Donald R. Nash

Mycobacterium immunogenum sp. nov., a novel species related to Mycobacterium abscessus and associated with clinical disease, pseudo- outbreaks and contaminated metalworking fluids: an international cooperative study on mycobacterial taxonomy

PCR-restriction enzyme pattern analysis of a 439 bp hsp65 gene segment identified 113 unique isolates among non-pigmented rapidly growing mycobacteria (RGM) from clinical and environmental sources that failed to match currently recognized species patterns. This group represented 40% of isolates recovered from bronchoscope contamination pseudo-outbreaks, 0% of disease-associated nosocomial outbreaks and 4% of routine clinical isolates of the Mycobacterium abscessus/Mycobacterium chelonae group submitted to the Mycobacteria/Nocardia laboratory for identification. It is grouped within the Mycobacterium fortuitum complex, with growth in less than 7 d, absence of pigmentation, positive 3-d arylsulfatase reaction and growth on MacConkey agar without crystal violet. It exhibited overlapping biochemical, antimicrobial susceptibility and HPLC characteristics of M. abscessus and M. chelonae. By 16S rRNA gene sequencing, these isolates comprised a homogeneous group with a unique hypervariable region A sequence and differed by 8 and 10 bp, respectively, from M. abscessus and M. chelonae. Surprisingly, this taxon contained two copies of the ribosomal operon, compared with single copies in the two related species. By DNA-DNA hybridization, this new group exhibited <30% homology with recognized RGM species. The name Mycobacterium immunogenum sp. nov. is proposed for this new taxon.

BRO beta-lactamases of Branhamella catarrhalis and Moraxella subgenus Moraxella, including evidence for chromosomal beta-lactamase transfer by conjugation in B. catarrhalis, M. nonliquefaciens, and M. lacunata.

Two closely related beta-lactamases, BRO-1 and BRO-2 (formerly called Ravasio and 1908), are found in Moraxella (Branhamella) catarrhalis. We screened strains of B. catarrhalis recovered in the United States since 1952 and identified the first beta-lactamase-positive isolate in August 1976. The prevalence of the enzymes among 394 clinical isolates from one Texas hospital has averaged 75% since testing began in 1983. Screening of isolates of Moraxella subgenus Moraxella revealed the BRO enzymes in two other human respiratory tract species, M. lacunata and M. nonliquefaciens, beginning in 1978. A different beta-lactamase with a pI of 6.4 predominated in other species of subgenus Moraxella. BRO-2 had a different isoelectric focusing pattern and was produced in lesser amounts than BRO-1, but the two enzymes were indistinguishable by substrate or inhibitor profile. BRO enzymes from B. catarrhalis, M. nonliquefaciens, and M. lacunata could be transferred by conjugation and, for B. catarrhalis, also by transformation to B. catarrhalis. Plasmid bands were demonstrated in 90% of M. nonliquefaciens and in one previously reported strain of B. catarrhalis, but no change in plasmid profiles was seen in beta-lactamase-positive recombinants, supporting previous studies that suggested the beta-lactamase genes are chromosomal. Images

Mycobacterial contamination of metalworking fluids: involvement of a possible new taxon of rapidly growing mycobacteria

Contamination of air and metalworking fluid (MWF) systems with a rapidly growing mycobacterium (RGM) was detected in 1995 in a single manufacturing plant with recent cases of hypersensitivity pneumonitis (HP). Extensive environmental sampling was performed to determine the extent of the contamination and its variability over time. RGM were present in multiple indoor air samples, 100% of the central MWF storage tanks, and 75% of the freestanding cutting, drilling, and grinding machines. With one exception, contamination was limited to a recently introduced formulation (brand) of semisynthetic MWF used in 95% of the facilitys machining operations. In general, the mycobacterial counts were stable over time, with the degree of contamination ranging from 10(2)-10(7) colony forming units (CFU)/mL. A few systems were culture positive for the mycobacterium (> 10(1) CFU/mL), changed to culture negative (< 10(1) CFU/mL), then changed back to culture positive without explanation. Samples obtained from diluted (5%) but unused MWF, a replenishment line with 2% unused MWF, an MWF pasteurizer, city water, and deionized water were culture negative for this species of mycobacterium. Inoculation and growth studies demonstrated that this mycobacterium does not grow in liquid samples of 5% unused MWF. By molecular techniques, the mycobacterial isolates consisted of a single strain and represented a previously undescribed taxon closely related to Mycobacterium chelonae/abscessus. The relationship of this mycobacterium to the cases of HP is unknown.

Incidence of Branhamella catarrhalis in the Sputa of Patients with Chronic Lung Disease

SummaryThe incidence of Branhamella catarrhalis in the respiratory tract of adults, especially in the United States, is not known. During the 30-month period from January 1983 to June 1985, 4180 sputum and endotracheal samples from patients in a hospital for chest diseases were evaluated. All samples were acceptable for Gram-stain analysis and/or culture based on published cellular criteria. Using primarily Gram-stain directed cultures, 220 isolates of B. catarrhalis were identified in 180 patients, being present in 5.3% of all sputum cultures and 11.5% of those positive for a pathogen. B. catarrhalis was the fourth most common pathogen identified. It was found in pure culture (124) and mixed culture (96), the latter usually in association with Haemophilus influenzae or Streptococcus pneumoniae. Of the 220 B. catarrhalis isolates, 158 (71.8%) were positive for β-lactamase. The number and incidence of B. catarrhalis varied, with the organism being most prevalent during the winter months. Despite its frequent presence in sputum, B. catarrhalis was not recovered from pleural fluid or blood during the same period. This study demonstrates the frequent presence of B. catarrhalis in the sputum of adults with chronic lung disease, although the role of this organism as a pathogen was not determined.

Isoelectric focusing of β-lactamases from sputum and middle ear isolates of Branhamella catarrhalis recovered in the United States

SummaryBranhamella catarrhalis obtained from the sputum of 146 patients with lower respiratory tract disease and from middle ear fluids of 26 children with otitis media were evaluated for β-lactamase activity and the enzymes were characterised by isoelectric focusing (IEF). 71% (103 of 146) of the sputum isolates and 77% (20 of 26) of the ear isolates produced β-lactamase. By IEF, the β-lactamases of 113 of 123 (92%) strains revealed patterns identical with the Ravasio type strain, having unique enzyme bands at pIs of 6.4 and 6.65. The remaining 10 isolates (8%) produced patterns similar to the 1908 type strain with a unique band of activity having a pI of 6.55. In addition, the 1908 types revealed a band of minor enzyme activity with a pI of 7.55 that was absent from the Ravasio types. All strains tested shared major enzyme bands with pIs of 5.1, 5.3, 5.55 and 6.1. These results indicate that the most common β-lactamase(s) produced by clinical isolates of B. catarrhalis in the United States are similar to those produced by the Belgian Ravasio type strain.

Disk diffusion susceptibility of Branhamella catarrhalis and relationship of beta-lactam zone size to beta-lactamase production.

We tested 231 isolates of Branhamella catarrhalis for beta-lactamase production and drug susceptibility by the National Committee for Clinical Laboratory Standards disk diffusion method. The nitrocephin disk (Cefinase) identified beta-lactamase in 98% of the enzyme-producing strains, and a zone diameter of inhibition of less than or equal to 29 mm for penicillin correctly predicted the presence of beta-lactamase in 99% of the isolates. No resistance to erythromycin, tetracycline, trimethoprim-sulfamethoxazole, or amoxicillin-clavulanic acid was observed.

Partial characterization of Nocardia farcinica beta-lactamases.

The beta-lactamases obtained from culture supernatants and cell extracts of 26 clinical strains and 5 reference strains of Nocardia farcinica were partially characterized. The enzymes exhibited two patterns on isoelectric focusing (IEF). beta-Lactamases from the majority of the 31 strains (87%) including the 5 reference strains exhibited two major bands with pIs of 4.56 and 4.49. The remaining strains had two similar major bands but with slightly higher pIs. Culture supernatants and cell extracts exhibited identical patterns. The two sets of enzymes were functionally indistinguishable by substrate and inhibitor profiles and lack of inducibility. By disk testing, ampicillin, amoxicillin, ticarcillin, amoxicillin-clavulanic acid, and imipenem were highly synergistic with cefotaxime. The enzymes were primarily penicillinases and hydrolyzed cephalosporins at rates of < or = 12% of those for penicillins. N. farcinica beta-lactamases were susceptible to inhibition by clavulanic acid and BRL 42715, exhibiting 50% inhibitory concentrations of 0.025 to 0.045 micrograms/ml (0.12 to 0.22 microM) and 0.05 to 0.1 micrograms/ml (0.31 to 0.63 microM), respectively, less susceptible to tazobactam, and least susceptible to sulbactam, cloxacillin, and imipenem. The beta-lactamases of N. farcinica are believed to mediate penicillin resistance and may play a secondary role in extended-spectrum cephalosporin resistance. The close similarity among N. farcinica beta-lactamases and their distinct differences from beta-lactamases of other Nocardia species support the taxonomic identity of this species. Images

Amplification and cloning of the Mycobacterium tuberculosis dnaA gene

To identify and subsequently clone the gene encoding the DnaA protein, degenerate oligodeoxyribonucleotide (oligo) primers targeted against two highly conserved domains of the eubacterial DnaA were used to amplify a 780-bp DNA region spanning the two primers from genomic DNA preparations of Mycobacterium tuberculosis (Mt), M. bovis (Mb) and M. avium (Ma). Nucleotide (nt) sequences and deduced amino acid (aa) sequences of these fragments revealed homologies with each other and with the corresponding regions from other bacteria. Using an oligo specific to Mt dnaA as a probe, the Mt genomic DNA cosmid libraries propagated in Escherichia coli were screened and a cosmid DNA clone hybridizing with the oligo was identified. Furthermore, a 5-kb DNA fragment containing the Mt dnaA was subcloned into a pUC18 vector.

Factors XWenatchee I and II: compound heterozygosity involving two variant proteins.

Variant factor X in an individual with a mild bleeding tendency was suspected based on deficient procoagulant activity (10-20% of normal) and antigen (30-35% of normal) levels of plasma factor X. Heteroduplex analysis of factor X gene exons indicated heterozygosity for mutations in both exons 6 and 4, confirmed by direct sequencing of the amplified exons. Substitution of C by T at nucleotide position 13,984 (Arg-139 to Cys) was found in the factor X gene exon 6 of the propositus. This mutation creates a BsmI site and the patient tested heterozygous for the BsmI cleavage involved, as did one of his two daughters. In addition, exon 4 was found to have the normal A and a novel C (Asn-57 to Thr) at nucleotide position 9338. The exon 4 mutation creates a BsaJI site, detectable after amplification mismatch to remove an existing BsaJI site. Both the patient and the second of his two daughters were heterozygous for this cleavage. The two variant proteins are called factors XWenatchee I (Arg-139 to Cys) and II (Asn-57 to Thr). A mixed variant isolate derived from the plasma of the propositus exhibited heavy/light chains of normal size, as well as an apparent single-chain molecule not dissociable by reducing agent. A single-chain molecule would be predicted for form I, if the mutation blocks processing cleavages that normally remove a tripeptide interposed between the heavy and light chains. A Western blot of partially purified factor X from the daughter who inherited the form I defect revealed a component migrating the same as the putative single-chain species. Based upon the factor X activity vs. antigen ratios for the propositus and both daughters, both forms I and II are probably dysfunctional molecules.

Primary immune responsiveness and other observations in mice given oral dimethyl sulfoxide

Mice were allowed to drink 5% DMSO in their drinking water ad libitum for a period of six weeks, during which time a number of variables were compared with age-, weight-, and sex-matched control mice given ordinary tap water. The amount of DMSO consumed increased from a daily average of 27 g/kg/mouse during the first week to 52 g/kg/mouse by the end of the third week. Significant decreases were observed in the treated animals for total body weight, percent spleen weight relative to total body weight, and serum volume, while serum immunoglobulin concentrations remained unchanged. Mice given 4.4 g/kg DMSO intraperitoneally for 7 days, however, revealed significant losses in serum IgG and IgA, but not IgM. The effect of DMSO on the primary humoral response was assessed following immunization of mice with sheep red blood cells either 16 days or 8 weeks after the start of DMSO. Compared with similarly immunized controls, test animals demonstrated moderate, but significant, reductions in both spontaneous and facilitated plaque-forming cells, hemagglutination titres, and serum concentrations of IgG1. Based on a comparison of the two DMSO-treated groups, the degree of immune inhibition increased and the peak of the response was delayed relative to the length of time of DMSO ingestion prior to immunization.