Audrey Wanger

Single Nucleotide Polymorphisms in Genes Associated with Isoniazid Resistance in Mycobacterium tuberculosis

ABSTRACT Isoniazid (INH) is a central component of drug regimens used worldwide to treat tuberculosis. Previous studies have identified resistance-associated mutations in katG, inhA, kasA, ndh, and the oxyR-ahpC intergenic region. DNA microarray-based experiments have shown that INH induces several genes in Mycobacterium tuberculosis that encode proteins physiologically relevant to the drugs mode of action. To gain further insight into the molecular genetic basis of INH resistance, 20 genes implicated in INH resistance were sequenced for INH resistance-associated mutations. Thirty-eight INH-monoresistant clinical isolates and 86 INH-susceptible isolates of M. tuberculosis were obtained from the Texas Department of Health and the Houston Tuberculosis Initiative. Epidemiologic independence was established for all isolates by IS6110 restriction fragment length polymorphism analysis. Susceptible isolates were matched with resistant isolates by molecular genetic group and IS6110 profiles. Spoligotyping was done with isolates with five or fewer IS6110 copies. A major genetic group was established on the basis of the polymorphisms in katG codon 463 and gyrA codon 95. MICs were determined by the E-test. Semiquantitative catalase assays were performed with isolates with mutations in the katG gene. When the 20 genes were sequenced, it was found that 17 (44.7%) INH-resistant isolates had a single-locus, resistance-associated mutation in the katG, mabA, or Rv1772 gene. Seventeen (44.7%) INH-resistant isolates had resistance-associated mutations in two or more genes, and 76% of all INH-resistant isolates had a mutation in the katG gene. Mutations were also identified in the fadE24, Rv1592c, Rv1772, Rv0340, and iniBAC genes, recently shown by DNA-based microarray experiments to be upregulated in response to INH. In general, the MICs were higher for isolates with mutations in katG and the isolates had reduced catalase activities. The results show that a variety of single nucleotide polymorphisms in multiple genes are found exclusively in INH-resistant clinical isolates. These genes either are involved in mycolic acid biosynthesis or are overexpressed as a response to the buildup or cellular toxicity of INH.

Comparison of Etest and National Committee for Clinical Laboratory Standards broth macrodilution method for antifungal susceptibility testing: enhanced ability to detect amphotericin B-resistant Candida isolates.

The National Committee for Clinical Laboratory Standards (NCCLS) proposed macrobroth reference method (M27P) for susceptibility testing of yeasts is technically difficult. We evaluated Etest, a simple agar-based MIC methodology, as a possible alternative. In studies of six yeast quality control strains, Etest yielded results identical to those obtained by the NCCLS reference method for both amphotericin B and fluconazole. In studies of 91 clinical Candida isolates, agreement +/- 2 dilutions between the two methods was 95% for fluconazole with phosphate-buffered RPMI 1640 agar and 96 to 97% for amphotericin B with either MOPS (morpholinepropanesulfonic acid)-buffered RPMI 1640 agar or antibiotic medium 3 agar. While the two methods had excellent general agreement, testing of a collection of amphotericin B-resistant isolates demonstrated that, unlike the NCCLS reference method, Etest readily identified the resistant isolates and could do so with a defined medium. Etest is equivalent to the NCCLS proposed method for susceptibility testing of yeasts and superior in its ability to detect amphotericin B resistance.

Disruption of the Genes Encoding Antigen 85A and Antigen 85B of Mycobacterium tuberculosis H37Rv: Effect on Growth in Culture and in Macrophages

ABSTRACT The mechanism of pathogenesis of Mycobacterium tuberculosis is thought to be multifactorial. Among the putative virulence factors is the antigen 85 (Ag85) complex. This family of exported fibronectin-binding proteins consists of members Ag85A, Ag85B, and Ag85C and is most prominently represented by 85A and 85B. These proteins have recently been shown to possess mycolyl transferase activity and likely play a role in cell wall synthesis. The purpose of this study was to generate strains of M. tuberculosis deficient in expression of the principal members of this complex in order to determine their role in the pathogenesis ofM. tuberculosis. Constructs of fbpA andfbpB disrupted with the kanamycin resistance marker ΩKm and containing varying amounts of flanking gene and plasmid vector sequences were then introduced as linear fragments into H37Rv by electroporation. Southern blot and PCR analyses revealed disruption of the homologous gene locus in one fbpA::ΩKm transformant and one fbpB::ΩKm transformant. The fbpA::ΩKm mutant, LAa1, resulted from a double-crossover integration event, whereas thefbpB::ΩKm variant, LAb1, was the product of a single-crossover type event that resulted in insertion of both ΩKm and plasmid sequences. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis confirmed that expression of the disrupted gene was not detectable in the fbpA andfbpB mutants. Analysis of growth rates demonstrated that the fbpB mutant LAb1 grew at a rate similar to that of the wild-type parent in enriched and nutrient-poor laboratory media as well as in human (THP-1) and mouse (J774.1A) macrophage-like cell lines. ThefbpA mutant LAa1 grew similarly to the parent H37Rv in enriched laboratory media but exhibited little or no growth in nutrient-poor media and macrophage-like cell lines. The targeted disruption of two genes encoding mycolyl transferase and fibronectin-binding activities in M. tuberculosis will permit the systematic determination of their roles in the physiology and pathogenesis of this organism.

Community-associated methicillin-resistant Staphylococcus aureus in pediatric patients.

Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) infections increased from 2000 to 2003 in hospitalized pediatric patients in Houston. CA-MRSA was associated with greater illness than was infection with methicillin-susceptible strains. Children with CA-MRSA were younger and mostly African American. Of MRSA isolates, 4.5% had the inducible macrolide-lincosamide-streptogramin B phenotype.

Correlation between Mutations in liaFSR of Enterococcus faecium and MIC of Daptomycin: Revisiting Daptomycin Breakpoints

ABSTRACT Mutations in liaFSR, a three-component regulatory system controlling cell-envelope stress response, were recently linked with the emergence of daptomycin (DAP) resistance in enterococci. Our previous work showed that a liaF mutation increased the DAP MIC of a vancomycin-resistant Enterococcus faecalis strain from 1 to 3 μg/ml (the DAP breakpoint is 4 μg/ml), suggesting that mutations in the liaFSR system could be a pivotal initial event in the development of DAP resistance. With the hypothesis that clinical enterococcal isolates with DAP MICs between 3 and 4 μg/ml might harbor mutations in liaFSR, we studied 38 Enterococcus faecium bloodstream isolates, of which 8 had DAP MICs between 3 and 4 μg/ml by Etest in Mueller-Hinton agar. Interestingly, 6 of these 8 isolates had predicted amino acid changes in the LiaFSR system. Moreover, we previously showed that among 6 DAP-resistant E. faecium isolates (MICs of >4 μg/ml), 5 had mutations in liaFSR. In contrast, none of 16 E. faecium isolates with a DAP MIC of ≤2 μg/ml harbored mutations in this system (P < 0.0001). All but one isolate with liaFSR changes exhibited DAP MICs of ≥16 μg/ml by Etest using brain heart infusion agar (BHIA), a medium that better supports enterococcal growth. Our findings provide a strong association between DAP MICs within the upper susceptibility range and mutations in the liaFSR system. Concomitant susceptibility testing on BHIA may be useful for identifying these E. faecium first-step mutants. Our results also suggest that the current DAP breakpoint for E. faecium may need to be reevaluated.

A mutant of Mycobacterium tuberculosis H37Rv that lacks expression of antigen 85A is attenuated in mice but retains vaccinogenic potential.

ABSTRACT The fbpA and fbpB genes encoding the 85A and 85B proteins of Mycobacterium tuberculosis H37Rv, respectively, were disrupted, the mutants were examined for their ability to survive, and the strain lacking 85A (ΔfbpA) was tested for its ability to immunize mice. The ΔfbpA mutant was attenuated in mice after intravenous or aerosol infection, while replication of the ΔfbpB mutant was similar to that of the wild type. Complementation of the fbpA gene in ΔfbpA restored its ability to grow in the lungs of mice. The ΔfbpA mutant induced a stronger expression of pulmonary mRNA messages in mice for tumor necrosis factor alpha, interleukin-1 beta (IL-1β), gamma interferon, IL-6, IL-2, and inducible nitric oxide (NO) synthase, which led to its decline, while H37Rv persisted despite strong immune responses. H37Rv and ΔfbpA both induced NO in macrophages and were equally susceptible to NO donors, although ΔfbpA was more susceptible in vitro to peroxynitrite and its growth was enhanced by NO inhibitors in mice and macrophages. Aerosol-infected mice, which cleared a low-dose ΔfbpA infection, resisted a challenge with virulent M. tuberculosis. Mice subcutaneously immunized with ΔfbpA or Mycobacterium bovis BCG and challenged with M. tuberculosis also showed similar levels of protection, marked by a reduction in the growth of challenged M. tuberculosis. The ΔfbpA mutant was thus attenuated, unlike ΔfbpB, but was also vaccinogenic against tuberculosis. Attenuation was incomplete, however, since ΔfbpA revived in normal mice after 370 days, suggesting that revival was due to immunosenescence but not compensation by the fbpB or fbpC gene. Antigen 85A thus affects susceptibility to peroxynitrite in M. tuberculosis and appears to be necessary for its optimal growth in mice.

Mycobacterial protein HbhA binds human complement component C3.

ABSTRACT Mycobacterium tuberculosis and Mycobacterium avium are facultative intracellular pathogens that are able to survive and replicate in mononuclear phagocytes. Human complement component C3 has previously been shown to mediate attachment and phagocytosis of these bacteria by mononuclear phagocytes. In this study, a C3 ligand affinity blot protocol was used to identify a 30-kDa C3-binding protein in M. tuberculosis andMycobacterium smegmatis and a 31-kDa C3-binding protein inM. avium. The C3-binding proteins in M. tuberculosis and M. avium localized to the cell membrane fraction and partitioned to the detergent fraction during Triton X-114 phase partitioning. The C3-binding protein from M. tuberculosis was partially purified using a cation exchange column and was shown to bind concanavalin A. The N terminus and an internal fragment of the partially purified C3-binding protein were subjected to amino acid sequence analysis. The resulting amino acid sequences matched the M. tuberculosis heparin-binding hemagglutinin (HbhA) protein. Recombinant full-length HbhA and the C terminus of HbhA fused to maltose-binding protein, but not recombinant HbhA lacking the C-terminal region, bound human C3. Recombinant full-length HbhA coated on polystyrene beads, was found to enhance the adherence and/or phagocytosis of the coated beads to J774.A1 cells in both the presence and absence of human serum. The presence of complement-sufficient serum increased the adherence of the HbhA-coated beads to the J774.A1 cells in a C3-dependent manner. If HbhA within the bacterial cell membrane functions similarly to isolated HbhA, this protein may enhance the adherence and phagocytosis of M. tuberculosis and M. avium to mononuclear phagocytes through the binding of C3 and interaction with C3 receptors on mononuclear phagocytes.

Evaluation of Vancomycin Susceptibility Testing for Methicillin-Resistant Staphylococcus aureus: Comparison of Etest and Three Automated Testing Methods

ABSTRACT We evaluated the ability of four commercial MIC testing systems (MicroScan, Vitek 2, Phoenix, and Etest) to detect vancomycin MIC values of ≤1 to ≥2 in 200 methicillin-resistant Staphylococcus aureus (MRSA) strains compared to the Clinical and Laboratory Standards Institute broth microdilution (BMD) reference methods. Compared to the BMD method, absolute agreement (0 ± dilution) was highest for the Phoenix system (66.2%) and the MicroScan turbidity method (61.8%), followed by the Vitek 2 system (54.3%). The Etest produced MIC values 1 to 2 dilutions higher than those produced by the BMD method (36.7% agreement). Of interest, the MicroScan system (prompt method) was more likely to overcall an MIC value of 1 mg/liter (74.1%), whereas the Phoenix (76%) and Vitek 2 (20%) systems had a tendency to undercall an MIC of 2 mg/liter. The ability to correctly identify vancomycin MIC values of 1 and 2 has clinical implications and requires further evaluation.

Clinical Characteristics, Outcomes, and Microbiologic Features Associated with Methicillin-Resistant Staphylococcus aureus Bacteremia in Pediatric Patients Treated with Vancomycin

ABSTRACT Vancomycin is the first-line therapy for methicillin-resistant Staphylococcus aureus (MRSA) bacteremia, but its efficacy in adult patients has been questioned. Less is known about the outcomes of MRSA bacteremia treated with vancomycin in pediatric patients. This study reviews the outcomes and clinical characteristics of MRSA bacteremia in children treated with vancomycin and characterizes the microbiologic and molecular features of the bloodstream isolates. A retrospective cohort study was conducted among pediatric patients with MRSA bacteremia treated with vancomycin for >5 days from 1 August 2005 to 31 May 2007 in a large tertiary care center. MRSA bloodstream isolates were characterized by antimicrobial susceptibility testing, PCR analysis of virulence genes, and Diversilab typing. Clinical records were reviewed for outcomes and comorbidities. A total of 22 pediatric patients with MRSA bacteremia were identified. Eleven cases (50.0%) were considered vancomycin treatment failures. Features significantly associated with vancomycin treatment failure were prematurity (P = 0.02) and isolates positive for Panton-Valentine leukocidin (PVL) (P = 0.008). Features typically associated with community-associated MRSA strains were identified in hospital-associated isolates. A dominant clone was not responsible for the high number of treatment failures. Further studies are needed to determine if vancomycin should be the first-line treatment for MRSA bacteremia in premature infants and for PVL-positive isolates.

Antimicrobial Susceptibility Testing: A Primer for Clinicians

Appropriate use of antimicrobials in health care continues to be a challenge. Reliable and reproducible antimicrobial susceptibility testing methods are necessary to provide the clinician with valuable information that can be translated into positive clinical outcomes at the bedside. However, there are nuances with these testing methods that, if unrecognized, could lead to misinterpretation of results and inappropriate antibiotic selection. This primer describes the common antimicrobial susceptibility tests used in the clinical microbiology laboratory and reviews how subtle differences in testing methods and technique can influence reported results. Clinicians who have a thorough understanding of qualitative and quantitative methods, automated susceptibility testing systems, and commonly used screening and confirmatory tests for antibiotic‐resistant organisms can strengthen institutional antibiotic stewardship programs and improve patient outcomes.