Association between mycobacterial genotypes and disease progression in Mycobacterium avium pulmonary infection.

BACKGROUND: Non-tuberculous mycobacterial lung disease, most commonly caused by Mycobacterium avium infection, tends to show variable disease progression, and significant disease predictors have not been adequately established. METHODS: Variable numbers of tandem repeats (VNTR) were evaluated in 16 mycobacterial interspersed repetitive unit (MIRU) loci from M avium isolates cultured from respiratory specimens obtained from 2005 to 2007. Specifically, the association between VNTR profiles and disease progression was assessed. RESULTS: Among the 37 subjects who provided positive respiratory cultures for M avium during the 2005-6 period, 15 subjects were treated within 10 months following a microbiological diagnosis of progressive M avium lung disease. Nine subjects underwent long-term follow-up (>24 months) without treatment for stable M avium lung disease. Based on a neighbour-joining cluster analysis used to classify M avium-positive subjects according to the VNTR profile, subjects with progressive versus stable lung disease were found to be grouped together in distinct clusters. Further analysis using logistic regression modelling showed that disease progression was significantly associated with the genetic distance of the M avium isolate from an appropriately selected reference (age-adjusted odds ratio 1.95; 95% confidence interval 1.16 to 3.30; p = 0.01 for the most significant model). A best-fit model could be used to predict the progression of M avium lung disease when subjects from the 2005-6 period were combined with those from 2007 (p = 0.003). CONCLUSION: Progressive lung disease due to M avium infection is associated with specific VNTR genotypes of M avium.

Increased levels of mitochondrial DNA in an etoposide-resistant human monocytic leukaemia cell line (THP-1/E).

Electron microscopic observations of THP-1/E (an etoposide-resistant human monocytic leukaemia cell line) showed a remarkable change of mitochondrial structure. Mitochondria were swollen and cristae were relatively intact. There was no difference in the activity of cytochrome oxidase, an enzyme which contains three subunits coded by mitochondrial DNA (mtDNA) between THP-1/E and THP-1 (the parent cell of THP-1/E). No measurable quantitative change of mitochondrial RNA was observed, but the level of mtDNA in THP-1/E was increased by a factor of about 4 compared with that of mtDNA in THP-1. These results suggest that, on acquisition of resistance to etoposide, some factors affect mitochondria, change its morphology and amplify its DNA.

Identification of mycobacteria by nonradioisotopic single-strand conformation polymorphism analysis.

Clinical isolates of mycobacteria were identified to species levels using nonradioisotopic single-strand conformation polymorphism (non-RI SSCP) analysis of 16S rRNA gene fragments amplified by polymerase chain reaction with primers common to all of mycobacterial species. The method is based on a hypervariable region within the 16S rRNA in mycobacteria, which is characterized by species-specific nucleotide sequences. A total of 92 mycobacterial strains (Mycobacterium tuberculosis, M. avium, M. gordonae, M. intracellulare, M. kansasii, M. chelonae, M. nonchromogenicum, M. xenopi, and unidentified strain) were studied. They were classified into nine types of pattern showing single-strand DNA bands having different mobilities. Each strain was shown in the species-specific mobility by non-RI SSCP analysis. The results of non-RI SSCP analysis were identical to those of standard biochemical methods and 16S rRNA sequencing.

Novel arbekacin- and amikacin-modifying enzyme of methicillin-resistant Staphylococcus aureus.

An aminoglycoside-modifying enzyme in arbekacin-resistant methicillin-resistant Staphylococcus aureus (MRSA), exhibiting 4'''-N-acetylation, was examined. Although the MRSA strain with AAC(4''') had no AAC(6')-APH(2") activity, a DNA fragment of the AAC(6')-APH(2") gene was amplified by PCR and the purified N-terminal 30-amino acid sequence of this AAC(4''') was identical to AAC(6')-APH(2"). Direct DNA sequencing of this 'silent' AAC(6')-APH(2") gene revealed a single point mutation leading to a substitution of Gly for Asp80, through which the secondary structure is affected. A change in protein conformation could lead to a cleavage and a change of the enzymatic activity. We propose a new aminoglycoside-resistance mediated by AAC(4''') is caused by a mutation-modified AAC(6')-APH(2").

DNA gyrase gyr A gene mutations in ofloxacin-resistant Staphylococcus aureus.

A total of 106 clinical isolates of Staphylococcus aureus were classified into nine pattern types correlated with gyrA mutations. In 62 strains, mutations were found at a single codon (84, 85, 86 and 88), while 22 strains showed double mutations either at codons 84 and 85 or at codons 84 and 88. The double mutations led to the highest levels of ofloxacin resistance (MIC, > or = 128 microg/ml). All isolates with a single Ser-84--> Leu change had an ofloxacin MIC of 8-128 microg/ml, whereas others showed an MIC range of 8-16 microg/ml. Twenty-two wild type strains and one strain with a single mutation at codon 86 (silent mutation) were ofloxacin-susceptible. Thus, gyrA mutations seem to play a definite role in the high-level of resistance to ofloxacin.

Reduced topoisomerase II-mediated DNA cleavage in VP-16 resistant human leukemic cell line.

Drug resistance in the VP-16 resistant human leukemic cell line (THP-1/E) was studied the possible relevance of topoisomerase II activity. Strand-passing activity in crude nuclear extract from sensitive and resistant cells was comparable and equally sensitive to inhibition by VP-16. However, it was demonstrated that VP-16-mediated pBR322 DNA cleavage in the presence of nuclear extract from resistant cells was reduced to one-tenth of that from sensitive cells. These results suggested that the resistance of THP-1/E cells to VP-16 was due to reduced DNA cleavage activity.

[Clinical studies on S 6472 granule preparation (sustained-release cefaclor) in chronic respiratory tract infections].

S 6472 granule preparation (sustained-release cefaclor) was orally administered to 15 patients with chronic respiratory tract infections (2 acutely exacerbated cases of chronic bronchitis, 13 cases of secondary infections consisting of 1 case of bronchial asthma, 2 cases of bronchial asthma/pulmonary emphysema, and 10 cases of bronchiectasis) at a daily dose of 750 mg divided into 2 doses administered after breakfast and dinner, for a duration of 14 days. The drug was ineffective in 3 of the 10 cases of bronchiectasis but was effective in the other 12 cases, with a rate of efficacy of 80%. There were no side effects of abnormal laboratory findings due to administration of this drug.

[In vitro activity of biapenem (BIPM) against clinically isolated respiratory pathogens in 1996-1998].

The in vitro antibacterial activity of biapenem (BIPM), a new carbapenem antibiotic, was compared with those of imipenem (IPM), panipenem (PAPM), meropenem (MEPM), ceftazidime (CAZ) and piperacillin (PIPC) against 280 isolates of 9 respiratory pathogens. The MIC90s of biapenem (BIPM) for methicillin-susceptible Staphylococcus aureus (MSSA), methicillin-resistant Staphylococcus aureus (MRSA), Streptococcus pneumoniae, Moraxella catarrhalis, Pseudomonas aeruginosa, and Haemophilus influenzae were 0.12, 32, 0.25, 0.06, 4 and 8 micrograms/ml, respectively. In comparison with other antibiotics, the activity of biapenem (BIPM) for P. aeruginosa was as potent as meropenem (MEPM), but for H. influenzae it was slightly less than those of other antibiotics, and for other respiratory pathogens it was as potent as those of other antibiotics. The MIC90s of biapenem (BIPM) for Escherichia coli, Klebsiella pneumoniae, Enterobacter cloacae and Serratia marcescens were 0.06, 1, 1, 0.5 microgram/ml, respectively, and which were equal to or somewhat lower than those of other antibiotics. Biapenem (BIPM) showed strong activity against Gram-positive and Gram-negative pathogens, especially P. aeruginosa in general. Based on these results, biapenem (BIPM) is seemed to be highly useful antibiotic for the treatment of respiratory infections with several organism.

[Susceptibilities of Enterococcus faecium, PRSP and MRSA to RP59500 and their correlations with those to other drugs].

Investigations on emergence of vancomycin-resistant Enterococcus faecium (VREF) which has recently been attracting attention, especially in the Western countries, have been conducted in Japan. A total of 1,239 isolates of E. faecium were collected from 19 institutions during the period of April 1995 and June 1996, in the purpose of evaluating susceptibilities to variety of antimicrobial agents, including RP59500 and vancomycin (VCM), and detecting vancomycin-resistant genes (van genes). Susceptibilities of penicillin-resistant Streptococcus pneumoniae (PRSP) and methicillin-resistant Staphylococcus aureus (MRSA) were also studied. As a result, 2 isolates of E. faecium were found to be moderately resistant to VCM showing MIC of 8 micrograms/ml though the final identification in species level and the detection of van genes by PCR method have not been completed. On the other hand there detected no MRSA nor PRSP showing moderately resistant or resistant to VCM. It was concluded that RP59500 and VCM possessed favorable activity against clinically isolated E. faecium, PRSP and MRSA. Among other species of enterococci, moderately resistant strains to VCM showing MIC of 8 micrograms/ml were detected; 10 isolates of E. gallinarum, 4 of E. casseliflavus and 2 of E. flavescens. In those isolates, vanC1 and vanC2 were detected by PCR, and vanB was also detected in a isolates of E. gallinarum simultaneously.

[Management of mycobacteriosis in general hospital without isolation ward for tuberculosis patients. Clinical study on pulmonary tuberculosis associated with lung cancer patients].

Sixteen of 758 lung cancer in patients (2.1%) were found to have coexisting pulmonary tuberculosis. Of the above 16 of 758 patients (fifteen men and one woman), 4 of 214 patients (1.9%) were found from 1988 to 1989, and 12 of 544 patients (2.2%) from 1991 to 1994. In six patients, pulmonary tuberculosis and lung cancer were found at the same time by clinical work up. In five cases each, pulmonary tuberculosis preceded lung cancer, and lung cancer preceded pulmonary tuberculosis, respectively. Ten patients had adenocarcinoma, 4 had squamous cell carcinoma, and one each had large cell carcinoma and small cell carcinoma, respectively. Five patients were in stage "II", one in "IIIa", two in "IIIb", and eight in "IV" of clinical stage of lung cancer. As regards extent of pulmonary tuberculosis, one patient was in category "II" of the classification of the Japanese Society for Tuberculosis, 13 were in "III", and two were in "IV". Among 544 lung cancer patients from 1991 to 1994, 9 of 151 patients (6.0%) with a past history of pulmonary tuberculosis, had active pulmonary tuberculosis, and 3 of 393 patients (0.8%) with no history of pulmonary tuberculosis, had active pulmonary tuberculosis (statistically significant; p < 0.005). Five smear-positive patients were transferred to a tuberculosis hospital or a tuberculosis ward, and the remaining 11 patients were treated in isolation in the ward where they were. The efficacy of anti-tuberculous chemotherapy was almost comparable to that in patients without lung cancer. However, prognosis was poor, in line with that of lung cancer. Main discussion was devoted to the reason why the incidence (in association with tuberculosis) of adenocarcinoma exceeded that of squamous cell carcinoma in our present study at variance with the studies of other investigators.

Detection of methicillin-resistant Staphylococcus aureus (MRSA) using polymerase chain reaction amplification.

Polymerase chain reaction (PCR) was applied for the detection of methicillin-resistant Staphylococcus aureus (MRSA). This procedure amplified a segment of MRSA-PBP (penicillin-binding protein) gene of DNA extract from the clinical isolates of S. aureus. A 1339-base-pair fragment of MRSA-PBP gene in DNA from S. aureus (MIC of methicillin, greater than or equal to 16 micrograms/ml) was amplified and detected by a specific oligonucleotide probe. Moreover, a 4.3 kb HindIII fragment containing MRSA-PBP gene was detected by using the same oligonucleotide probe. On the other hand, no PCR-amplified product was detected in DNA from methicillin-sensitive S. aureus (MIC of methicillin, less than 16 micrograms/ml).

Comparison of a polymerase chain reaction assay and a conventional microbiologic method for detection of methicillin-resistant Staphylococcus aureus.

The presence or absence of a methicillin resistance gene in 58 clinical isolates of Staphylococcus aureus was examined by the polymerase chain reaction (PCR) and Southern blot analyses. The results were analyzed in relation to those of the MIC assay of methicillin and oxacillin. PCR assay results were identical to those of Southern blot analysis of genomic DNA digested with HindIII (positive, 28 strains; negative, 30 strains). Among the 28 PCR-positive strains, 6 strains showed methicillin susceptibility by the conventional susceptibility test (MICs, less than or equal to 8 micrograms/ml). Culturing of the six strains with ceftizoxime led to an increase in the phenotypic level of resistance to methicillin and oxacillin, indicating that these strains should be classified as methicillin-resistant S. aureus (MRSA). The PCR assay was found to be a sensitive and reliable procedure for the rapid diagnosis of MRSA infection, even in cases in which the conventional MIC assay failed to detect MRSA.

Characterization of resistance to VP-16 in human leukemic cell line.

Resistance mechanism was studied in the VP-16-resistant human leukemic cell line (THP-1/E) which was developed by continuous drug exposure. The drug uptake and efflux studies revealed no decrease in net cellular drug accumulation. VP-16-induced DNA single- and double-strand breaks in the whole THP-1/E cells decreased significantly compared to the sensitive counterpart as assessed by alkaline elution methods. Decrease in DNA SSBs was also observable in the isolated nuclei from the THP-1/E cells. The resistance to VP-16 in THP-1/E appeared to be independent of altered membrane permeability, and more likely to be associated with decreased VP-16-mediated DNA cleavage.

Detection of novel mutations in the gyrA gene of Staphylococcus aureus by nonradioisotopic single-strand conformation polymorphism analysis and direct DNA sequencing.

A total of 36 clinical isolates of Staphylococcus aureus (29 fluoroquinolone-resistant strains and 7 fluoroquinolone-susceptible strains) were studied for the presence of point mutations in the gyrA gene by nonradioisotopic single-strand conformation polymorphism (Non-RI SSCP) analysis with silver stain. Direct DNA sequencing analysis of the PCR-amplified DNA fragments confirmed the results obtained by Non-RI SSCP analysis and revealed that fluoroquinolone resistance is closely associated with six types of mutations in the gyrA gene, of which three types of mutations were newly identified: (i) Ser-84-->Leu and Glu-88-->Gly, (ii) Ser-84-->Leu and Glu-88-->Lys, and (iii) Glu-88-->Gly. Furthermore, the novel ATT-->ATC mutation at codon 86 (silent mutation) was seen in only one fluoroquinolone-susceptible strain. All seven mutational types were separated from the wild type in a single electrophoretic step within 3 h after PCR amplification. Thus, we conclude that this new technique is a rapid, simple, and useful screening method for the genotyping of gyrA mutations associated with fluoroquinolone resistance.

Specificity of 4'''-acetylation by an aminoglycoside-modifying enzyme in arbekacin-resistant strains of methicillin-resistant Staphylococcus aureus.

Four arbekacin (ABK)-resistant clinical isolates of methicillin-resistant Staphylococcus aureus (MRSA) were studied regarding their mechanism of aminoglycoside-resistance. Metabolites of amikacin (AMK) and gentamicin(GM) were obtained by reaction with excess amounts of crude enzyme preparation extracted from the ABK-resistant MRSA strains. Then the metabolites were then isolated and analyzed by means of 1H, 13C-nuclear magnetic resonance. The AMK-modification was 4'''-N-acetylation. However, the site of GM-acetylation may be 6'-N-position by these strains.

CD44 variant isoform expression and breast cancer prognosis.

We examined the expression of CD44 isoforms in samples of breast cancer tissues from 95 patients by reverse transcription-polymerase chain reaction and immunohistochemistry, and tried to correlate the results with survival period. At the RNA level, expression of exon v2 was observed in 33 (35%) and that of v6 in 69 (73%) of the 95 specimens. Patients with CD44v2 mRNA expression had significantly shorter survival times than those with v2-negative tumors (P = 0.05), but there was only a weak correlation, if any, between v6 mRNA expression and overall survival (P = 0.06). Tumor tissue from 22 (23%) and 72 (76%) patients showed positive immunoreactivity with monoclonal antibody (mAb) M23.6.1. (CD44v2) and mAb 2F10 (CD44v6), respectively. Immunohistochemical evidence of CD44v2 peptide expression correlated with overall survival (P = 0.02), but there was no such association with CD44v6 expression in these tumors (P = 0.67). There were significant correlations between v2 immunoreactivity and higher histological grade and lower levels of estrogen and progesterone receptor. There was no significant correlation between v6 immunoreactivity and such clinicopathological characteristics. Although the expression of v2 was significantly associated with reduced overall survival, it was not an independent prognostic factor because it also correlated with progesterone receptor status. These findings suggest that v2 isoform expression might have more value than v6 expression for clinical use.