Comparative evaluation of the in vitro antimycobacterial activities of six aminoglycoside antibiotics using an agar dilution method.

Given the increasing prevalence of mycobacterial resistance to aminoglycoside antibiotics, we examined the susceptibility of 76 clinical isolates of mycobacteria to arbekacin, amikacin, gentamicin, kanamycin, tobramycin and streptomycin using an agar dilution method. Only arbekacin and amikacin showed excellent therapeutic potential (minimum inhibitory concentrationis (MICs) < or =0.25-4 microg/ml) against 30 isolates of rapidly growing mycobacteria, including Mycobacterium fortuitum, M. chelonae and a related organism, Nocardia asteroides. The MIC(90)of tobramycin against 23 isolates of M. avium complex was 8 microg/ml, while that of the other 5 aminoglycosides ranged from 64-256 microg/ml. Of the 23 M. tuberculosis isolates tested, 5 showed aminoglycoside resistance (MICs 128 to > or =1,024 microg/ml), while the others were variably susceptible (MICs < or =0.25-32 microg/ml) to all 6 aminoglycosides. The chemotherapeutic potential of arbekacin, amikacin and streptomycin as treatment of tuberculosis was apparent; however, proper patient management would be required to control against the emergence of the drug-resistant strains during prolonged treatment.

K-ras mutation in the endometrium of tamoxifen-treated breast cancer patients, with a comparison of tamoxifen and toremifene.

The putative presence of a mutation in codon 12 of the K-ras gene was investigated in the endometrium of tamoxifen (TAM) and toremifene (TOR)-treated breast cancer patients. DNA was extracted from fresh cytologic samples of the endometrium in 86 TAM and 21 TOR-treated breast cancer patients. Mutations were detected by enriched PCR and an enzyme-linked mini-sequence assay (ELMA). K-ras mutation was found in 35 TAM-treated endometrial samples, and in only one TOR-treated endometrium (P<0.003). In 24 premenopausal patients, K-ras mutation was found in seven (43.8%) of 16 patients with less than 47 months of TAM treatment, while none was found in eight patients with more than 48 months of TAM treatment (P<0.03). In 62 postmenopausal-amenorrheic patients, K-ras mutation was found in three (15.8%) of 19 patients with less than 23 months of TAM treatment, while it was found in 16 (61.5%) of 26 patients with 24-47 months of TAM treatment and nine (52.9%) of 17 patients with more than 48 months of TAM treatment (P=0.002). The presence of K-ras mutation is significantly influenced by the duration of TAM treatment and menstrual status of the patients. TOR may have a lower potential genotoxicity than TAM.

[Functional characterization of a multiple-antibiotic resistant plasmid from clinical isolates of methicillin-resistant Staphylococcus aureus].

During surveillance done as part of the investigation of nosocominal infections due to methicillin-resistant Staphylococcus aureus (MRSA), we have been aware of the close relationship between the presence of certain plasmids and the characteristic patterns of antibiotic resistance. The hypothesis that a mechanism for the rapid and widespread dissemination of resistance to multiple aminoglycosides in clinical isolates of MRSA at our university hospital was the result of transfer of a single plasmid among these strains was examined. About 45% of the total isolates of MRSA (91/200 isolates) carried a 35.5 kb plasmid (designated pCL4) and these isolates always showed resistance to gentamicin, tobramycin, kanamycin, amikacin, astromicin, and arbekacin. Mating experiments between a susceptible strain and resistant MRSA isolates carrying pCL4 showed high frequency transfer of the plasmid. The aminoglycoside resistance patterns of all the transconjugants obtained corresponded well with those of parental strains. However, the plasmid could not necessarily be detected in the transconjugants, whereas the transformants obtained by means of electroporation usually possessed the plasmid. The plasmid-encoded aminoglycoside-resistance determinant, which has been identified as the gene aacA/aphD that encodes the bifunctional enzyme AAC (6')/APH(2"), either in the transconjugants and transformants could be transposed to their chromosomes in the absence of whole-plasmid integration resulting from a recombination event. Southern hybridization analysis using an aacA/aphD specific probe demonstrated that there are multiple sites of the insertions indistinguishable among the chromosomes of plasmid-free transconjugants and transformants. These results indicate that rapid dissemination of multiple-aminoglycoside-resistance in nosocominal strains of MRSA resulted from transfer of a conjugative plasmid and has been facilitated by translocations of the resistance gene to the chromosome.

[Emergence of new-risk factors associated with nosocominal infection].

During the last several decades, we have experienced the emerging and reemerging of infectious pathogens and diseases. The outbreaks of infection due to these pathogens sometimes occur via the sources and/or routes which have not been recognized during previous studies on epidemiology and pathogenesis of the diseases. There are many factors contributing to the increase in infectious diseases. Namely, medical progress often results in an increasing number of immunocompromised patients. Common, usually avirulent, commensal and environmental organisms become pathogens to these patients. The evolution of organisms acquiring resistance to antimicrobial agents, disinfectants, and environmental stimuli also relates to the high incidence of nosocominal (hospital-acquired) infections as well as to the clinical situation of the patients. Because of these tendencies, as risks of nosocominal infections, are characteristic especially in major health care centers such as large teaching hospitals, most health care workers are obliged to participate actively in control and preventive efforts in addition to their traditional roles. In this review I discuss the recent characteristics of nosocominal pathogens and the environments surrounding the hospitalized patients to design effective strategies for and to facilitate the infection control activities.

Extracellular hemolytic activity in rapidly growing mycobacteria.

Little is known about virulence factors associated with rapidly growing mycobacteria. We evaluated 42 clinical isolates of Mycobacterium fortuitum and Mycobacterium chelonae and 4 reference strains of Mycobacterium smegmatis for the production of hemolysin (or hemolytic substance) as a possible contributor to the pathogenesis of disease caused by these organisms. All the strains tested possessed extracellular hemolytic activity that was stable after heating and proteinase treatment, and the active substance had a molecular weight less than 10,000. The activity accumulated in culture medium during the late exponential to mid stationary phase of growth. Hemolysis in vitro was relatively slow; incubation for 10 h at 35 degrees C was required to obtain maximal activity. Some specificity of the hemolysis with regard to red blood cells from different animals was observed.

Development of a new host vector system in mycobacteria.

The hybrid plasmid pYT72/pYT92 constructed from an Escherichia coli plasmid pACYC177 and mycobacterial plasmid pMSC262 isolated from Mycobacterium scroflaceum strain W262 transformed both E. coli and BCG. Phage-sensitive mutants S-10 and S-20 isolated from BCG Tokyo strain showed higher frequency of transformation than the wild-type strain. Frequency of transformation was dependent on age of the culture and the electroporation condition used. Several deletion mutants were generated from pYT72/92 to determine the minimum region for the replication in the mycobacteria. A 2.3-kb fragment of pMSC262 was found to contain an essential region. Using this fragment and pACYC177, a small shuttle vector pYT937 containing two drug-resistance markers, kanamycin- and ampicillin-resistance, was constructed. pYT937 contains AatII, BamHI, BbvII, GsuI, HincII, PstI, ScaI and XbaI cloning sites.

[Establishment of a host-vector system in Mycobacterium bovis BCG].

The recombinant plasmids, pYT72 and pYT92, were generated from a mycobacterial plasmid, pMSC262, and a Escherichia coli plasmid, pACYC177. These plasmids were capable of replication, and of stable maintainance in Mycobacterium bovis BCG when introduced by electroporation technique. Efficiency of transformation was about 10(4) transformants/micrograms DNA, and was the highest in the phage sensitive mutants (S-10, S-20) isolated from BCG Tokyo strain. We have also isolated transformable mutants from rapidly growing bacterium, M. smegmatis strains Jucho and TMC1533. By isolating deletion mutants from pYT72/92, we could determine the location of replication region of pMSC262 within a 2.3 kb Pst I-Hind III fragment. Using this fragment, we constructed "mini" shuttle plasmid pYT937 (5.9 kb in size) which possesses kanamycin and ampicillin resistance markers and replicates in both E. coli and Mycobacterium. Nucleotide sequence analysis of the replication region revealed that there are 2 potential coding regions which contain more than 200 amino acids. The largest one (ORF1) which codes 311 amino acids, however, lacks Shine-Dalgarno like sequence in the upstream and therefore may not be functional. The other coding region (ORF2) contains 260 amino acids and was preceded by Shine-Dalgarno like sequence. Upstream of the ORF2, there were several repeat sequences which may be important in the plasmid replication. GC content of the 2.3 kb fragment was 69.8%.

Does aminoglycoside-acetyltransferase in rapidly growing mycobacteria have a metabolic function in addition to aminoglycoside inactivation?

All the rapidly growing mycobacteria tested, Mycobacterium fortuitum complex, M. smegmatis, M. phlei, and M. vaccae, contained one of two characteristics, but were different from previously recognized aminoglycoside-acetyltransferases. The acetylation reaction of both the enzymes from M. fortuitum and Pseudomonas aeruginosa (3-N-acetyltransferase-III) with radiolabeled acetyl coenzyme A was inhibited severely by oxalacetate. It was suggested that the inhibitory effect of oxalacetate is due to the condensation reaction between oxalacetate and acetyl coenzyme A resulting in the generation of citrate.

Patterns and distribution of aminoglycoside-acetylating enzymes in rapidly growing mycobacteria.

Cell-free extracts from clinical, laboratory, and environmental isolates of Mycobacterium fortuitum, Mycobacterium smegmatis, Mycobacterium phlei, and Mycobacterium vaccae were tested for the presence of aminoglycoside-acetylating enzyme and compared with enzymes from gram-negative organisms. Acetylating activity was detected in all strains examined despite variable levels of aminoglycoside susceptibility. Substrate profiles revealed 2 different patterns of 3-N-acetyltransferase. One pattern exhibited broad substrate specificity including significant activity to fortimicin and was specific for Mycobacterium fortuitum strains, whereas the second pattern showed a much narrower substrate range and was observed for the other 3 environmental species. Both types of enzymes inactivated the antimicrobial activity of drug in vitro. The acetylation reaction of mycobacterial enzyme with radiolabeled acetyl coenzyme A was significantly inhibited by malonyl- (34.7%), propionyl- (21.3%), and butyryl- (12.5%) coenzyme A in the presence of adenosine-5'-triphosphate, whereas no inhibition could be observed for the type enzyme (3-N-acetyltransferase-III) from Pseudomonas aeruginosa suggesting the two enzymes are different. Thus all species of rapidly growing mycobacteria probably contain one of several different types of aminoglycoside acetyltransferases including some isolates and species without a resistance phenotype. The origin and specific function of these enzymes are not known.

Characterization of beta-lactamases in Mycobacterium fortuitum including a role in beta-lactam resistance and evidence of partial inducibility.

The beta-lactamases from the 3 biovariants of M. fortuitum were compared on the basis of substrate profiles, susceptibility to enzyme inhibitors, and inducibility in the presence of selected beta-lactams. Despite differences in the distribution of beta-lactamase bands observed when enzymes from different isolates were subjected to isoelectric focusing, substrate profiles for the 3 biovariants were similar. All demonstrated a comparable broad spectrum hydrolytic activity for both cephalosporins and penicillins. The MIC for amoxicillin were reduced 4- to 16-fold when combined with the beta-lactamase inhibitor clavulanic acid, but not to a clinically susceptible range. The degree of reduction in MIC for amoxicillin correlated well with the susceptibility of enzyme to inhibition by clavulanic acid as determined in an in vitro assay. Although all M. fortuitum strains produce beta-lactamase under routine growth conditions, 90% of strains demonstrated an increase in the amount of this enzyme when cultured in the presence of selected beta-lactams as potential inducers. Quantitative assays and isoelectric focusing further indicated that this apparent induction of beta-lactamase is a simple enhancement of the same enzyme(s) produced in the absence of a known inducer. This is the first demonstration of any inducibility among mycobacterial beta-lactamases and suggests that synthesis of these enzymes in M. fortuitum is under some form of regulatory control. These results indicate that the beta-lactamases have a role in resistance of M. fortuitum to the beta-lactams. Other factors, such as permeability and penicillin-binding proteins, were not evaluated.

Biochemical mechanisms of antibiotic resistance in a clinical isolate of Mycobacterium fortuitum. Presence of beta-lactamase and aminoglycoside-acetyltransferase and possible participation of altered drug transport on the resistance mechanism.

Mycobacterium fortuitum was isolated from specimens of bone marrow from a patient with chronic osteomyelitis. This isolate was resistant to most antimycobacterial drugs, aminoglycosides, and beta-lactam antibiotics. Cell-free extracts from the clinical isolate contained an aminoglycoside acetyltransferase and beta-lactamase. On the basis of substrate specificity, the former enzyme was identified as the acetyltransferase (3), subtype III or IV. However, no positive correlation could be observed between resistance levels (minimal inhibitory concentrations) and the degree of inactivation of aminoglycosides. In vitro, the enzyme reaction required 40 to 60 min to completely inactivate kanamycin. Protein synthesis by ribosomes prepared from this clinical isolate was inhibited by one tenth the concentration of aminoglycosides required to inhibit growth of whole cells. These results suggest that some crypticity factor(s), such as the decreased permeability of drugs, may participate in the intrinsic resistance of the organism.

Salt-induced filamentous growth of a Salmonella strain isolated from blood.

A strain of Salmonella choleraesuis subsp. choleraesuis serovar paratyphi-A isolated from the blood of a febrile patient grew into filaments on a nutrient agar containing various salts, such as NaCl, KCl, MgCl2, NH4Cl, (NH4)2SO4, or (NH4)2HPO4, at concentrations of 50 to 400 mM. The filamentous cells were nonseptate and multinucleate, and they had colony-forming ability. This mutant strain, however, did not show filamentous growth in liquid media which contained the same salts. On nutrient agar containing 20% sucrose but no salts, some of the cells formed large spheroplasts. Both ampicillin treatment and in vivo environment may in part be responsible for the induction of the mutant strain.

Isoelectric focusing of beta-lactamases in Mycobacterium fortuitum. Association of a single enzyme pattern with cefoxitin resistance.

The uninduced culture supernatants and cell extracts from 58 strains of the 3 biovariants (biovar) of Mycobacterium fortuitum were all positive for beta-lactamase with the chromogenic cephalosporin substrate. By analytical isoelectric focusing (IEF), 29 of 30 strains of biovar fortuitum exhibited an identical beta-lactamase pattern with 1 major band. In contrast, the beta-lactamases of biovar peregrinum and the unnamed third biovar were heterogeneous, with multiple bands and a variety of patterns. The pH range of isoelectric points for the beta-lactamases was relatively narrow, however, with most bands appearing between pH 4.3 and 5.2. Although additional genetic studies are required, these enzymes appear to be chromosomal, as they are present in all strains including some without detectable plasmids. Repeat isolates from the same patient obtained up to six months apart always had the same beta-lactamase pattern by IEF. Of the third biovar complex, 30% are cefoxitin resistant with minimal inhibitory concentrations greater than 32 micrograms/ml. All 9 cefoxitin-resistant isolates tested had the same unique beta-lactamase pattern by IEF, although this enzyme failed to hydrolyze cefoxitin while hydrolyzing cephalothin and benzylpenicillin. Thus, despite the association of cefoxitin-resistance with a single enzyme pattern, the role of this beta-lactamase in resistance is not known.

Morphological changes induced by beta-lactam antibiotics in Mycobacterium avium-intracellulare complex.

In vitro activity of seven beta-lactam antibiotics against strains of Mycobacterium avium-intracellulare was evaluated by the agar dilution method. The activity was influenced by the presence or absence of Tween 80 in Dubos medium, and cephazolin and cefotaxime were effective against most strains in the presence of Tween 80. beta-Lactam antibiotics at low concentrations induced long filamentous cells with branching. In contrast to the filaments induced by ampicillin, in which septation was rarely observed, filaments induced by cephazolin had many septa, suggesting that the mechanisms of filament induction were different from the drugs used. At high concentrations, ampicillin and cephazolin induced osmotically sensitive cells with bulging at polar end of the cells. Analysis of penicillin binding proteins (PBPs) of the organism showed that there were at least nine PBPs with molecular weights between 32,000 and 94,000 in the cytoplasmic membrane. Ampicillin showed the highest affinity for PBPs 1a or 1b, or both, and also PBPs 3a or 3b, or both. In contrast, there was very little specificity of binding of cephazolin for any of the PBPs.

Isolation and characterization of a filamentous phage, Vf33, specific for Vibrio parahaemolyticus.

Phage Vf33, a filamentous phage about 1,400 nm long and 7 nm wide, specific for Vibrio parahaemolyticus, was isolated and characterized. The buoyant density of Vf33 in CsCl was 1.292 g/cm3. As with other filamentous phages, the lytic activity of Vf33 was resistant to heating below 80 C and to treatment with diethylether, acetone or methanol but sensitive to chloroform. The nucleic acid of this phage is single-stranded circular DNA 8.4 kb in size. The viral genome was converted to a double-stranded replicative form in the host-cell. Among the strains tested, only V. parahaemolyticus strains possessing K38 antigen was sensitive to the phage.

Mechanism of antibiotic resistance in Mycobacterium intracellulare.

The mechanism of resistance of Mycobacterium intracellulare strain 103 and other clinical isolates to a variety of drugs including aminoglycoside and peptide antibiotics was investigated. Enzymatic inactivation of aminoglycoside and peptide antibiotics could not be demonstrated. Ribosomes of the strain were found to be sensitive to the antibiotics. The levels of resistance of strain 103 and other clinical isolates decreased dramatically when the culture medium was changed from Dubos agar to Tween 80-containing agar. These results suggest that a permeability barrier is the reason for naturally occurring resistance in M. intracellulare.

An improved method for the preparation of mycobacterial spheroplasts and the mechanism involved in the reversion to bacillary form: electron microscopic and physiological study.

An efficient method is described for preparing spheroplasts and protoplasts by treating bacillary cells of Mycobacterium smegmatis with precise concentrations of L-glycine (followed by lysozyme). This improved procedure was widely applicable to many rapidly growing mycobacteria by selecting the concentrations of glycine suitable for the individual strains used. The process of reversion of spheroplasts to original bacillary form on solid and in liquid media, as revealed by electron microscopy, appeared to involve the formation of an internal elementary or initial body with subsequent budding from the spheroplast. The internal membrane systems appeared to function in the induction of initial bodies and in the maturation of elementary bodies to become dividing forms. Possible mechanisms involved in the development of bacilli from spheroplasts are discussed.

Spheroplast formation of Mycobacterium smegmatis and morphological aspects of their reversion to the bacillary form.

Cell wall-deficient forms (spheroplasts) of Mycobacterium smegmatis strain P53 were prepared by combined treatment with glycine, lysozyme, and lytic enzyme no. 2 as the spheroplasting agents. Quantitative mass conversion to spherical forms was effected by pretreatment of the intact cells with 1.2% glycine in nutrient broth, followed by transfer to spheroplasting medium containing the above agents. Two apparent modes of reversion to the bacillary form were observed under electron microscopy. The first one was initiated by budding from the spheroplasts. The buds gradually elongated to become the mycelial form, which showed branching, septation, and fragmentation. The second resulted from the intracellular formation of tiny cells, possibly the elementary bodies, and their release from the spheroplasts.

Effect of sodium chloride on the activity and production of staphylococcal exonuclease.

Extracellular nuclease activity in Staphylococcus aureus was enhanced about fourfold by 1% (w/v) NaCl, KCl, CsCl or LiCl. The pH and concentration of Ca2+ for optimum activity varied with the NaCl concentration; with an increased NaCl concentration, a higher Ca2+ concentration and a lower pH were required. Vmax, but not Km, varied with the concentration of NaCl. The addition of 3% (w/v) NaCl to growing cultures of S. aureus increased nuclease production fivefold.

Effect of sodium chloride on the respiratory function of Staphylococcus aureus.

Sodium chloride at concentrations below 0.5 M, enhanced the respiratory activity (O2-consumption) of Staphylococcus aureus under endogenous and sugar-supported conditions, but did not overcome the inhibitory action of sodium azide. Several sugars, including the glucose analogue alpha-methylglucoside, and their metabolites enhanced bacterial O2-consumption, but acetylmethylcarbinol was ineffective.