Screening a transposon insertion library of Mycobacterium marinum identified kdpA, whose inactivation reduced the fraction of persisters after exposure to rifampicin. kdpA encodes a transmembrane protein that is part of the Kdp-ATPase, an ATP-dependent high-affinity potassium (K +) transport system. We found that kdpA is induced under low K + conditions and is required for pH homeostasis and growth in media with low concentrations of K +. The inactivation of the Kdp system in a kdpA insertion mutant caused hyperpolarization of the cross-membrane potential, increased proton motive force (PMF) and elevated levels of intracellular ATP. The KdpA mutant phenotype could be complemented with a functional kdpA gene or supplementation with high K + concentrations. Taken together, our results suggest that the Kdp system is required for ATP homeostasis and persister formation. The results also confirm that ATP-mediated regulation of persister formation is a general mechanism in bacteria, and suggest that K + transporters could play a role in the regulation of ATP levels and persistence. These findings could have implications for the development of new drugs that could either target persisters or reduce their presence.

The Cell Surface, 2021

HAL is a multidisciplinary open access archive for the deposit and dissemination of scientific re... more HAL is a multidisciplinary open access archive for the deposit and dissemination of scientific research documents, whether they are published or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. L'archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. Distributed under a Creative Commons Attribution-NonCommercial-NoDerivatives| 4.0 International License

Name: Biotecnología …, 2011

The direct detection of pyrazinamide resistance in Mycobacterium tuberculosis is not routinely pe... more The direct detection of pyrazinamide resistance in Mycobacterium tuberculosis is not routinely performed in many laboratories in the world because the drug is active only at acid pH, which also affects the growth of Mycobacterium tuberculosis. The pyrazinamidase enzyme, encoded by pncA gene, is necessary to convert prodrug pyrazinamide to its active form. Taking into account that nicotinamide, a structural analogue of pyrazinamide, converted in its active forms by the pyrazinamidase enzyme at a physiological pH does not affect bacterial growth, the aim of this research was to evaluate two colorimetric methods: Nitrate reductase and malachite green microtube assays, using nicotinamide to perform susceptibility testing in 102 Mycobacterium tuberculosis strains. The results were compared with those obtained by the classic Wayne assay. Mutations in the pncA gene were identified by sequencing the pncA gene from all isolates in which pyrazinamide resistance was detected by any of the three methods. Both the nitrate reductase and malachite green microtube assays showed sensitivities of 93.75% and specificities of 97.67%. Mutations in the pncA gene were found in 14 of 16 strains (87.5%) that were pyrazinamide resistant.

Revista Argentina de Microbiología, 2013

The relation of ethambutol resistance to embB mutations remains unclear, and there are no reports... more The relation of ethambutol resistance to embB mutations remains unclear, and there are no reports on ethambutol resistance from the caribbean. We examined the sequence of embB in 57 distinct Multi-Drug Resistant (MDR) and non-MDR strains of Mycobacterium tuberculosis, mostly from Cuba and the Dominican Republic. embB306 codon mutations were found exclusively in MDR-TB, but in both ethambutol sensitive and resistant strains. Valine substitutions predominated in ethambutol resistant strains, while isoleucine replacements were more common in sensitive strains. Three ethambutol resistant MDR strains without embB306 substitutions had replacements in embB406 or embB497, but these were also found in ethambutol sensitive MDR strains. The results confirm previous findings that amino acid substitutions in EmbB306, EmbB406 and EmbB497 are found only in MDR-TB strains but in both phenotypically resistant and sensitive strains. One ethambutol resistant non-MDR strain did not have any embB mutati...

Oligonucleotides used for PCR of carbapenemase-resistant Klebsiella pneumoniae isolates from a Ho... more Oligonucleotides used for PCR of carbapenemase-resistant Klebsiella pneumoniae isolates from a Hospital in Venezuela. (DOC 77 kb)

While fluoroquinolones (FQs) have been successful in helping cure multidrug-resistant tuberculosi... more While fluoroquinolones (FQs) have been successful in helping cure multidrug-resistant tuberculosis (MDR TB), studies in mice have suggested that if used as first-line agents they might reduce the duration of therapy required to cure drug-sensitive TB. The results of phase II trials with FQs as first-line agents have been mixed, but in at least three studies where moxifloxacin substituted for ethambutol, there was an increase in the early percentage of sputa that converted to negative for bacilli. Phase III trials are in progress to test the effective-ness of 4-month FQ-containing regimens, but there is concern that the widespread use of FQs for other infections could engender a high prevalence of FQ-resistant TB. However, several studies suggest that despite wide FQ use, the prevalence of FQ-resistant TB is low, and the majority of the resistance is low-level. The principal risk for resistance may be when FQs are used to treat nonspecific respiratory symptoms that are in fact TB, so...

While fluoroquinolones (FQ) have been successful in helping cure Multi-Drug Resistant tuberculosi... more While fluoroquinolones (FQ) have been successful in helping cure Multi-Drug Resistant tuberculosis, studies in mice have suggested that if used as first-line agents they might reduce the duration of therapy required to cure drug-sensitive TB. The results of phase II trials with FQs as first-line agents have been mixed, but in at least three studies where moxifloxacin substituted for ethambutol there was an increase in the early percentage of sputa that converted to negative for bacilli. Phase III trials are in progress to test the effectiveness of four-month FQ-containing regimens, but there is concern that the widespread use of FQs for other infections could engender a high prevalence of FQ resistant TB. However, several studies suggest that despite wide FQ use the prevalence of FQ-resistant TB is low, and the majority of the resistance is low-level. The principal risk for resistance may be when FQs are used to treat nonspecific respiratory symptoms that are in fact TB, so curtaili...

Investigacion clinica, 2014

Sucre municipality is a large, densely populated marginal area in the eastern part of Caracas, Ve... more Sucre municipality is a large, densely populated marginal area in the eastern part of Caracas, Venezuela that consistently has more cases of tuberculosis than other municipalities in the country. To identify the neighborhoods in the municipality with the highest prevalence of tuberculosis, and determine whether the Mycobacterium tuberculosis strain distribution in this municipality is different from that previously found in the western part of Caracas and the rest of Venezuela, we collected data on all tuberculosis cases in the municipality diagnosed in 2005-6. We performed two separate molecular epidemiological studies, spoligotyping 44 strains in a first study, and spoligotyping 131 strains, followed by MIRU-VNTR 15 on 21 clustered isolates in the second. With spoligotyping, the most common patterns were Shared International Type SIT17 (21%); SIT42 (15%); SIT93 (11%); SIT20 (7%); SIT53 (6%), a distribution similar to other parts of Venezuela, except that SIT42 and SIT20 were more ...

Background: Tuberculosis remains an endemic public health problem, but the ecology of the TB stra... more Background: Tuberculosis remains an endemic public health problem, but the ecology of the TB strains prevalent, and their transmission, can vary by country and by region. We sought to investigate the prevalence of Mycobacterium tuberculosis strains in different regions of Venezuela. A previous study identified the most prevalent strains in Venezuela but did not show geographical distribution nor identify clonal genotypes. To better understand local strain ecology, we used spoligotyping to analyze 1298 M. tuberculosis strains isolated in Venezuela from 1997 to 2006, predominantly from two large urban centers and two geographically distinct indigenous areas, and then studied a subgroup with MIRU-VNTR 24 loci. Results: The distribution of spoligotype families is similar to that previously reported for Venezuela and other South American countries: LAM 53%, T 10%, Haarlem 5%, S 1.9%, X 1.2%, Beijing 0.4%, and EAI 0.2%. The six most common shared types (SIT's 17, 93, 605, 42, 53, 20) ...

La tuberculose (TB), que beaucoup ont pense etre une maladie en voie d'eradication, a fait pr... more La tuberculose (TB), que beaucoup ont pense etre une maladie en voie d'eradication, a fait preuve d'une dramatique resurgence et a ete etiquetee par l'OMS comme une urgence mondiale Les elements clefs du programme de controle de la tuberculose sont un diagnostic precoce et le traitement des cas, ce qui reduit la transmission de la maladie. La cuti reaction est un e ement de diagnostic simple, economique et essentiel de la tuberculose, car il peut quantifier l'absence d'infection du patient. Cependant, elle n est ni specifique ni sensible puisque n'identifiant que seulement 50 a 70% des patients tuberculeux, et etant positive en presence de toutes les mycobacteries, pas seulement avec Mycobacterium tuberculosis l'agent causal de la tuberculose. La sensibilite du diagnostic peut atteindre 80% en cultivant les echantillons cliniques en milieu solide, et jusqu'a 90% avec les cultures en milieu liquide. Plusieurs systemes commerciaux de culture emploient d...

International Journal of Microbiology

Fluoroquinolones (FQs) are antibiotics useful in the treatment of drug-resistant tuberculosis, bu... more Fluoroquinolones (FQs) are antibiotics useful in the treatment of drug-resistant tuberculosis, but FQ-resistant mutants can be selected rapidly. Although mutations in the DNA gyrase are the principal cause of this resistance, pentapeptide proteins have been found to confer low-level FQ resistance in Gram-negative bacteria. MfpA is a pentapeptide repeat protein conserved in mycobacterial chromosomes, where it is adjacent to a group of four highly conserved genes termed a conservon. We wished to characterize the transcriptional regulation of the mfpA gene and relate its expression to ciprofloxacin resistance in M. smegmatis. Reverse transcription PCR showed that mfpA gene is part of an operon containing the conservon genes. Using a transcriptional fusion, we showed that a promoter was located 5′ to the mfpEA operon. We determined the promoter activity under different growth conditions and found that the expression of the operon increases slightly in late growth phases in basic pH and ...

Journal of clinical microbiology

Progress in understanding the basis of resistance to isoniazid (INH) and rifampin (RMP) has allow... more Progress in understanding the basis of resistance to isoniazid (INH) and rifampin (RMP) has allowed molecular tests for the detection of drug-resistant tuberculosis to be developed. Consecutive isolates (n = 95) of Mycobacterium tuberculosis, from a Spanish reference laboratory investigating outbreaks of multidrug-resistant tuberculosis, were coded and sent to two external laboratories for genotypic analysis of INH and RMP resistance by PCR-single-strand conformation polymorphism (SSCP) analysis of specific regions of four genes: part of the coding sequence of katG and the promoter regions of inhA and ahpC for INH and the RMP resistance region of rpoB. After correction for the presence of outbreak strains and multiple isolates from single patients, RMP resistance was detected successfully by PCR-SSCP in > 96% of the RMP-resistant strains. PCR-SSCP had a sensitivity of 87% for INH resistance detection, and mutations in katG, inhA, katG-inhA, ahpC, and katG-ahpC were identified in ...

Journal of Virology

We have studied the DNAs of fastidious enteric adenoviruses recovered from the stools of infants ... more We have studied the DNAs of fastidious enteric adenoviruses recovered from the stools of infants with gastroenteritis. By endonuclease analysis, the strains examined represent candidate adenovirus types 40 and 41, which are thought to comprise new adenovirus subgroups F and G. Cloning of DNA from representative enteric adenovirus isolates, together with hybridization and subcleavage analysis, permitted the mapping of restriction enzyme cleavage sites. Although the restriction profiles are different for the two strains, they appear to have several cleavage sites in common. Cross hybridization studies show considerable homology between the subgroup F and G strains but much less homology to adenovirus 2. In addition, regions on both ends of enteric adenovirus genomes (map units, 2.9 to 11.3 and 75 to 100) possess little or no homology to adenovirus 2. Restriction enzyme digests reveal submolar fragments that map to the terminal regions of the genome. Electron micrographic studies of de...

Journal of Clinical Microbiology

The aim of this study was to assess the efficacy of PCR methodology in establishing the diagnosis... more The aim of this study was to assess the efficacy of PCR methodology in establishing the diagnosis of cutaneous leishmaniasis in patients from areas of endemicity in Venezuela. Biopsies from 233 patients with cutaneous ulcers suggestive of leishmaniasis were analyzed by PCR, employing oligonucleotides directed against conserved regions of kinetoplast DNA (kDNA), and the PCR products were then hybridized to nonradioactively labeled, species-specific, cloned kDNA fragments. The ability of PCR to detect Leishmania cells was compared with those of the conventional methodologies: skin testing with killed promastigotes (Montenegro test), examination of Giemsa-stained biopsy smears, and in vitro culture of biopsy tissue. The PCR-hybridization technique detected the presence of Leishmania cells in 98% of patients clinically diagnosed as having leishmaniasis and also positive by the Montenegro skin test. In comparison, leishmania positivity was found in only 42% of cultures and 64% of biopsy ...

Scientific Reports

An amendment to this paper has been published and can be accessed via a link at the top of the pa... more An amendment to this paper has been published and can be accessed via a link at the top of the paper.

International Journal of Systematic and Evolutionary Microbiology

Three strains, CLM-U50 T , CLM-R50 and IVIC-Bov1, belonging to the genus Leptospira, were isolate... more Three strains, CLM-U50 T , CLM-R50 and IVIC-Bov1, belonging to the genus Leptospira, were isolated in Venezuela from a patient with leptospirosis, a domestic rat (Rattus norvegicus) and a cow (Bos taurus), respectively. The initial characterisation of these strains based on the rrs gene (16S rRNA) suggested their designation as a novel species within the 'intermediates' group of the genus Leptospira. Further phylogenomic characterisation based on single copy core genes was consistent with their separation into a novel species. The average nucleotide identity between these three strains was >99 %, but below 89 % with respect to any previously described leptospiral species, also supporting their designation as a novel species. Given this evidence, these three isolates were considered to represent a novel species, for which the name Leptospira venezuelensis sp. nov. is proposed, with CLM-U50 T (=CIP 111407 T =DSM 105752 T) as the type strain.

Scientific reports, Jan 7, 2018

The city of Shenzhen has recently experienced extraordinary economic growth accompanied by a huge... more The city of Shenzhen has recently experienced extraordinary economic growth accompanied by a huge internal migrant influx. We investigated the local dynamics of tuberculosis (TB) epidemiology in the Nanshan District of Shenzhen to provide insights for TB control strategies for this district and other rapidly developing regions in China. We analyzed the age-specific incidence and number of TB cases in the Nanshan District from 2011 to 2016. Over all, the age-standardized incidence of TB decreased at an annual rate of 3.4%. The incidence was lowest amongst the age group 0-14 and showed no increase in this group over the six-year period (P = 0.587). The fastest decreasing incidence was among the 15-24 age group, with a yearly decrease of 13.3% (β = 0.867, P < 0.001). In contrast, the TB incidence increased in the age groups 45-54, 55-54, and especially in those aged ≥65, whose yearly increase was 13.1% (β = 1.131, P < 0.001). The peak time of TB case presentation was in April, Ma...

Emerging microbes & infections, Jan 6, 2018

Compensatory mutations have been suggested to promote multidrug-resistant tuberculosis (MDR-TB) t... more Compensatory mutations have been suggested to promote multidrug-resistant tuberculosis (MDR-TB) transmission, but their role in facilitating the recent transmission of MDR-TB is unclear. To investigate the epidemiological significance of compensatory mutations, we analyzed a four-year population-based collection of MDR-TB strains from Shanghai (the most populous city in China) and 1346 published global MDR-TB strains. We report that MDR-TB strains with compensatory mutations in the rpoA, rpoB, or rpoC genes were neither more frequently clustered nor found in larger clusters than those without compensatory mutations. Our results suggest that compensatory mutations are not a major contributor to the current epidemic of MDR-TB.

Journal of Bacteriology

The mini-Tn10 transposon (delta 16 delta 17Tn10) confers tetracycline resistance. When inserted b... more The mini-Tn10 transposon (delta 16 delta 17Tn10) confers tetracycline resistance. When inserted between a gene and its promoter, it blocks transcription and prevents expression of that gene. Tetracycline in the medium induces divergent transcription of the tetA and tetR genes within the transposon, and this transcription extends beyond the transposon in both directions into the bacterial genes. If the mini-Tn10 inserts between an essential bacterial gene and its promoter, the insertion mutation can cause conditional growth which is dependent on the presence of tetracycline. Two essential genes in adjacent operons of Escherichia coli have been detected by screening for tetracycline dependence among tetracycline-resistant insertion mutants. These essential genes are the era gene in the rnc operon and the dpj gene in the adjacent pdxJ operon. The pdxJ operon has not been described previously. It consists of two genes, pdxJ and dpj. Whereas the dpj gene is essential for E. coli growth i...