Fluoroquinolone antibiotics are among the most potent second-line drugs used for treatment of multidrug-resistant tuberculosis (MDR TB), and resistance to this class of antibiotics is one criterion for defining extensively drug resistant tuberculosis (XDR TB). Fluoroquinolone resistance in Mycobacterium tuberculosis has been associated with modification of the quinolone resistance determining region (QRDR) of gyrA. Recent studies suggest that amino acid substitutions in gyrB may also play a crucial role in resistance, but functional genetic studies of these mutations in M. tuberculosis are lacking. In this study, we examined twenty six mutations in gyrase genes gyrA (seven) and gyrB (nineteen) to determine the clinical relevance and role of these mutations in fluoroquinolone resistance. Transductants or clinical isolates harboring T80A, T80A+A90G, A90G, G247S and A384V gyrA mutations were susceptible to all fluoroquinolones tested. The A74S mutation conferred low-level resistance to moxifloxacin but susceptibility to ciprofloxacin, levofloxacin and ofloxacin, and the A74S+D94G double mutation conferred cross resistance to all the fluoroquinolones tested. Functional genetic analysis and structural modeling of gyrB suggest that M330I, V340L, R485C, D500A, D533A, A543T, A543V and T546M mutations are not sufficient to confer resistance as determined by agar proportion. Only three mutations, N538D, E540V and R485C+T539N, conferred resistance to all four fluoroquinolones in at least one genetic background. The D500H and D500N mutations conferred resistance only to levofloxacin and ofloxacin while N538K and E540D consistently conferred resistance to moxifloxacin only. Transductants and clinical isolates harboring T539N, T539P or N538T+T546M mutations exhibited low-level resistance to moxifloxacin only but not consistently. These findings indicate that certain mutations in gyrB confer fluoroquinolone resistance, but the level and pattern of resistance varies among the different mutations. The results from this study provide support for the inclusion of the QRDR of gyrB in molecular assays used to detect fluoroquinolone resistance in M. tuberculosis .

Since the discovery of streptomycin's bactericidal activity against Mycobacterium tuberculosis, aminoglycosides have been utilized to treat tuberculosis (TB). Today, the aminoglycosides kanamycin and amikacin are used to treat multidrug-resistant (MDR) TB, and resistance to any of the second-line injectable antibiotics, including kanamycin, amikacin, or capreomycin, is a defining characteristic of extensively drug-resistant (XDR) TB. Resistance to kanamycin and streptomycin is thought to be due to the acquisition of unlinked chromosomal mutations. However, we identified eight independent mutations in the 5= untranslated region of the transcriptional activator whiB7 that confer low-level resistance to both aminoglycosides. The mutations lead to 23-to 145-fold increases in whiB7 transcripts and subsequent increased expression of both eis (Rv2416c) and tap (Rv1258c). Increased expression of eis confers kanamycin resistance in these mutants, while increased expression of tap, which encodes an efflux pump, is a previously uncharacterized mechanism of low-level streptomycin resistance. Additionally, high-level resistance to streptomycin arose at a much higher frequency in whiB7 mutants than in a wild-type (WT) strain. Although whiB7 is typically associated with intrinsic antibiotic resistance in M. tuberculosis, these data suggest that mutations in an uncharacterized regulatory region of whiB7 contribute to cross-resistance against clinically used second-line antibiotics. As drug resistance continues to develop and spread, understanding the mechanisms and molecular basis of antibiotic resistance is critical for the development of rapid molecular tests to diagnose drug-resistant TB strains and ultimately for designing regimens to treat drug-resistant cases of TB.

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Mycobacterium tuberculosis is an example of a bacterial pathogen with a specialized SecA2-dependent protein export system that contributes to its virulence. Our understanding of the mechanistic basis of SecA2-dependent export and the role(s) of the SecA2 pathway in M. tuberculosis pathogenesis has been hindered by our limited knowledge of the proteins exported by the pathway.

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Summary Mycobacterium tuberculosis proteins that are exported out of the bacterial cytoplasm are ideally positioned to be virulence factors; however, the functions of individual exported proteins remain largely unknown. Previous studies identified Rv0199 as an exported membrane protein of unknown function. Here, we characterized the role of Rv0199 in M. tuberculosis virulence using an aerosol model of murine infection. Rv0199 appears to be a member of a Mce-associated membrane (Mam) protein family leading us to rename it OmamA, for orphaned Mce-associated membrane protein A. Consistent with a role in Mce transport, we showed OmamA is required for cholesterol import, which is a Mce4-dependent process. We further demonstrated a function for OmamA in stabilizing protein components of the Mce1 transporter complex. These results indicate a function of OmamA in multiple Mce transporters and one that may be analogous to the role of VirB8 in stabilizing Type IV secretion systems, as structural similarities between Mam proteins and VirB8 proteins are predicted by the Phyre 2 program. In this study, we provide functional information about OmamA and shed light on the function of Mam family proteins in Mce transporters.

Resistance to key first-line drugs is a major hurdle to achieve the global end tuberculosis (TB) targets. A prodrug, pyrazinamide (PZA) is the only drug, effective in latent TB, recommended in drug resistance and susceptible Mycobacterium tuberculosis (MTB) isolates. The prodrug conversion into active form, pyrazinoic acid (POA), required the activity of pncA gene encoded pyrazinamidase (PZase). Although pncA mutations have been commonly associated with PZA resistance but a small number of resistance cases have been associated with mutationss in RpsA protein. Here in this study a total of 69 PZA resistance isolates have been sequenced for pncA mutations. However, samples that were found PZA resistant but pncA wild type ( pncA WT ), have been sequenced for rpsA and panD genes mutation. We repeated a drug susceptibility testing according to the WHO guidelines on 18 pncA WT MTB isolates. The rpsA and panD genes were sequenced. Out of total 69 PZA resistant isolates, 51 harbored 36 mutations in pncA gene (GeneBank Accession No. MH46111) while, fifteen different mutations including seven novel, were detected in the fourth S1 domain of RpsA known as C-terminal (MtRpsA CTD ) end. We did not detect any mutations in panD gene. Among the rpsA mutations, we investigated the molecular mechanism of resistance behind mutations, D342N, D343N, A344P, and I351F, present in the MtRpsA CTD through molecular dynamic simulations (MD). WT showed a good drug binding affinity as compared to mutants (MTs), D342N, D343N, A344P, and I351F. Binding pocket volume, stability, and fluctuations have been altered whereas the total energy, protein folding, and geometric shape analysis further explored a significant variation between WT and MTs. In conclusion, mutations in MtRpsA CTD might be involved to alter the RpsA activity, resulting in drug resistance. Such molecular mechanism behind resistance may provide a better insight into the resistance mechanism to achieve the global TB control targets.

Methicillin-resistant staphylococci (MRS) pose a challenge to clinicians and health administrators in human medicine, but MRS infections in cats and dogs are not perceived as a problem in veterinary medicine. Ten methicillin-resistant staphylococcal isolates obtained from healthy and diseased cats and dogs were subjected to partial DNA sequencing of the mecA gene. Sequence analysis shows that MRS isolates from both healthy and diseased cats and dogs can harbor the mecA gene. The mecA genes of animal isolates were identical to that found in human MRS strains, and therefore the possibility of zoonotic transfer must be considered.Methicillin-resistant Staphylococcus aureus (MRSA) isolates have been identified as a serious cause of nosocomial infections, and more recently, community-acquired MRSA has been recognized as an emerging problem in a number of countries. The resultant infections include a range of conditions from minor skin infections to severe life-threatening conditions. Staphylococcal resistance to methicillin is attributable to multiple mechanisms (6), but the most important mechanism from the standpoint of nosocomial infections and treatment appears to be intrinsic resistance and is due to the expression of low-affinity penicillin-binding protein 2A (11,22,30). Penicillin-binding protein 2A is highly conserved among staphylococci and is encoded by the chromosomal gene mecA (3).Cats and dogs have become an integral part of modern society, especially in developed countries, and attention is given to their care and welfare. A large proportion of the human population is in contact with cats and dogs on a daily basis, and so there is the potential for transfer of bacteria or resistance genes between companion animals and humans (5, 9). Studies conducted in recent years have shown that staphylococci isolated from cats and dogs have become resistant to methicillin (8,16,19,20,24,25,26,31,33). However, none of these studies on methicillin-resistant staphylococci have compared the mecA gene from these animals' isolates with that from human MRSA strains. Although methicillin or other antistaphylococcal penicillins such as oxacillin and cloxacillin are not commonly used antibiotics in the treatment of staphylococcal infections in cats and dogs, there are clear public health implications related to the presence of methicillin-resistant staphylococci on the skin of cats and dogs. In this study, we report the isolation, antibiotic susceptibility, and partial nucleotide sequence determination of 2 kbp of the mecA gene of staphylococci isolated from diseased and healthy cats and dogs. MATERIALS AND METHODS Isolation of staphylococci.A total of 55 healthy cats and 51 healthy dogs undergoing desexing or boarding at two veterinary clinics in Adelaide were sampled. One hundred forty-one diseased dogs and five diseased cats with various skin infections were also sampled. Staphylococci were isolated from swabs taken from the skin and lesions of the animals. A selective medium, GiolitiCantoni broth (Oxoid, Basingstoke, Engl...