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 .

Background Human coronaviruses (HCoVs) have been detected in children with upper and lower respiratory symptoms but little is known about their relationship with severe respiratory illness. Objective To compare the prevalence of HCoV species among children hospitalized for acute respiratory illness and/or fever with asymptomatic controls and to assess the severity of outcomes among hospitalized children with HCoV compared with other respiratory viruses. Methods From December 2003–April 2004 and October 2004–April 2005, we conducted prospective, population-based surveillance of children <5 years of age hospitalized for ARI/fever in three U.S. counties. Asymptomatic outpatient controls were enrolled concurrently. Nasal/throat swabs were tested for HCoV species HKU1, NL63, 229E, and OC43 by RT-PCR. Specimens from hospitalized children were also tested for other common respiratory viruses. Demographic and medical data were collected by parent/guardian interview and medical chart review. Results Overall, HCoV was detected in 113 (7.6%) of 1,481 hospitalized children (83 [5.7%] after excluding 30 cases coinfected with other viruses) and 53 (7.1%) of 742 controls. The prevalence of HCoV or individual species was not significantly higher among hospitalized children than controls. Hospitalized children testing positive for HCoV alone tended to be less ill than those infected with other viruses, while those coinfected with HCoV and other viruses were clinically similar to those infected with other viruses alone. Conclusion In this study of children hospitalized for acute respiratory illness and/or fever, HCoV infection was not associated with hospitalization or with increased severity of illness.

The attachment organelle of Mycoplasma pneumoniae is a polar, tapered cell extension containing an intracytoplasmic, electron-dense core. This terminal structure is the leading end in gliding motility, and its duplication is thought to precede cell division, raising the possibility that mutations affecting cytadherence also confer a defect in motility or cell development. Mycoplasma surface protein P30 is associated with the attachment organelle, and P30 mutants II-3 and II-7 do not cytadhere. In this study, the recombinant wild-type but not the mutant II-3 p30 allele restored cytadherence when transformed into P30 mutants by recombinant transposon delivery. The mutations associated with loss of P30 in mutant II-3 and reacquisition of P30 in cytadhering revertants thereof were identified by nucleotide sequencing of the p30 gene. Morphological abnormalities that included ovoid or multilobed cells having a poorly defined tip structure were associated with loss of P30. Digital image analysis confirmed quantitatively the morphological differences noted visually. Transformation of the P30 mutants with the wild-type p30 allele restored a normal morphology, as determined both visually and by digital image analysis, suggesting that P30 plays a role in mycoplasma cell development. Finally, the P30 mutants localized the adhesin protein P1 to the terminal organelle, indicating that P30 is not involved in P1 trafficking but may be required for its receptor-binding function.

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%...