Incidence of moxifloxacin resistance in clinical Mycobacterium tuberculosis isolates in Houston, Texas.

Comprehensive data on the prevalence of quinolone resistance in Mycobacterium tuberculosis clinical isolates in the United States are scarce. By use of a systematic population-based approach, M. tuberculosis strains from tuberculosis (TB) cases were collected in Harris County, TX, in 2007 to 2008. The susceptibilities of M. tuberculosis isolates to moxifloxacin and ofloxacin were determined by the agar proportion indirect susceptibility method. Spoligotyping and 12-locus mycobacterial interspersed repetitive unit (MIRU12)-based genotyping of M. tuberculosis isolates were performed, and the gyrA, gyrB, Rv2686c, Rv2687c, and Rv2688c genes in quinolone-resistant and year-of-diagnosis-matched M. tuberculosis isolates were sequenced. Susceptibility testing was performed on 557 M. tuberculosis isolates, of which 10 (1.8%) were resistant to moxifloxacin. There was 100% concordance between ofloxacin and moxifloxacin susceptibilities. A quinolone was prescribed to at least 5 (50%) patients in the period preceding TB diagnosis. Multidrug-resistant TB (MDR-TB) was significantly associated with quinolone resistance (P = 0.01). Mutations in the quinolone resistance-determining region of gyrA were found for 50% of the resistant isolates. No other presumptive quinolone resistance-associated mutations were identified. We conclude that the incidence of moxifloxacin-resistant TB is low in Harris County and is associated with MDR-TB. Previous exposure to quinolones is common among patients with moxifloxacin resistance and warrants more careful evaluation.

Does directly observed therapy (DOT) reduce drug resistant tuberculosis?

BACKGROUND: Directly observed therapy (DOT) is a widely recommended and promoted strategy to manage tuberculosis (TB), however, there is still disagreement about the role of DOT in TB control and the impact it has on reducing the acquisition and transmission of drug resistant TB. This study compares the portion of drug resistant genotype clusters, representing recent transmission, within and between communities implementing programs differing only in their directly observed therapy (DOT) practices. METHODS: Genotype clusters were defined as 2 or more patient members with matching IS6110 restriction fragment length polymorphism (RFLP) and spoligotype patterns from all culture-positive tuberculosis cases diagnosed between January 1, 1995 and December 31, 2001. Logistic regression was used to compute maximum-likelihood estimates of odds ratios (ORs) and 95% confidence intervals (CIs) comparing cluster members with and without drug resistant isolates. In the universal DOT county, all patients received doses under direct observation of health department staff; whereas in selective DOT county, the majority of received patients doses under direct observation of health department staff, while some were able to self-administer doses. RESULTS: Isolates from 1,706 persons collected during 1,721 episodes of tuberculosis were genotyped. Cluster members from the selective DOT county were more than twice as likely than cluster members from the universal DOT county to have at least one isolate resistant to isoniazid, rifampin, and/or ethambutol (OR = 2.3, 95% CI: 1.7, 3.1). Selective DOT county isolates were nearly 5 times more likely than universal DOT county isolates to belong to clusters with at least 2 resistant isolates having identical resistance patterns (OR = 4.7, 95% CI: 2.9, 7.6). CONCLUSIONS: Universal DOT for tuberculosis is associated with a decrease in the acquisition and transmission of resistant tuberculosis.

Multiphasic approach reveals genetic diversity of environmental and patient isolates of Mycobacterium mucogenicum and Mycobacterium phocaicum associated with an outbreak of bacteremias at a Texas hospital.

Between March and May 2006, a Texas hospital identified five Mycobacterium mucogenicum bloodstream infections among hospitalized oncology patients using fluorescence high-performance liquid chromatography analysis of mycolic acids. Isolates from blood cultures were compared to 16 isolates from environmental sites or water associated with this ward. These isolates were further characterized by hsp65, 16S rRNA, and rpoB gene sequencing, hsp65 PCR restriction analysis, and molecular typing methods, including repetitive element PCR, random amplified polymorphic DNA PCR, and pulsed-field gel electrophoresis (PFGE) of large restriction fragments. Three of five patient isolates were confirmed as M. mucogenicum and were in a single cluster as determined by all identification and typing methods. The remaining two patient isolates were identified as different strains of Mycobacterium phocaicum by rpoB sequence analysis. One of these matched an environmental isolate from a swab of a hand shower in the patient's room, while none of the clinical isolates of M. mucogenicum matched environmental strains. Among the other 15 environmental isolates, 11 were identified as M. mucogenicum and 4 as M. phocaicum strains, all of which were unrelated by typing methods. Although the 16S rRNA gene sequences matched for all 14 M. mucogenicum isolates, there were two each of the hsp65 and rpoB sequevars, seven PCR typing patterns, and 12 PFGE patterns. Among the seven M. phocaicum isolates were three 16S rRNA sequevars, two hsp65 sequevars, two rpoB sequevars, six PCR typing patterns, and six PFGE patterns. This outbreak represents the first case of catheter-associated bacteremia caused by M. phocaicum and the first report of clinical isolates from a U.S. hospital. The investigation highlights important differences in the available typing methods for mycobacteria and demonstrates the genetic diversity of these organisms even within narrow confines of time and space.

Forty years of disinfectant failure: outbreak of postinjection Mycobacterium abscessus infection caused by contamination of benzalkonium chloride.

Benzalkonium chloride (BC) continues to be used as an antiseptic and contributes to serious outbreaks of disease. In July 1999, 6 postinjection joint infections caused by Mycobacterium abscessus were reported to the Texas Department of Health (Austin). We investigated this outbreak and identified 12 case patients who had been seen by the same physician and who had received an intra-articular or periarticular steroid injection during the period of 1 April through 31 July 1999. M. abscessus was cultured from either joint fluid or periarticular soft-tissue specimens obtained from 10 patients. We cultured environmental samples, and we compared isolates recovered from case patients with environmental isolates by pulsed-field gel electrophoresis and randomly amplified polymorphic DNA polymerase chain reaction (RAPD-PCR). Four environmental samples containing diluted BC yielded M. abscessus. Clinical and environmental strains of M. abscessus were indistinguishable by RAPD-PCR. The case patients' strain was resistant to BC. The use of BC as an antiseptic should be discontinued.

Using GIS technology to identify areas of tuberculosis transmission and incidence.

BACKGROUND: Currently in the U.S. it is recommended that tuberculosis screening and treatment programs be targeted at high-risk populations. While a strategy of targeted testing and treatment of persons most likely to develop tuberculosis is attractive, it is uncertain how best to accomplish this goal. In this study we seek to identify geographical areas where on-going tuberculosis transmission is occurring by linking Geographic Information Systems (GIS) technology with molecular surveillance. METHODS: This cross-sectional analysis was performed on data collected on persons newly diagnosed with culture positive tuberculosis at the Tarrant County Health Department (TCHD) between January 1, 1993 and December 31, 2000. Clinical isolates were molecularly characterized using IS6110-based RFLP analysis and spoligotyping methods to identify patients infected with the same strain. Residential addresses at the time of diagnosis of tuberculosis were geocoded and mapped according to strain characterization. Generalized estimating equations (GEE) analysis models were used to identify risk factors involved in clustering. RESULTS: Evaluation of the spatial distribution of cases within zip-code boundaries identified distinct areas of geographical distribution of same strain disease. We identified these geographical areas as having increased likelihood of on-going transmission. Based on this evidence we plan to perform geographically based screening and treatment programs. CONCLUSION: Using GIS analysis combined with molecular epidemiological surveillance may be an effective method for identifying instances of local transmission. These methods can be used to enhance targeted screening and control efforts, with the goal of interruption of disease transmission and ultimately incidence reduction.

Geographic differences in time to culture conversion in liquid media: Tuberculosis Trials Consortium study 28. Culture conversion is delayed in Africa.

BACKGROUND: Tuberculosis Trials Consortium Study 28, was a double blind, randomized, placebo-controlled, phase 2 clinical trial examining smear positive pulmonary Mycobacterium tuberculosis. Over the course of intensive phase therapy, patients from African sites had substantially delayed and lower rates of culture conversion to negative in liquid media compared to non-African patients. We explored potential explanations of this finding. METHODS: In TBTC Study 28, protocol-correct patients (n = 328) provided spot sputum specimens for M. tuberculosis culture in liquid media, at baseline and weeks 2, 4, 6 and 8 of study therapy. We compared sputum culture conversion for African and non-African patients stratified by four baseline measures of disease severity: AFB smear quantification, extent of disease on chest radiograph, cavity size and the number of days to detection of M. tuberculosis in liquid media using the Kaplan-Meier product-limit method. We evaluated specimen processing and culture procedures used at 29 study laboratories serving 27 sites. RESULTS: African TB patients had more extensive disease at enrollment than non-African patients. However, African patients with the least disease by the 4 measures of disease severity had conversion rates on liquid media that were substantially lower than conversion rates in non-African patients with the greatest extent of disease. HIV infection, smoking and diabetes did not explain delayed conversion in Africa. Some inter-site variation in laboratory processing and culture procedures within accepted practice for clinical diagnostic laboratories was found. CONCLUSIONS: Compared with patients from non-African sites, African patients being treated for TB had delayed sputum culture conversion and lower sputum conversion rates in liquid media that were not explained by baseline severity of disease, HIV status, age, smoking, diabetes or race. Further investigation is warranted into whether modest variation in laboratory processes substantially influences the efficacy outcomes of phase 2 TB treatment trials or if other factors (e.g., nutrition, host response) are involved. TRIAL REGISTRATION: ClinicalTrials.gov NCT00144417.

Rapid, standardized method for determination of Mycobacterium tuberculosis drug susceptibility by use of mycolic acid analysis.

Multidrug-resistant (MDR) Mycobacterium tuberculosis and extrensively drug-resistant (XDR) M. tuberculosis are emerging public health threats whose threats are compounded by the fact that current techniques for testing the susceptibility of M. tuberculosis require several days to weeks to complete. We investigated the use of high-performance liquid chromatography (HPLC)-based quantitation of mycolic acids as a means of rapidly determining drug resistance and susceptibility in M. tuberculosis. Standard susceptibility testing and determination of the MICs of drug-susceptible (n = 26) and drug-resistant M. tuberculosis strains, including MDR M. tuberculosis strains (n = 34), were performed by using the Bactec radiometric growth system as the reference method. The HPLC-based susceptibilities of the current first-line drugs, isoniazid (INH), rifampin (RIF), ethambutol (EMB), and pyrazinamide (PZA), were determined. The vials were incubated for 72 h, and aliquots were removed for HPLC analysis by using the Sherlock mycobacterial identification system. HPLC quantitation of total mycolic acid peaks (TMAPs) was performed with treated and untreated cultures. At 72 h, the levels of agreement of the HPLC method with the reference method were 99.5% for INH, EMB, and PZA and 98.7% for RIF. The inter- and intra-assay reproducibilities varied by drug, with an average precision of 13.4%. In summary, quantitation of TMAPs is a rapid, sensitive, and accurate method for antibiotic susceptibility testing of all first-line drugs currently used against M. tuberculosis and offers the potential of providing susceptibility testing results within hours, rather than days or weeks, for clinical M. tuberculosis isolates.

Mycobacterium arupense sp. nov., a non-chromogenic bacterium isolated from clinical specimens.

Several Mycobacterium-like organisms related to the Mycobacterium terrae complex have been isolated from clinical samples. In the clinical microbiology laboratory, partial 16S rRNA gene sequencing (approximately the first 500 bp) rather than full 16S rRNA gene sequencing is often used to identify Mycobacterium species. Partial 16S rRNA gene sequence analysis revealed 100 % similarity between 65 clinical isolates and Mycobacterium sp. MCRO 6 (GenBank accession no. X93032). Even after sequencing the nearly full-length 16S rRNA gene, closest similarity was only 99.6 % to Mycobacterium nonchromogenicum ATCC 19530(T). Sequencing of the nearly full-length 16S rRNA gene, the 16S-23S internal transcribed spacer region and the hsp65 gene did not reveal genotypic identity with the type strains of M. nonchromogenicum, M. terrae or Mycobacterium triviale. Although sequence analysis suggested that these clinical isolates represented a novel species, mycolic acid analysis by HPLC failed to distinguish them from M. nonchromogenicum. Therefore, phenotypic analysis including growth characterization, antibiotic susceptibility testing and biochemical testing was performed. These strains from clinical samples should be recognized as representing a novel species of the genus Mycobacterium, for which the name Mycobacterium arupense sp. nov. is proposed. The type strain is AR30097(T) (=ATCC BAA-1242(T) = DSM 44942(T)).

Polyphasic characterization reveals that the human pathogen Mycobacterium peregrinum type II belongs to the bovine pathogen species Mycobacterium senegalense.

Mycobacterium peregrinum consists of two taxa: types I and II. We evaluated 43 clinical type II strains from throughout the United States. They were responsible for soft-tissue and bone infections, catheter-related infections, and possible pneumonitis. By carbohydrate utilization, they were indistinguishable from type I strains, being D-mannitol and trehalose positive. However, they had a distinct susceptibility pattern that included intermediate ciprofloxacin MICs but low clarithromycin and doxycycline MICs of < or =1 microg/ml. These features were also shared by reference isolates of Mycobacterium senegalense from African bovine cases of "farcy." By 16S rRNA gene sequencing, the type II isolates shared 100% sequence identity with M. senegalense. Partial sequencing of the type II hsp65 gene (441 bp) revealed four sequevars showing > or =98.4% identity with each other and > or =98.6% identity with the sequence of five bovine strains of M. senegalense. There was < or =97.1% identity with M. peregrinum type I isolates and other Mycobacterium fortuitum group species. Sequencing of additional gene targets including the 16S-23S rDNA internal transcribed spacer region and the rpoB gene (partial sequence) revealed a similar phylogenetic grouping. DNA-DNA hybridization showed 76 to 99% relatedness between the bovine and human strains. These studies demonstrate that type II isolates are not isolates of M. peregrinum but represent human strains of M. senegalense. This study is the first to demonstrate this species as a human pathogen. Representative human M. senegalense strains include ATCC 35755 and newly submitted strains ATCC BAA-849, ATCC BAA-850, and ATCC BAA-851.

Sequence-based identification of aerobic actinomycetes.

We investigated the utility of 500-bp 16S rRNA gene sequencing for identifying clinically significant species of aerobic actinomycetes. A total of 28 reference strains and 71 clinical isolates that included members of the genera Streptomyces, Gordonia, and Tsukamurella and 10 taxa of Nocardia were studied. Methods of nonsequencing analyses included growth and biochemical analysis, PCR-restriction enzyme analysis of the 439-bp Telenti fragment of the 65 hsp gene, susceptibility testing, and, for selected isolates, high-performance liquid chromatography. Many of the isolates were included in prior taxonomic studies. Sequencing of Nocardia species revealed that members of the group were generally most closely related to the American Type Culture Collection (ATCC) type strains. However, the sequences of Nocardia transvalensis, N. otitidiscaviarum, and N. nova isolates were highly variable; and it is likely that each of these species contains multiple species. We propose that these three species be designated complexes until they are more taxonomically defined. The sequences of several taxa did not match any recognized species. Among other aerobic actinomycetes, each group most closely resembled the associated reference strain, but with some divergence. The study demonstrates the ability of partial 16S rRNA gene sequencing to identify members of the aerobic actinomycetes, but the study also shows that a high degree of sequence divergence exists within many species and that many taxa within the Nocardia spp. are unnamed at present. A major unresolved issue is the type strain of N. asteroides, as the present one (ATCC 19247), chosen before the availability of molecular analysis, does not represent any of the common taxa associated with clinical nocardiosis.

Transmission of drug-resistant tuberculosis in Texas and Mexico.

To examine the transmission of drug-resistant (DR) tuberculosis between Texas and Mexico, Mycobacterium tuberculosis isolates resistant to one or more of the first-line antimycobacterial drugs were obtained from 606 patients who resided in Texas and 313 patients who resided in Mexico, primarily within the state of Tamaulipas. The isolates were genotyped by IS6110-based restriction fragment length polymorphism (RFLP) analysis and spoligotyping. Of the 919 isolates genotyped, 413 (45%) grouped into 105 clusters containing 2 or more isolates with identical genotypes. In addition to having identical genotypes, identical drug resistance patterns were identified in 250 isolates in 78 clusters (DR clusters). Twenty DR clusters, containing isolates from 32% of the total number of patients infected with DR strains, were geographically distributed across Mexico and Texas. Within this population of 919 patients infected with DR isolates, the probability of being in a DR cluster was the same for residents of Mexico and Texas. In Texas, the significant independent predictors of clustering within DR clusters as opposed to genotype clusters were found to be race, age, country of birth, human immunodeficiency virus (HIV) infection status, and resistance to more than one drug. Specifically, isolates from African Americans, individuals under age 65, individuals born in the United States, and HIV-positive individuals were each more likely to be associated with a DR cluster. By contrast, no significant independent predictors of clustering in a DR cluster were identified in Mexico. Although some DR M. tuberculosis strains are geographically restricted, this study suggests that a number of strains are transmitted between Mexico and the United States.

Clinical and laboratory features of Mycobacterium mageritense.

Six clinical isolates of the nonpigmented, rapidly growing species Mycobacterium mageritense were recovered from sputum, bronchial wash, blood, sinus drainage, and two surgical wound infections from separate patients in Texas, New York, Louisiana, and Florida. The isolates matched the ATCC type strain by PCR restriction enzyme analysis of the 65-kDa hsp gene sequence of Telenti, high-performance liquid chromatography, biochemical reactions, and partial 16S rRNA gene sequencing. These are the first isolates of this species to be described in the United States and the first isolates to be associated with clinical disease. Susceptibility testing of all known isolates of the species revealed all isolates to be susceptible or intermediate to amikacin, cefoxitin, imipenem, and the fluoroquinolones and sulfonamides but resistant to clarithromycin. Because of their phenotypic and clinical similarity to isolates of the Mycobacterium fortuitum third biovariant complex (sorbitol positive), isolates of M. mageritense are likely to go undetected unless selected carbohydrate utilization or molecular identification methods are used.

Clinical and laboratory features of Mycobacterium porcinum.

Recent molecular studies have shown Mycobacterium porcinum, recovered from cases of lymphadenitis in swine, to have complete 16S rDNA sequence identity and >70% DNA-DNA homology with human isolates within the M. fortuitum third biovariant complex. We identified 67 clinical and two environmental isolates of the M. fortuitum third biovariant sorbitol-negative group, of which 48 (70%) had the same PCR restriction enzyme analysis (PRA) profile as the hsp65 gene of M. porcinum (ATCC 33776(T)) and were studied in more detail. Most U.S. patient isolates were from Texas (44%), Florida (19%), or other southern coastal states (15%). Clinical infections included wound infections (62%), central catheter infections and/or bacteremia (16%), and possible pneumonitis (18%). Sequencing of the 16S rRNA gene (1,463 bp) showed 100% identity with M. porcinum ATCC 33776(T). Sequencing of 441 bp of the hsp65 gene showed four sequevars that differed by 2 to 3 bp from the porcine strains. Clinical isolates were positive for arylsulfatase activity at 3 days, nitrate, iron uptake, D-mannitol, i-myo-inositol, and catalase at 68 degrees C. They were negative for L-rhamnose and D-glucitol (sorbitol). Clinical isolates were susceptible to ciprofloxacin, sulfamethoxazole, and linezolid and susceptible or intermediate to cefoxitin, clarithromycin, imipenem, and amikacin. M. porcinum ATCC 33776(T) gave similar results except for being nitrate negative. These studies showed almost complete phenotypic and molecular identity between clinical isolates of the M. fortuitum third biovariant D-sorbitol-negative group and porcine strains of M. porcinum and confirmed that they belong to the same species. Identification of M. porcinum presently requires hsp65 gene PRA or 16S rRNA or hsp65 gene sequencing.

Characterization of a novel group of mycobacteria and proposal of Mycobacterium sherrisii sp. nov.

We describe here the characterization of five isolates of Mycobacterium simiae-like organisms representing a novel group based on whole-cell fatty acid analysis and genotypic evaluation. Two of the five isolates in this study, W55 and W58, were previously considered to belong to M. simiae serotype 2. Analysis of cellular fatty acids by gas-liquid chromatography indicated a close clustering of this group, which was well differentiated from the other M. simiae-like species. Molecular characterization was performed by nucleic acid sequencing of the small subunit rRNA gene and the gene encoding the 65-kDa heat shock protein and genomic DNA hybridization. Sequence analysis of the entire 16S rRNA gene showed a unique sequence most closely related to those of M. triplex and M. simiae. The hsp65 partial gene sequence was identical for the five isolates, with 97% identity to the M. simiae type strain. However, qualitative whole genomic DNA hybridization analysis confirmed that this group is genetically distinct from M. simiae and M. triplex. Antimicrobial susceptibilities for this group resemble those of M. simiae and M. lentiflavum. We conclude that this group represents a unique Mycobacterium species for which we propose the name Mycobacterium sherrisii sp. nov.