c Sequencing of the Mycobacterium tuberculosis pncA gene allows for pyrazinamide susceptibility testing. We summarize data on pncA polymorphisms that do not confer resistance at a susceptibility breakpoint of 100 g/ml pyrazinamide in MGIT within a cohort of isolates from South Africa and the U.S. Centers for Disease Control and Prevention. C ulture-based drug susceptibility testing (DST) using Bactec MGIT 960 PZA medium (Becton Dickinson, Sparks, MD) at 100 g/ml is the current gold standard test for pyrazinamide (PZA) resistance (1). False resistance results are known to occur with this assay (1, 2), which may be the result of alkalinization of the medium due to a high inoculum size or the presence of bovine serum albumin (3). The uses of alternative susceptibility breakpoint concentrations or different media are additional factors that may contribute to disparities in PZA susceptibility results (4-6). A further limitation of culture-based methods is the long turnaround time, which can exceed 20 days (7-9). Molecular methods offer an alternative strategy for the detection of PZA susceptibility. These methods detect polymorphisms in the 561-bp pncA gene, which encodes the pyrazinamidase (PZase) enzyme that is responsible for conversion of PZA (prodrug) to pyrazinoic acid (active form) (10). More than 325 polymorphisms (single nucleotide polymorphisms [SNPs], insertions, and deletions) throughout the length of the pncA gene and in the promoter region have been described, complicating molecular detection (11)(12)(13)(14). A good correlation (sensitivity of Ͼ90%) between pncA polymorphisms in circulating isolates and phenotypic susceptibility results have been observed for PZA (15)(16)(17)(18). Incomplete correlation of pncA molecular results with culture-based PZA testing has been ascribed to poor reproducibility of the phenotypic test or the presence of alternative genetic mechanisms for resistance, including polymorphisms in the rpsA gene (19,20). Additionally, a few pncA mutations have been reported in the absence of phenotypic resistance (15), but the role of such mutations in PZA resistance has not been thoroughly investigated. This study aimed to collate data on pncA polymorphisms in clinical isolates that do not confer resistance at a susceptibility breakpoint of 100 g/ml PZA. To capture the spectrum of pncA mutations not associated with phenotypic PZA resistance, we performed a comprehensive literature search. In the 77 papers reporting genotypic and phenotypic PZA susceptibility (see Table S1 in the supplemental material), 77 different pncA polymorphisms in 71 different codons were reported to have a PZA-susceptible phenotype using either Bactec MGIT 960 PZA (Becton Dickinson, Sparks, MD), Bactec 460 PZA (Becton Dickinson, Sparks, MD) or the Wayne assay (21). Forty-seven (61%) of these polymorphisms have also been reported in PZA-resistant isolates. These inconsistent phenotypic results may be due to technical difficulties of phenotypic PZA assays or MICs that are close to the breakpoint. Another 26 (33.7%...

Neisseria gonorrhoeae is an obligate human pathogen that causes the common sexually transmitted infection gonorrhea. Gonococcal infections cause significant morbidity, particularly among women, as the organism ascends to the upper reproductive tract, resulting in pelvic inflammatory disease, ectopic pregnancy, and infertility. In the last few years, antibiotic resistance rates have risen dramatically, leading to severe restriction of treatment options for gonococcal disease. Gonococcal infections do not elicit protective immunity, nor is there an effective vaccine to prevent the disease. Thus, further understanding of the expression, function, and regulation of surface antigens could lead to better treatment and prevention modalities in the future. In the current study, we determined that an iron-repressed regulator, MpeR, interacted specifically with the DNA sequence upstream of fetA and activated FetA expression. Interestingly, MpeR was previously shown to regulate the expression of gonococcal antimicrobial efflux systems. We confirmed that the outer membrane transporter FetA allows gonococcal strain FA1090 to utilize the xenosiderophore ferric enterobactin as an iron source. However, we further demonstrated that FetA has an extended range of substrates that encompasses other catecholate xenosiderophores, including ferric salmochelin and the dimers and trimers of dihydroxybenzoylserine. We demonstrated that fetA is part of an iron-repressed, MpeR-activated operon which putatively encodes other iron transport proteins. This is the first study to describe a regulatory linkage between antimicrobial efflux and iron transport in N. gonorrhoeae. The regulatory nidus that links these systems, MpeR, is expressed exclusively by pathogenic neisseriae and is therefore expected to be an important virulence factor.

Previous studies have shown that the MpeR transcriptional regulator produced by Neisseria gonorrhoeae represses the expression of mtrF, which encodes a putative inner membrane protein (MtrF). MtrF works as an accessory protein with the Mtr efflux pump, helping gonococci to resist high levels of diverse hydrophobic antimicrobials. Regulation of mpeR has been reported to occur by an iron-dependent mechanism involving Fur (ferric uptake regulator). Collectively, these observations suggest the presence of an interconnected regulatory system in gonococci that modulates the expression of efflux pump protein-encoding genes in an iron-responsive manner. Herein, we describe this connection and report that levels of gonococcal resistance to a substrate of the mtrCDE-encoded efflux pump can be modulated by MpeR and the availability of free iron. Using microarray analysis, we found that the mtrR gene, which encodes a direct repressor (MtrR) of mtrCDE, is an MpeR-repressed determinant in the late logarithmic phase of growth when free iron levels would be reduced due to bacterial consumption. This repression was enhanced under conditions of iron limitation and resulted in increased expression of the mtrCDE efflux pump operon. Furthermore, as judged by DNA-binding analysis, MpeR-mediated repression of mtrR was direct. Collectively, our results indicate that both genetic and physiologic parameters (e.g., iron availability) can influence the expression of the mtr efflux system and modulate levels of gonococcal susceptibility to efflux pump substrates.

Resistance to the first-line antituberculosis (TB) drug isoniazid (INH) is widespread, and the mechanism of resistance is unknown in approximately 15% of INH-resistant (INH-R) strains. To improve molecular detection of INH-R TB, we used whole-genome sequencing (WGS) to analyze 52 phenotypically INH-R complex (MTBC) clinical isolates that lacked the common S315T or promoter mutations. Approximately 94% (49/52) of strains had mutations at known INH-associated loci that were likely to confer INH resistance. All such mutations would be detectable by sequencing more DNA adjacent to existing target regions. Use of WGS minimized the chances of missing infrequent INH resistance mutations outside commonly targeted hotspots. We used recombineering to generate 12 observed clinical mutations in the pansusceptible H37Rv reference strain and determined their impact on INH resistance. Our functional genetic experiments have confirmed the role of seven suspected INH resistance mutations and discovered five novel INH resistance mutations. All recombineered mutations conferred resistance to INH at a MIC of ≥0.25 μg/ml and should be added to the list of INH resistance determinants targeted by molecular diagnostic assays. We conclude that WGS is a useful tool for detecting uncommon INH resistance mutations that would otherwise be missed by current targeted molecular testing methods and suggest that its use (or use of expanded conventional or next-generation-based targeted sequencing) may provide earlier diagnosis of INH-R TB.