M. Donald Cave

Global Phylogeny of Mycobacterium tuberculosis Based on Single Nucleotide Polymorphism (SNP) Analysis: Insights into Tuberculosis Evolution, Phylogenetic Accuracy of Other DNA Fingerprinting Systems, and Recommendations for a Minimal Standard SNP Set

We analyzed a global collection of Mycobacterium tuberculosis strains using 212 single nucleotide polymorphism (SNP) markers. SNP nucleotide diversity was high (average across all SNPs, 0.19), and 96% of the SNP locus pairs were in complete linkage disequilibrium. Cluster analyses identified six deeply branching, phylogenetically distinct SNP cluster groups (SCGs) and five subgroups. The SCGs were strongly associated with the geographical origin of the M. tuberculosis samples and the birthplace of the human hosts. The most ancestral cluster (SCG-1) predominated in patients from the Indian subcontinent, while SCG-1 and another ancestral cluster (SCG-2) predominated in patients from East Asia, suggesting that M. tuberculosis first arose in the Indian subcontinent and spread worldwide through East Asia. Restricted SCG diversity and the prevalence of less ancestral SCGs in indigenous populations in Uganda and Mexico suggested a more recent introduction of M. tuberculosis into these regions. The East African Indian and Beijing spoligotypes were concordant with SCG-1 and SCG-2, respectively; X and Central Asian spoligotypes were also associated with one SCG or subgroup combination. Other clades had less consistent associations with SCGs. Mycobacterial interspersed repetitive unit (MIRU) analysis provided less robust phylogenetic information, and only 6 of the 12 MIRU microsatellite loci were highly differentiated between SCGs as measured by GST. Finally, an algorithm was devised to identify two minimal sets of either 45 or 6 SNPs that could be used in future investigations to enable global collaborations for studies on evolution, strain differentiation, and biological differences of M. tuberculosis.

Population Genetics Study of Isoniazid Resistance Mutations and Evolution of Multidrug-Resistant Mycobacterium tuberculosis

ABSTRACT The molecular basis for isoniazid resistance in Mycobacterium tuberculosis is complex. Putative isoniazid resistance mutations have been identified in katG, ahpC, inhA, kasA, and ndh. However, small sample sizes and related potential biases in sample selection have precluded the development of statistically valid and significant population genetic analyses of clinical isoniazid resistance. We present the first large-scale analysis of 240 alleles previously associated with isoniazid resistance in a diverse set of 608 isoniazid-susceptible and 403 isoniazid-resistant clinical M. tuberculosis isolates. We detected 12 mutant alleles in isoniazid-susceptible isolates, suggesting that these alleles are not involved in isoniazid resistance. However, mutations in katG, ahpC, and inhA were strongly associated with isoniazid resistance, while kasA mutations were associated with isoniazid susceptibility. Remarkably, the distribution of isoniazid resistance-associated mutations was different in isoniazid-monoresistant isolates from that in multidrug-resistant isolates, with significantly fewer isoniazid resistance mutations in the isoniazid-monoresistant group. Mutations in katG315 were significantly more common in the multidrug-resistant isolates. Conversely, mutations in the inhA promoter were significantly more common in isoniazid-monoresistant isolates. We tested for interactions among mutations and resistance to different drugs. Mutations in katG, ahpC, and inhA were associated with rifampin resistance, but only katG315 mutations were associated with ethambutol resistance. There was also a significant inverse association between katG315 mutations and mutations in ahpC or inhA and between mutations in kasA and mutations in ahpC. Our results suggest that isoniazid resistance and the evolution of multidrug-resistant strains are complex dynamic processes that may be influenced by interactions between genes and drug-resistant phenotypes.

Identification of Risk Factors for Extrapulmonary Tuberculosis

The proportion of extrapulmonary tuberculosis cases in the United States has increased from 16% of tuberculosis cases, in 1991, to 20%, in 2001. To determine associations between the demographic, clinical, and life style characteristics of patients with tuberculosis and the occurrence of extrapulmonary tuberculosis, a retrospective case-control study was conducted. This study included 705 patients with tuberculosis, representing 98% of the culture-proven cases of tuberculosis in Arkansas from 1 January 1996 through 31 December 2000. A comparison between 85 patients with extrapulmonary tuberculosis (case patients) and 620 patients with pulmonary tuberculosis (control patients) showed women (OR, 1.98; 95% CI, 1.25-3.13), non-Hispanic blacks (OR, 2.38; 95% CI, 1.42-3.97), and HIV-positive persons (OR, 4.93; 95% CI, 1.95-12.46) to have a significantly higher risk for extrapulmonary tuberculosis than men, non-Hispanic whites, and HIV-negative persons. This study expands the knowledge base regarding the epidemiology of extrapulmonary tuberculosis and enhances our understanding of the relative contribution of host-related factors to the pathogenesis of tuberculosis.

IS6110: Conservation of sequence in the Mycobacterium tuberculosis complex and its utilization in DNA fingerprinting

Multiple copies of an insertion sequence, IS6110, were shown to be present in the genome of members of the Mycobacterium tuberculosis complex (M. tuberculosis and M. bovis). Ten to 12 copies are present in various strains of M. tuberculosis, while strains of M. bovis contain only one to three copies. IS6110 was not detected in the DNA of other species of mycobacteria. Restriction endonuclease analysis indicated that the sequence of IS6110 is conserved across strain and species lines. Hybridization to the insertion sequence can be used to detect restriction fragment length polymorphism reflecting divergence in the sequence of regions flanking the various copies of IS6110. These differences were used to fingerprint various strains of the M. tuberculosis complex.

The Home Environment and Salmonellosis in Children

Objective.  To explore the role of foods and the home environment in the development ofSalmonella infections in infants and children. Methods.  Home investigations were conducted of patients younger than 4 years of age infected with Salmonella. Cultures were obtained from foods, persons residing in the home, animals/pets/insects, and environmental sources. Like serotypes encountered in the index patients and isolates from the home underwent typing with pulsed-field gel electrophoresis. Results.  Home inspections were conducted in ∼66% of eligible homes on the average of 3.4 days after the confirmation of theSalmonella isolate. A total of 526 cultures from 50 homes were obtained from foods (120), household members (73), refrigerators (52), water (47), countertops (46), soil (42), can-openers (36), vacuum cleaners (34), animals/pets/insects (26), and others (50). Isolates with a serotype identical to those in the index patient were found in 16 homes, 3 of which included an isolate of a second serotype, and an isolate of a different serotype was recovered in 3 homes. The pulsed-field gel electrophoresis patterns of the isolates of identical serotypes from the subjects and from their environment were indistinguishable in all but 2 patients. Among isolates of the same serotype encountered in different homes, all patterns were different. The identical serotype was found in multiple locations (4), dirt surrounding front doors (4), household members (3), vacuum cleaner (1), animals/pets/insects (1), and a refrigerator shelf (1). Conclusions.  These data illustrate the importance of the childs environment in the development of salmonellosis. Clinicians should concentrate on educating the parents about the environmental spread of Salmonella. Contaminated foods in the home play a less significant role in the infection of infants and children.

Interpretation of Restriction Fragment Length Polymorphism Analysis of Mycobacterium tuberculosis Isolates from a State with a Large Rural Population

Epidemiologic relatedness of Mycobacterium tuberculosis isolates from Arkansas residents diagnosed with tuberculosis in 1992-1993 was assessed using IS6110- and pTBN12-based restriction fragment length polymorphism (RFLP) and epidemiologic investigation. Patients with isolates having similar IS6110 patterns had medical records reviewed and were interviewed to identify epidemiologic links. Complete RFLP analyses were obtained for isolates of 235 patients; 78 (33%) matched the pattern of > or = 1 other isolate, forming 24 clusters. Epidemiologic connections were found for 33 (42%) of 78 patients in 11 clusters. Transmission of M. tuberculosis likely occurred many years in the past for 5 patients in 2 clusters. Of clusters based only on IS6110 analyses, those with > or = 6 IS6110 copies had both a significantly greater proportion of isolates that matched by pTBN12 analysis and patients with epidemiologic connections, indicating IS6110 patterns with few bands lack strain specificity. Secondary RFLP analysis increased specificity, but most clustered patients still did not appear to be epidemiologically related. RFLP clustering in rural areas may not represent recent transmission.

Role of embB Codon 306 Mutations in Mycobacterium tuberculosis Revisited: a Novel Association with Broad Drug Resistance and IS6110 Clustering Rather than Ethambutol Resistance

ABSTRACT Mutations at position 306 of embB (embB306) have been proposed as a marker for ethambutol resistance in Mycobacterium tuberculosis; however, recent reports of embB306 mutations in ethambutol-susceptible isolates caused us to question the biological role of this mutation. We tested 1,020 clinical M. tuberculosis isolates with different drug susceptibility patterns and of different geographical origins for associations between embB306 mutations, drug resistance patterns, and major genetic group. One hundred isolates (10%) contained a mutation in embB306; however, only 55 of these mutants were ethambutol resistant. Mutations in embB306 could not be uniquely associated with any particular type of drug resistance and were found in all three major genetic groups. A striking association was observed between these mutations and resistance to any drug (P < 0.001), and the association between embB306 mutations and resistance to increasing numbers of drugs was highly significant (P < 0.001 for trend). We examined the association between embB306 mutations and IS6110 clustering (as a proxy for transmission) among all drug-resistant isolates. Mutations in embB306 were significantly associated with clustering by univariate analysis (odds ratio, 2.44; P = 0.004). In a multivariate model that also included mutations in katG315, katG463, gyrA95, and kasA269, only mutations in embB306 (odds ratio, 2.14; P = 0.008) and katG315 (odds ratio, 1.99; P = 0.015) were found to be independently associated with clustering. In conclusion, embB306 mutations do not cause classical ethambutol resistance but may predispose M. tuberculosis isolates to the development of resistance to increasing numbers of antibiotics and may increase the ability of drug-resistant isolates to be transmitted between subjects.

Simultaneous Infection with Multiple Strains of Mycobacterium tuberculosis

Drug-susceptible and drug-resistant isolates of Mycobacterium tuberculosis were recovered from 2 patients, 1 with isoniazid-resistant tuberculosis (patient 1) and another with multidrug-resistant tuberculosis (patient 2). An investigation included patient interviews, record reviews, and genotyping of isolates. Both patients worked in a medical-waste processing plant. Transmission from waste was responsible for at least the multidrug-resistant infection. We found no evidence that specimens were switched or that cross-contamination of cultures occurred. For patient 1, susceptible and isoniazid-resistant isolates, collected 15 days apart, had 21 and 19 restriction fragments containing IS6110, 18 of which were common to both. For patient 2, a single isolate contained both drug-susceptible and multidrug-resistant colonies, demonstrating 10 and 11 different restriction fragments, respectively. These observations indicate that simultaneous infections with multiple strains of M. tuberculosis occur in immunocompetent hosts and may be responsible for conflicting drug-susceptibility results, though the circumstances of infections in these cases may have been unusual.

Intracellular Macrophage Growth Rates and Cytokine Profiles of Mycobacterium tuberculosis Strains with Different Transmission Dynamics

Mycobacterium tuberculosis strains associated with IS6110 restriction fragment-length polymorphism (RFLP) pattern clusters and strains demonstrating unique IS6110 RFLP patterns were investigated in interferon- gamma -activated THP-1 cells by measurement of binding, intracellular growth rate, and cytokine production. Binding was the same for all strains; however, strains from clusters grew significantly more rapidly than did unique strains. Maximal concentration of tumor necrosis factor (TNF)-alpha was detected at 2 days after infection, with unique strains eliciting significantly greater amounts than did strains from clusters. Interleukin (IL)-10 levels peaked at 1 day after infection with strains from clusters, whereas they peaked at 5 days after infection with unique strains. Rapid growth demonstrated by strains from clusters was highly correlated with rapid production of IL-10 and suppression of TNF-alpha in THP-1 cells during the early stages of infection. Characterization of this phenotype will further advance the investigation of virulence factors in M. tuberculosis.

Diversity of Campylobacter Isolates from Retail Poultry Carcasses and from Humans as Demonstrated by Pulsed-Field Gel Electrophoresis

Campylobacter spp. are a major contaminant of poultry. Eating undercooked chicken and handling raw poultry have been identified as risk factors for campylobacteriosis in humans. Previous studies have found Campylobacter spp. on 90% of poultry carcasses. In the present study, pulsed-field gel electrophoresis (PFGE) was used to assess the genetic diversity of strains on retail poultry carcasses. PFGE patterns of isolates from campylobacteriosis cases were compared to those from the poultry isolates. Over a 1-year study period (March 2000 through February 2001), whole fresh young chickens (n = 72) were obtained from three retail outlets in an urban community in the south-central United States. Campylobacter spp. were isolated from 82% of these carcasses. Strains (n = 70) were defined on the basis of their PFGE pattern. Sixty-seven percent of the carcasses from which Campylobacter spp. were isolated were contaminated with more than one PFGE-distinguishable strain. During the 1-year study period, most of the PFGE patterns (59%) were limited to isolates obtained from a single carcass. Forty-one percent of the PFGE-distinguishable strains were recovered from more than one carcass. Ninety-seven percent of the carcasses contaminated with the same strain were purchased at the same time from the same store. To examine the degree of genetic stability, four strains were followed in vitro over an estimated 1,000 doublings. The PFGE pattern of one of these isolates underwent minor changes during in vitro growth. The data indicate extensive variability in the PFGE patterns of Campylobacter spp. isolated from humans and from poultry carcasses. In spite of difficulties caused by such diversity and the fact that some carcasses are contaminated with more than one strain, the pattern variation provides a useful method for linking a particular strain to its source.