The Mycobacterium tuberculosis mmpL7 gene, encoding a hypothetical resistance nodulation division transporter, confers a high resistance level to isoniazid when overexpressed in Mycobacterium smegmatis. The resistance level decreased in the presence of the efflux pump inhibitors reserpine and CCCP (carbonyl cyanide m-chlorophenylhydrazone). Energy-dependent efflux of isoniazid from M. smegmatis cells expressing the mmpL7 gene was observed.The intrinsic resistance of Mycobacterium tuberculosis to most antibiotics is generally attributed to the low permeability of the mycobacterial cell wall, due to its specific lipid-rich composition and structure (2). However, since the intracellular concentration of a given drug depends on the balance between its influx and efflux, along with cell wall permeability, active efflux systems also provide resistance by extruding the drug molecules that enter the cell.Bacterial drug efflux pumps have been classified into five families (11). The genome of M. tuberculosis contains genes encoding drug efflux transporters from all of these families (http://www.membranetransport.org), and, as described in a recent review, several mycobacterial drug efflux pumps have been identified and characterized experimentally (8).Resistance nodulation division (RND) transporters have been found in all major kingdoms of living organisms, but they seem to be involved in drug efflux only in gram-negative bacteria. The AcrAB/TolC drug efflux pump of Escherichia coli provides a prototype for such export systems, with AcrB constituting the membrane pump itself, AcrA the membrane fusion protein, and TolC the outer membrane component (12).Interestingly, the genome sequence of M. tuberculosis revealed the presence of 13 putative transmembrane proteins, predicted to be transport proteins of the RND superfamily (5). Since these proteins appear to be confined to mycobacteria, they have been designated MmpL (mycobacterial membrane proteins, large). The hydrophobic nature of the MmpL proteins and the close association of four of their genes with those involved in lipid metabolism suggest that they may be naturally involved in the transport of fatty acids (13). Indeed, MmpL8 is involved in sulfolipid-1 biosynthesis by transporting a precursor of this molecule (6, 9), while the MmpL7 protein catalyzes the export of phthiocerol dimycocerosate (PDIM) in M. tuberculosis (3). A mutant lacking the mmpL7 gene was severely attenuated for growth in the lungs (7).Given the similarities with other RND transporters, it is possible that the MmpL proteins can also act in drug efflux.In this paper, we demonstrate that the mmpL7 gene from M. tuberculosis confers a high level of resistance to isoniazid (INH) when overexpressed in Mycobacterium smegmatis. The resistance level significantly decreases in the presence of efflux inhibitors. We also observed energy-dependent efflux of INH from M. smegmatis cells expressing the mmpL7 gene.Transformation of the M. tuberculosis cosmid library into M. smegmatis and selection for INH resistan...

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To evaluate the water compartment antibiotic-resistance contamination rates, 11 wells, five streams, and four treatment plants located in the Oltrepò Pavese area were screened for the presence of third generation cephalosporins resistant Gram-negative bacteria. Enterobacteriaceae were also characterized for the Extended-Spectrum-β-Lactamases (ESBLs), carbapenemases, and mcr-1 genes presence. From December 2014 to November 2015, 246 water samples were filtered, plated on Plate Count Agar, MacConkey Agar, and MacConkey Agar with cefotaxime. Isolates were species identified using AutoSCAN-4-System and ESBLs, carbapenemases, and colistin resistance determinants were characterized by PCR, sequencing, and microarray. Plasmid conjugative transfer experiments, PCR-based Replicon typing, Pulsed-Field Gel Electrophoresis, Multi-Locus-Sequence-Typing, and in-silico plasmid characterization were performed. A total of 132 enterobacteria isolates grew on MacConkey agar with cefotaxime: 82 (62.1%) were obtained from streams, 41 (31.1%) from treatment plants, and 9 (6.8%) from wells. Thirty out of 132 (22.7%) isolates, mainly belonging to Escherichia coli (n = 15) species, showed a synergic effect with piperacillin-tazobactam. A single ESBL gene of blaCTX−M-type was identified in 19/30 isolates. In further two E. coli strains, a blaCTX−M−1 gene co-existed with a blaSHV-type ESBL determinant. A blaSHV−12 gene was detected in two isolates of E. coli (n = 1) and Klebsiella oxytoca (n = 1), while any ESBL determinant was ascertained in seven Yersinia enterocolitica strains. A blaDHA-type gene was detected in a cefoxitin resistant Y. enterocolitica from a stream. Interestingly, two Klebsiella pneumoniae strains of ST307 and ST258, collected from a well and a wastewater treatment plant, resulted KPC-2, and KPC-3 producers, respectively. Moreover, we report the first detection of mcr-1.2 ST10 E. coli on a conjugative IncX4 plasmid (33.303 bp in size) from a stream of Oltrepò Pavese (Northern Italy). Both ESBLs E. coli and ESBLs/carbapenemase-producing K. pneumoniae strains showed clonal heterogeneity by Pulsed-Field Gel Electrophoresis and Multi-Locus-Sequence-Typing. During one-year study and taking in account the whole Gram-negative bacterial population, an average percentage of cefotaxime resistance of 69, 32, and 10.3% has been obtained for the wastewater treatment plants, streams, and wells, respectively. These results, of concern for public health, highlight the need to improve hygienic measures to reduce the load of discharged bacteria with emerging resistance mechanisms.

The rapid spread of multidrug-resistant Mycobacterium tuberculosis (MDR-TB) and extensively drug-resistant M. tuberculosis (XDR-TB) strains demands new antitubercular drugs that overcome the problem of drug-resistant strains (4). Recently, two different classes of promising new antitubercular agents, the benzothiazinones (BTZ) and dinitrobenzamides (DNB), were found to be highly active against M. tuberculosisresistant and -sensitive strains, including XDR and MDR strains (2, 3). The lead compound belonging to the BTZ class, 2-[2-S-methyl-1,4-dioxa-8-azaspiro[4.5]dec-8-yl]-8-nitro-6-(trifluoromethyl)-4H-1,3-benzothiazin-4-one (or BTZ043), has been used in this study. Interestingly, both BTZ and DNB drugs have the same target, the heterodimeric decaprenylphosphoryl-␤-D-ribose 2Ј-epimerase, encoded by the dprE1 (Rv3790) and dprE2 (Rv3791) genes (3). DprE1 and DprE2 are involved in the transformation of decaprenylphosphoryl-D-ribose to decaprenylphosphoryl-D-arabinose, which is the sole precursor for arabinogalactan and lipoarabinomannan synthesis in the mycobacterial cell wall (3, 7). Moreover, dprE1 is essential for M. tuberculosis growth (5).In all the spontaneous BTZ-resistant laboratory mutants, the Cys387 codon within dprE1 was replaced by a Ser or Gly codon. Furthermore, the Cys387 codon was shown to be highly conserved in several actinobacterial species, with the exception of Mycobacterium avium, which was found to be naturally resistant to BTZ043, due to replacement of the Cys387 by an Ala codon (3).The pharmaceutical industry usually estimates the likelihood of the development of resistance against a new drug by focusing on the mutation resistance rate, on the assumption that this rate is a major determinant of resistance development in clinical settings (1). Therefore, drug targets for which the resistance mechanisms have the most negative effect on fitness are expected to show a low resistance development rate (1). It is noteworthy that in vitro M. tuberculosis BTZ-resistant mutants were rare, arising at a frequency of Ͻ10 Ϫ8 (3). The aim of this work is to investigate whether any of the present M. tuberculosis clinical isolates are BTZ resistant.A total of 240 M. tuberculosis clinical isolates (including MDR-and XDR-TB strains) from four different European hospitals were screened for mutations in the Cys387 codon of dprE1 and for BTZ sensitivity. Seventy-eight M. tuberculosis

ObjectiveLingual orthodontics is becoming more popular in dental practice. The purpose of the present investigation was to compare plaque formation on teeth bonded with the same bracket onto buccal or lingual surface, with non-bonded control teeth, via an in vivo growth experiment over a 30-day period. Material and MethodsA randomized controlled trial with split-mouth design was set up enrolling 20 dental students. Within each subject sites with buccal and lingual brackets and control sites were followed. Clinical periodontal parameters (periodontal pocket depth: PPD; bleeding on probing: BOP) were recorded at baseline and on days 1, 7 and 30. Microbiological samples were taken from the brackets and the teeth on days 1, 7 and 30 to detect colony-forming units (CFU). Total CFU, streptococci CFU and anaerobe CFU were measured. ResultsNo significant differences (P>0.05) were found between buccal and lingual brackets in terms of clinical periodontal parameters and microbiological values. ConclusionBracket position does not have significant impact on bacterial load and on periodontal parameters.