Analysis of DNA curvature distribution in mycobacterial promoters using theoretical models

In this paper, 125 different mycobacterial promoters are analyzed for their DNA curvature distribution using several di- and tri-nucleotide dependent models of DNA curvature. Different models give similar behavior and therefore qualitative validation of the results. Mycobacterial promoters resembling the E. coli sigma(70) type have almost 81% (85%) sequences having medium and high curvature profiles using dinucleotide-dependent models. Non-E. coli sigma(70) type mycobacterial promoters have comparatively higher percent of low curvature profiles. Very few extended -10 promoters have low curvature profiles. Mycobacterial promoters having A(n)T(m) (n+m > or =3) tract in the upstream region of -35 box and repeated in phase with each other have high curvature profiles. M. smegmatis promoters have high curvature profiles compared to M. tuberculosis promoters.     

Prevalence of Escherichia coli O157 in cattle feeds in Midwestern feedlots

Comparisons of enrichment methods (with or without antibiotics and with or without a preenrichment step) using gram-negative (GN) broth or tryptic soy broth (TSB) were conducted with feeds inoculated with Escherichia coli O157:H7. TSB was more sensitive than GN broth, and TSB with a preenrichment step followed by TSB with antibiotics was more sensitive than plain TSB enrichment, in detecting E. coli O157 in inoculated feeds. Feed samples were collected from feed bunks from 54 feedlots to determine the prevalence of E. coli O157 in cattle feeds. TSB preenrichment followed by TSB with antibiotics and the standard GN broth enrichment were used for each feed sample. All samples underwent immunomagnetic separation and were plated onto sorbitol MacConkey agar with cefixime and potassium tellurite. Identification of E. coli O157 was based on indole production, positive latex agglutination for O157 antigen, API 20E test strip results, PCR for the eaeA gene, and the presence of at least one Shiga toxin. E. coli O157 was detected in 52 of 504 feed samples (10.3%) by using GN broth enrichment and in 46 of 504 feed samples (9.1%) by using TSB followed by TSB supplemented with cefixime and vancomycin. E. coli O157 was detected in 75 of 504 feed bunk samples (14.9%) by one or both methods. There was no correlation between E. coli O157 prevalence and generic coliform counts in feeds. The prevalence of E. coli O157 in cattle feed warrants further studies to increase our knowledge of the on-farm ecology of E. coli O157 in order to develop strategies to prevent food-borne disease in humans.     

Selective toxicity of engineered lentivirus lytic peptides in a CF airway cell model

Lentivirus lytic peptides (LLPs) are derived from HIV-1 and have antibacterial properties. LLP derivatives (eLLPs) were engineered for greater potency against Staphylococcus aureus (SA) and Pseudomonas aeruginosa (PA). Minimum bactericidal concentration (MBC) was determined in low and physiologic salt concentrations. MBC was decreased against SA and equivalent against PA in physiologic salt when compared to the parent compound LLP1. In a novel cystic fibrosis (CF) airway cell model, one derivative, WLSA5, reduced the number of adherent PA and only moderately affected CF cell viability. Overall, eLLPs are selectively toxic to bacteria and may be useful against CF airway infections.     

Deletion of vapA encoding Virulence Associated Protein A attenuates the intracellular actinomycete Rhodococcus equi

Virulent strains of the facultative intracellular bacterium Rhodococcus equi isolated from young horses (foals) with R. equi pneumonia, carry an 80-90 kb virulence plasmid and express a highly immunogenic 15-17 kDa protein of unknown function called VapA (Virulence Associated Protein A). Recent sequencing of the virulence plasmid identified a putative pathogenicity island encoding a novel family of seven Vap proteins including VapA. These proteins exhibit a significant sequence similarity to each other but have no homologues in other organisms. In this study, we describe the construction of an R. equi mutant lacking a 7.9 kb DNA region spanning five vap genes (vapA, -C, -D, -E and -F ). This vap locus mutant was attenuated for virulence in mice as it was unable to replicate in vivo and was rapidly cleared in comparison to the virulent wild-type strain. Complementation analysis of the vap locus mutant showed that expression of vapA alone could restore full virulence, whereas expression of vapC, -D and -E could not. We subsequently constructed an R. equi strain lacking only the vapA gene and found that it was attenuated for growth in vivo to the same degree as the vap locus mutant. Unlike wild-type R. equi which replicates intracellularly, both of the mutant strains exhibited a growth defect in macrophages although their attachment to the macrophages was unaffected. These studies provide the first proof of a role for vapA in the virulence of R. equi, and demonstrate that its presence is essential for intracellular growth in macrophages.     

The additional 165 amino acids in the B protein of Escherichia coli DNA gyrase have an important role in DNA binding

DNA gyrase is the only enzyme known to negatively supercoil DNA. The enzyme is a heterotetramer of A(2)B(2) subunit composition. Alignment of the primary sequence of gyrase B (GyrB) from various species shows that they can be grouped into two classes. The GyrB of Gram-negative eubacteria has a stretch of about 165 amino acids in the C-terminal half, which is lacking in other GyrB subunits and type II topoisomerases. In Escherichia coli, no function has so far been attributed to this stretch. In this study, we have tried to assess the function of this region both in vivo and in vitro. A deletant (GyrBDelta160) lacking this region is non-functional in vivo. The holoenzyme reconstituted from gyrase A (GyrA) and GyrBDelta160 shows reduced but detectable supercoiling and quinolone-induced cleavage activity in vitro. GyrBDelta160 retains its ability to bind to GyrA and novobiocin. However, when reconstituted with GyrA, the deletant shows greatly impaired DNA binding. The intrinsic ATPase activity of the GyrBDelta160 is comparable to that of wild type GyrB, but this activity is not stimulated by DNA. These studies indicate that the additional stretch present in GyrB is essential for the DNA binding ability of E. coli gyrase.     

Linkage analysis of X-linked cone-rod dystrophy: localization to Xp11.4 and definition of a locus distinct from RP2 and RP3.

Progressive X-linked cone-rod dystrophy (COD1) is a retinal disease affecting primarily the cone photoreceptors. The COD1 locus originally was localized, by the study of three independent families, to a region between Xp11.3 and Xp21.1, encompassing the retinitis pigmentosa (RP) 3 locus. We have refined the COD1 locus to a limited region of Xp11.4, using two families reported elsewhere and a new extended family. Genotype analysis was performed by use of eight microsatellite markers (tel-M6CA, DXS1068, DXS1058, DXS993, DXS228, DXS1201, DXS1003, and DXS1055-cent), spanning a distance of 20 cM. Nine-point linkage analysis, by use of the VITESSE program for X-linked disorders, established a maximum LOD score (17.5) between markers DXS1058 and DXS993, spanning 4.0 cM. Two additional markers, DXS977 and DXS556, which map between DXS1058 and DXS993, were used to further narrow the critical region. The RP3 gene, RPGR, was excluded on the basis of two obligate recombinants, observed in two independent families. In a third family, linkage analysis did not exclude the RPGR locus. The entire coding region of the RPGR gene from two affected males from family 2 was sequenced and was found to be normal. Haplotype analysis of two family branches, containing three obligate recombinants, two affected and one unaffected, defined the COD1 locus as distal to DXS993 and proximal to DXS556, a distance of approximately 1.0 Mb. This study excludes COD1 as an allelic variant of RP3 and establishes a novel locus that is sufficiently defined for positional cloning.     

TB Treatment Delays in Odisha,India: Is It Expected Even after These Many Years of RNTCP Implementation?

Background

In India, the Revised National TB Control Programme (RNTCP) envisages initiation of TB treatment within seven days of diagnosis among smear-positive patients. After nearly two decades of RNTCP implementation, treatment delays are usually not expected.

Objectives

To determine the proportion of sputum smear-positive TB patients who were initiated on treatment after seven days and their associated risk factors.

Methods

The study was conducted in Cuttack and Rayagada districts of Odisha. It was a retrospective cohort study that involves review of TB treatment registers and laboratory registers for 2013.

Results

Among 1,800 pulmonary TB (PTB) patients, 1,074 (60%) had been initiated on treatment within seven days of diagnosis, 721 (40%) had been initiated on treatment more than seven days, and 354 (20%) had delays of more than 15 days. The mean duration between TB diagnosis and treatment initiation was 21 days with a range of 8–207 days (median = 14 days). Odds of treatment delay of more than seven days were 4.9 times (95% confidence interval [CI] 3.3-6.6) among those who had been previously treated, 6.2 times (95% CI 1.3-29.7) among those infected with HIV, and 1.8 times (95% CI 1.1-2.9) among those diagnosed outside district DMC.

Conclusion

Delay in initiation of TB treatment occurred in majority of the smear-positive patients. The RNTCP should focus on core areas of providing quality TB services with time-tested strategies. To have real-time monitoring mechanisms for diagnosed smear-positive TB patients is expected to be the way forward.