Positive selection of allelic exchange mutants in Mycobacterium bovis BCG

Abstract A resistant mutant with vancomycin MIC of 100 μg/ml was isolated relatively easily through step pressure in the laboratory from a Staphylococcus aureus strain with initial MIC of 1.5 μg/ml for the antibiotic. Upon addition of vancomycin (50 μg/ml) to the growth medium mass increase of the culture and peptidoglycan synthesis continued but cell division (daughter cell separation), cell wall turnover and autolysis were inhibited, resulting in the production of multicellular clumps of bacteria. Parallel with the increase of culture density, the concentration of vancomycin measured both by biological activity and by HPLC gradually declined in the culture medium. Cell division and wall turnover of the culture resumed with the production of cells of normal morphology at the time when the concentration of the drug in the medium decreased below 0.5–1.0 μg/ml. There was no detectable change in the antibiotic concentration in the culture medium during growth of a vancomycin-resistant ( vanA -positive) strain of Enterococcus faecium and an intrinsically vancomycin-resistant strain of Leuconostoc . The vancomycin-resistant staphylococcal mutant gave no signal with the vanA or vanB DNA probes and contained no detectable d-lactate terminating cell wall precursors. The biochemical mechanism and clinical significance of such glycopeptide-resistant mutants remains to be established.     

Antibiotics and the Oral Streptococci of Man

The effects of 3 antibiotics, phenoxymethylpenicillin, cephalexin and clindamycin on the normal oral streptococcal flora in the region of the gingival crevice were investigated because these organisms are able to cause subacute bacterial endocarditis. Secretion of these antibiotics into the oral cavity was also examined. Penicillin and clindamycin exerted marked effects on the normal oral streptococci, whereas cephalexin did not cause any obvious change in the total flora. Following penicillin therapy, streptococci resistant to 1.5 μg/ml penicillin were observed and these organisms could be detected at least 8 weeks after the last dose of the antibiotic. They probably arose by selection from the mixed flora. Following cephalexin therapy, a much lower proportion of streptococci resistant to 15 μg/ml was found. The proportion of resistant strains fluctuated appreciably, however, probably due to their transient nature. Streptococci resistant to 1 μg/ml clindamycin were not observed in 10 out of 11 treated subjects.
Penicillin and clindamycin could be detected in the pooled saliva and gingival fluid after administering single doses of 500 mg and 300 mg, respectively. The peak levels were obtained between half and 1 h. The concentration of penicillin dropped rapidly within 3 h but clindamycin could be detected at significant levels for at least 6 h. No cephalexin could be detected in the pooled saliva or gingival fluid after a 500 mg dose. The implications of these findings in the prevention of subacute bacterial endocarditis are discussed.     

The effect of growth-promoting antibiotics on the faecal enterococci of healthy young chickens

Small groups of chickens were given feed containing either avoparcin, nitrovin, vir-giniamycin or zinc bacitracin from the day of their purchase as day-olds. Differences between the birds receiving growth promoters and the untreated controls were observed during the last third of the 23 d survey period. The enterococcal population of the 'dosed' birds contained a greater proportion of Enterococcus faecium than did that of the control birds while the converse was true for Ent. gallinarum . This apparent selection of Ent. faecium by the growth-promoting antibiotics had an influence on the incidence of resistance to therapeutic antibiotics among the enterococcal population as a whole. This was because this species was generally more resistant than Ent. gallinarum to cephalothin, the MLS antibiotics (erythromycin, lincomycin and tylosin) and tetracycline.     

The effect of ampicillin and tylosin on the faecal enterococci of healthy young chickens

Enterococcal isolates from young chickens were differentiated into one of three species, namely Enterococcus faecalis, Ent. faecium and Ent. gallinarum . The proportion of each species among the enterococcal population changed with time in birds not dosed with antibiotics. This pattern of change was modified in birds dosed with either tylosin or ampicillin even though ampicillin did not select for ampicillin resistance among the enterococcal population. A gradual increase in tylosin resistance was recorded with time among the enterococci of the 'undosed' control birds. This was associated with an increase in the proportion of Ent. faecium , a species commonly resistant to tylosin, among the enterococci of the birds as they grew older.     

The effect of ampicillin and tylosin on the faecal enterococci of healthy young chickens

Enterococcal isolates from young chickens were differentiated into one of three species, namely Enterococcus faecalis, Ent. faecium and Ent. gallinarum. The proportion of each species among the enterococcal population changed with time in birds not dosed with antibiotics. This pattern of change was modified in birds dosed with either tylosin or ampicillin even though ampicillin did not select for ampicillin resistance among the enterococcal population. A gradual increase in tylosin resistance was recorded with time among the enterococci of the 'undosed' control birds. This was associated with an increase in the proportion of Ent. faecium, a species commonly resistant to tylosin, among the enterococci of the birds as they grew older.     

Effect of raw-milk cheese consumption on the enterococcal flora of human feces

Enterococci are one of the major facultative anaerobic bacterial groups that reside in the human gastrointestinal tract. In the present study, the composition of the enterococcal fecal flora in three healthy humans was analyzed before, during, and after the daily consumption of approximately 125 g of a raw-milk Cheddar-type cheese containing 3.2 x 10(4) enterococci/g of cheese. Enterococcal counts ranged between 1.4 x 10(2) and 2.5 x 10(8) CFU/g of feces and differed from subject to subject and from week to week. The cheese contained mainly Enterococcus casseliflavus and a small population of Enterococcus faecalis. Clonal relationships were determined by pulsed-field gel electrophoresis. Before and after consumption of the cheese, samples from humans contained mainly Enterococcus faecium, with some of the clones being resident. During consumption of the cheese, one particular transient clone of E. faecalis, clone Fs2, which was present in small numbers in the cheese, largely dominated the feces. Two clones of E. casseliflavus from the cheese were also found in the feces of one of the subjects during cheese consumption. These results suggest that a clone need not be present in a food in high numbers to establish itself in the intestine.     

Nucleotide sequence of the structural gene for the penicillin-binding protein 2 of Staphylococcus aureus and the presence of a homologous gene in other staphylococci

Abstract During growth of Streptomyces niveus wild-type in the novobiocin production medium CDM the resistance of mycelia to novobiocin rises from about 25 μg/ml to over 200 μg/ml. ( S. lividans , a novobiocin-sensitive strain, is resistant to approx. 10 μg/ml novobiocin.) The initial period of low level resistance extends from the time of inoculation of the culture until approx. 70 h when the culture is still in the growth phase. High level resistance is initiated before the start of novobiocin production and rises rapidly to a maximum level beyond the end of the growth phase. The rise in pH of the unbuffered CDM medium which occurs during S. niveus fermentation was shown not to be the cause of the change in novobiocin resistance. However, mycelia-free CDM from S. niveus cultures expressing high level novobiocin resistance was shown to contain a factor which induced high level novobiocin resistance in germinating S. niveus spores. Kinetic studies revealed that the inducer first appears in the culture medium before the switch to high level resistance begins and reaches its highest concentration before resistance reaches its maximum level.     

A Note on the Sensitivity of Chlorine-stressed Bifidobacteria to Nalidixic Acid

Bifidobacterium adolescentis , normally resistant to 200 μg/ml nalidixic acid, became sensitive to 10 μg/ml nalidixic acid after 15 min exposure to chlorinated sewage effluent (0–08 mg/1000 ml of residual chlorine). After exposure to 0.7 mg/1000 ml of residual chlorine, Bifidobacterium adolescentis became sensitive to 5 μg/ml nalidixic acid.     

Effect of Raw-Milk Cheese Consumption on the Enterococcal Flora of Human Feces

Enterococci are one of the major facultative anaerobic bacterial groups that reside in the human gastrointestinal tract. In the present study, the composition of the enterococcal fecal flora in three healthy humans was analyzed before, during, and after the daily consumption of ~125 g of a raw-milk Cheddar-type cheese containing 3.2 × 10⁴ enterococci/g of cheese. Enterococcal counts ranged between 1.4 × 10² and 2.5 × 10⁸ CFU/g of feces and differed from subject to subject and from week to week. The cheese contained mainly Enterococcus casseliflavus and a small population of Enterococcus faecalis. Clonal relationships were determined by pulsed-field gel electrophoresis. Before and after consumption of the cheese, samples from humans contained mainly Enterococcus faecium, with some of the clones being resident. During consumption of the cheese, one particular transient clone of E. faecalis, clone Fs2, which was present in small numbers in the cheese, largely dominated the feces. Two clones of E. casseliflavus from the cheese were also found in the feces of one of the subjects during cheese consumption. These results suggest that a clone need not be present in a food in high numbers to establish itself in the intestine.     

OHIO-1 β-lactamase resistant to mechanism-based inactivators

Abstract Spontaneous mutants of OHIO-1 β-lactamase, an SHV-1 family enzyme, resistant to inactivation by clavulanic acid, sulbactam and tazobactam, have been isolated. The resistant mutant (M4) was inhibited by 100 μg/ml ampicillin plus 32 μg/ml clavulanic acid compared to ≤2 μg/ml clavulanic acid required for the parent strain. The pI of the mutant beta-lactamase was 7.0, identical to the parent enzyme. Kinetic parameters showed that the M4 enzyme had an increased V_max/K_m ratio for all beta-lactam substrates compared to the parent enzyme. The apparent K _i for clavulanic acid, sulbactam and tazobactam was 15.1, 182 and 18 μM, respectively, up to 70-fold higher than the parent enzyme. Partial nucleotide sequencing revealed that the mutant enzyme had a predicted methionine₆₉→ isoleucine₆₉ substitution accounting for the observed changes in substrate specificity.     

The Effect of Monensin on Pure and Mixed Cultures of Rumen Bacteria

The antibiotic monensin was added to pure cultures of Bacteroides ruminicola, Selenomonas ruminantium, Anaerovibrio lipolytica and Megasphaera elsdenii. These organisms, representing succinate- and propionate-producing rumen bacteria, were not affected by monensin up to 10 μg/ml. Methanobacterium ruminantium was slightly inhibited by monensin, Butyrivibrio fibrisolvens, Ruminococcus albus and Streptococcus bovis were inhibited to differing extents by monensin at concentrations between 0.1 and 10 μg/ml. Bacteroides succinogenes was inhibited at first by monensin at >0.5 μg/ml but after a prolonged lag phase adapted to grow in the presence of monensin at concentrations below 5 μg/ml.
Monensin (1 μg/ml) almost completely stopped the digestion of chopped straw and dewaxed cotton fibres by rumen contents incubated in vitro. The digestion of grass and powdered filter paper was not significantly reduced under these conditions, but when the concentration of monensin was increased to between 3 and 5 μg/ml, the digestion of these substrates was reduced.     

Antibiotic susceptibility of potentially probiotic Bifidobacterium isolates from the human gastrointestinal tract

Sixteen Bifidobacterium isolates from the human gastrointestinal tract were assayed for susceptibility to 44 antibiotics by soft agar overlay disc diffusion on TPY agar. Five isolates (3/7 B. bifidum and 2/3 B. breve ) exhibited atypical antibiotic susceptibility profiles. Poor growth in the agar overlay accounted for susceptibility of B. bifidum but not B. breve isolates. All other isolates were resistant to cefoxitin (30 μg), aztreonam (30 μg), vancomycin (30 μg), amikacin (30 μg), gentamicin (10 μg), kanamycin (30 μg), streptomycin (10 μg), fusidic acid (10 μg), trimethoprim (5 μg), norfloxacin (10 μg), nalidixic acid (30 μg), metronidazole (5 μg), polymyxin B (300 μg) and colistin sulphate (10 μg), and they were susceptible to the six penicillins studied, cephalothin (30 μg), cefuroxime (30 μg), cefaclor (30 μg), ceftizoxime (30 μg), cefotaxime (30 μg), bacitracin (10 μg), chloramphenicol (30 μg), erythromycin (15 μg), clindamycin (2 μg), rifampicin (5 μg) and nitrofurantoin (300 μg). In addition, they varied in their susceptibility to cephradine (30 μg), cephazolin (30 μg), cefoperazone (75 μg), ceftriaxone (30 μg), ofloxacin (5 μg) and furazolidone (15 μg). They were resistant, or only marginally moderately susceptible, to ceftazidime (30 μg), netilmicin (10 μg), sulphamethoxazole (100 μg), cotrimoxazole (25 μg) and ciprofloxacin (5 μg), and susceptible or marginally moderately susceptible to tetracycline (30 μg). All B. bifidum isolates were susceptible to cefixime (5 μg). Four micro-organism-drug combinations were evaluated for β-lactamase activity but its absence suggested that cell wall impermeability was responsible for cephalosporin resistance among bifidobacteria. The antibiotic susceptibility of B. animalis 25527T was similar to that of the human isolates.     

Efficacy of Novel Antimicrobials Against Clinical Isolates of Opportunistic Amebas

ABSTRACT We examined the effects of the macrolide antimicrobial agent azithromycin and phenothiazine compounds against clinical isolates of Acanthamoeba spp. and Balamuthia mandrillaris , opportunistic pathogens of human beings and other animals. Acanthamoeba growth was inhibited in vitro at 1,5, and 10 μg/ml of azithromycin, but not the macrolides, erythromycin, and clarithromycin. In experiments attempting to simulate in vivo conditions, azithromycin protected monolayers of rat glioma cells from destruction by Acanthamoeba at a concentration of 0.1 μg/ml, and delayed destruction at concentrations of 0.001 and 0.01 μg/ml. We concluded that the minimal inhibitory concentration of azithromycin was 0.1 μg/ml. Our results, however, suggested that the drug was amebastatic but not amebicidal, since ameba growth eventually resumed after drug removal. The phenothiazines (chlorpromazine, chlorprothixene, and triflupromazine) inhibited Acanthamoeba growth by 70-90% at 5 and 10 μg/ml, but some of these compounds were toxic for rat glioma cells at 10 μg/ml. Azithromycin was not very effective against B. mandrillaris in an in vitro setting, but was amebastatic in tissue culture monolayers at concentrations of 0.1 μg/ml and higher. Balamuthia amebas showed in vitro sensitivity to phenothiazines. Ameba growth was inhibited 30-45% at 5 μg/ml in vitro, but completely at 5 μg/ml in the rat glioma model. In spite of their potential as antiamebic drugs in Balamuthia infections, toxicity of phenothiazines limits their use in clinical settings.     

Cytokinesis of Candida albicans by Diethylstilbestrol

In vitro effects of the synthetic oestrogenic hormone diethylstilbestrol (DS) and diethylstilbestrol dipropionate (DSP) on Candida albicans have been assessed. At a concentration of 5–20 μg/ml. these compounds suppressed the growth of C. albicans even though the multiplication of the organism was not influenced immediately. When C. albicans was grown for approximately 4 h in tryptic soy broth, its multiplication was rapidly retarded by these two substances. Since C. albicans must grow on a suitable culture medium in order to absorb DS and DSP, it was not surprising that respiration, the uptake, and incorporation of nutrients by C. albicans was not influenced when the cells were 'resting'. Such plasma steroids as androsterone (0·5 μg/ml), 5α-androstan-3 β-diol (0·25 μg/ml), dehydroisoandrosterone (2 μg/ml), epiandrosterone (0·1 μg/ml), oestrone (0·1 μg/ml), progesterone (0·4 μg/ml), cortisol (0·2 μg/ml), cholesterol (10 μg/ml) in combination with DSP did not antagonize the retardive action of DSP for C. albicans .     

Mutants of Acanthamoeba castellanii Resistant to Erythromycin, Chloramphenicol, and Oligomycin

SYNOPSIS. Cell lines of Acanthamoeba castellanii resistant to erythromycin (Ery^R), chloramphenicol (Cap^R), and oligomycin (Oli^R) have been isolated. These may be the first such mutants for A. castellanii. These mutants have been phenotypically stable for 2 years, surviving storage and vegetative multiplication in the absence of drugs. Resistance was specific for each drug, but double mutants (e.g. Ery^RCap^R) were obtained by stepwise selection. Mutant frequencies were determined in multiwell plates; <10 colony forming units (CFU/10⁵ amebas were observed in wild-type populations 12 days after incubation in 500 μg Ery/ml, 2.5 mg Cap/ml, or 15 μg Oli/ml. After 30 days, averages of 100 CFU/10⁵ amebas were observed in Ery and Cap, whereas, frequencies for Oli remained unchanged. Frequencies for Ery^R and Cap^R were consistent with rates of recovery from these drugs in batch cultures. We were unable to obtain spontaneous mutants resistant to cycloheximide, emetine, 5-fluorodeoxyuridine, or ethidium bromide. Ery^R, Cap^R and Oli^R could be mitochondrial mutants.     

Anaerobic expression of the gyrA gene demonstrated by β-galactosidase activity in gyrA::Mu-lac fusion derivatives of Escherichia coli

Abstract E. coli C600Δ lac was infected with a Mu- lac defective phage. Lysogens resistant to ampicillin (25 μg/ml) and nalidixic acid (20 μg/ml) were unable to grow anaerobically and insensitive to nalidixic acid (150 μg/ml), indicating formation of gyrA ::Mu- lac fusions. Mapping of the Mu- lac by P1 phage cotransduction with two different gyrA linked Tn 10 transposon loci suggests that the insertion is located in the gyrA gene. These fusion derivatives form small uncolored colonies on lactose MacConkey agar plates aerobically. When these plates are placed in an anaerobic jar for about 4 h, the colonies turn red indicating that the inserted lac gene is expressed only under anaerobic environment. Thus the gyrA promoter is activated under the anaerobic environment and loss of gyrA activity prevents anaerobic cell growth.     

Antibiotic Susceptibility of Helicobacter pylori Clinical Isolates: Comparative Evaluation of Disk-Diffusion and E-Test Methods

Antimicrobial susceptibility of 25 Helicobacter pylori strains isolated from patients with acid peptic diseases were tested for in vitro sensitivity to commonly used antibiotics using disk-diffusion and E-test, methods. All strains tested were susceptible to tetracycline by E-test, with the minimum inhibitory concentration (MIC) values being <0.125 μg/ml for all strains except for 6 (<0.023 μg/ml). However 1 strain was resistant by disk-diffusion method. One strain was resistant to clarithromycin both by disk diffusion and E-test (MIC <48 μg/ml), and 1 strain was resistant only by disk diffusion. Only one strain was resistant to amoxicillin by disk diffusion and E-test (MIC >256 μg/ml). For ciprofloxacin, three strains were resistant by disk diffusion and two by E-test (MIC <32 μg/ml). Sixteen strains were resistant to metronidazole by disk diffusion and E-test (MIC ≥ 8 μg/ml), and 1 was resistant only by E-test (MIC <48 μg/ml). Overall, 64% of the strains were resistant to metronidazole. The MIC for metronidazole was also tested by agar-dilution method, and metronidazole resistant strains had an, MIC >8 μg/ml. The disk-diffusion method showed excellent correlation with E-test results; there was 100% agreement for amoxicillin a other antibiotics showed 90% to 95% accuracy. Disk diffusion is cheaper than E-test (approximately 2.6 cents vs. US$2.60), is easy to perform, and is a reliable method for testing H. pylori susceptibility to antimicrobial agents in the clinical microbiology laboratory.     

The toxicity of a number of different bactericides to Clavibacter michiganense subsp. michiganense (Smith 1910) Jensen 1934 comb. nov. [basonym Corynebacterium michiganense pv. michiganense (AL)] and to the tomato plant, Lycopersicon esculentum

Twenty-seven proprietary products and pure chemicals were tested in vitro against cells of Clavibacter michiganense subsp. michiganense (Smith 1910) Jensen 1934 comb. nov. [basonym Corynebacterium michiganense pv. michiganense (AL)] (the cause of bacterial canker of tomato) and also for their phytotoxicity to tomato plants. The most bactericidal of these, with a minimum cidal concentration (MCC) range of > 10-< 100 μg/ml, were a phenolic product called Applied 3–78, two quaternary ammonium compounds (benzalkonium chloride and cetrimide), and a silver colloid compound. Of these, only Applied 3–78 was not phytotoxic at values of 10 μg/ml or less, although it was phytotoxic at 10000 μg/ml. Copper oxychloride and sodium hypochlorite were amongst the group with a middle range of bactericidal properties, their MCC range being from > 1000 to < 10000 μg/ml. They were phytotoxic at 1000 μg/ml or less. When organic matter, a dead yeast suspension, was added to Applied 3–78, Kohrsolin and Panacide, only the activity of Applied 3–78 was relatively unchanged. The MCC ranges were: Applied 3–78, >80–< 100 μg/ml; Kohrsolin, > 800-< 1000 μg/ml; and Panacide, > 1000 μg/ml. Phytotoxicity tests on 10 different tomato cultivars confirmed that Applied 3–78 was the least phytotoxic of these three products. Field trials on tomato crops showed that when Applied 3–78 was sprayed on the plants once, and Kohrsolin was either sprayed on or they were drenched with it once at 1000 μg/ml, no phytotoxicity symptoms developed.     

β-Lactamase production and in vitro antimicrobial susceptibility of Porphyromonas gingivalis

Abstract β-Lactamase production by 98 Porphyromonas strains was investigated by the nitrocefin (chromogenic cephalosporin) test. Human isolates of P. gingivalis (91), P. endodontalis (2), and P. asaccharolytica (1) were tested, with four closely related Porphyromonas spp. of animal origin and four reference strains. The in vitro susceptibility of 64 P. gingivalis strains was investigated on Brucella blood agar by the E test. None of the human Porphyromonas isolates tested produced β-lactamase, but one Porphyromonas strain of animal origin, most closely resembling P. endodontalis , produced β-lactamase. P. gingivalis was susceptible to almost all of the drugs tested: benzylpenicillin, ampicillin, cefaclor, cefuroxime, erythromycin, clindamycin, tetracycline, doxycycline, metronidazole and ciprofloxacin; all strains were inhibited at 0.016 μg/ml, 0.023 μg/ml, 0.315 μg/ml, 0.064 μg/ml, 0.19 μg/ml, 0.016 μg/ml, 0.094 μg/ml, 0.047 μg/ml, 0.023 μg/ml, and 0.75 μg/ml of these drugs, respectively. Cotrimoxazole exhibited variable efficacy against P. gingivalis ; the range of MICs was 0.1095-32.0 μg/ml. The results indicate that β-lactamase production is currently not a problem amongst clinical isolates of P. gingivalis and strains are susceptible to most antimicrobial agents.     

Mercury Tolerance of Thermophilic <Emphasis Type="Italic">Bacillus</Emphasis> sp. and <Emphasis Type="Italic">Ureibacillus</Emphasis> sp

Although resistance of microorganisms to Hg(II) salts has been widely investigated and resistant strains have been reported from many eubacterial genera, there are few reports of mercuric ion resistance in extremophilic microorganisms. Moderately thermophilic mercury resistant bacteria were selected by growth at 62 °C on Luria agar containing HgCl₂. Sequence analysis of 16S rRNA genes of two isolates showed the closest matches to be with Bacillus pallidus and Ureibacillus thermosphaericus. Minimum inhibitory concentration (MIC) values for HgCl₂ were 80 μg/ml and 30 μg/ml for these isolates, respectively, compared to 10 μg/ml for B. pallidus H12 DSM3670, a mercury-sensitive control. The best-characterised mercury-resistant Bacillus strain, B. cereus RC607, had an MIC of 60 μg/ml. The new isolates had negligible mercuric reductase activity but removed Hg from the medium by the formation of a black precipitate, identified as HgS by X-ray powder diffraction analysis. No volatile H₂S was detected in the headspace of cultures in the absence or presence of Hg²⁺, and it is suggested that a new mechanism of Hg tolerance, based on the production of non-volatile thiol species, may have potential for decontamination of solutions containing Hg²⁺ without production of toxic volatile H₂S.