Vincent A. Steingrube

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.

Mycobacterium wolinskyi sp. nov. and Mycobacterium goodii sp. nov., two new rapidly growing species related to Mycobacterium smegmatis and associated with human wound infections : a cooperative study from the international working group on mycobacterial taxonomy

Previous investigations demonstrated three taxonomic groups among 22 clinical isolates of Mycobacterium smegmatis. These studies were expanded to 71 clinical isolates, of which 35 (49%) (group 1) were identical to five ATCC reference strains including the type strain ATCC 19420T. Twenty-eight isolates (39%) were group 2, and eight isolates (11%) were group 3. Isolates of groups 2 and 3 were most often associated with post-traumatic or post-surgical wound infections including osteomyelitis, were susceptible to sulfamethoxazole, amikacin, imipenem and the tetracyclines, variably resistant to clarithromycin, and susceptible (group 1), intermediately resistant (group 2) or resistant (group 3) to tobramycin. The three groups were similar by routine biochemical and growth characteristics, but had different mycolic acid dimethoxy-4-coumarinylmethyl ester elution patterns by HPLC and different PCR-restriction enzyme patterns of a 439 bp fragment of the hsp-65 gene. Group 3 isolates differed from group 1 by 18 bp by 16S rRNA sequencing and exhibited < 25% homology by DNA-DNA hybridization, being most closely related to Mycobacterium mageritense. The 16S rRNA of group 1 and group 2 isolates differed by only 3 bp, but by DNA-DNA hybridization they exhibited only 40% homology. The following names are proposed: Mycobacterium goodii sp. nov. for group 2 isolates (type strain ATCC 700504T = MO69T), Mycobacterium wolinskyi sp. nov. for group 3 isolates (type strain ATCC 700010T = MO739T) and Mycobacterium smegmatis sensu stricto for group 1 isolates.

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.

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.

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.

Induction of aryl hydrocarbon hydroxylase by lymphocytes from women taking oral contraceptives.

The peripheral blood lymphocytes (PBL) from two female subjects were assayed for AHH induction 40 days prior to and 30 days during ingestion of progesterone and estrogen analogues as oral contraceptives. Three habitual users of oral contraceptives were also studied. No in vitro inhibition of AHH induction was observed as a consequence of the use of these hormone analogues. Values obtained for enzyme activity suggest a slight increase in AHH induction resulting from the use of oral contraceptives. Further studies with larger numbers of subjects are required before the apparent increase in enzyme inducibility can be considered significant.