Antibiotic and deoxycholate resistance in Campylobacter jejuni following freezing or heating

H umphrey , T.J. & C ruickshank , J.G. 1985. Antibiotic and deoxycholate resistance in Campylobacter jejuni following freezing or heating. Journal of Applied Bacteriology 59 , 65–71.
The surviving populations of Campylobacter jejuni serotypes following freezing or heat were found to be more sensitive to rifampicin and sodium deoxycholate on subsequent culture. Thus while control cultures had an IC₅₀ of greater than 20 μg/ml rifampicin those of injured cells were <5 μg/ml. Treatment with EDTA caused almost identical changes in resistance suggesting that the altered resistance pattern of injured cells was due to loss of the barrier properties of the bacterial outer membrane.     

Two groups of Mycobacterium avium complex strains determined according to the susceptibility to rifampicin and ansamycin

Mycobacterium avium complex strains previously not exposed to any antituberculosis agents could be divided into two groups according to their susceptibility to rifampicin and ansamycin; one group susceptible to 80 micrograms/ml rifampicin and to 1.25 micrograms/ml ansamycin, and another resistant to these concentrations. In each group, the ratio of the minimal inhibitory concentration of ansamycin against that of rifampicin was greatly different depending on the strain. This naturally occurring resistance to rifampicin and ansamycin was frequently correlated to naturally occurring resistance to ethambutol, kanamycin, enviomycin, kitasamycin, and minocycline, but not correlated to that to isoniazid and sulfadimethoxine. Ansamycin was more active than rifampicin against M. bovis, M. kansasii, M. marinum, M. xenopi, and M. haemophilum.     

Effects of rifampicin and doxycycline on the production of hydrogen peroxide by macrophages]

The influence of rifampicin and doxycycline on oxidative metabolism of macrophages was estimated in vitro by production of hydrogen peroxide. It was shown that low concentrations of rifampicin and doxycycline stimulated production of hydrogen peroxide by macrophages of guinea pigs. In concentrations of 1 to 10 micrograms/ml corresponding to the mean therapeutic ones doxycycline increased both the spontaneous and zymosan-induced production of hydrogen peroxide by the macrophages. The potentiating activity of doxycycline on the cells activated by opsonized zymosan was higher. The maximum increase in the induced production of hydrogen peroxide (by 40 per cent) was observed when the antibiotic concentration was 1 microgram/ml. Rifampicin in concentrations of 0.1 to 1 microgram/ml corresponding to the mean therapeutic ones stimulated the zymosan-induced production of hydrogen peroxide by the macrophages. The maximum increase in the production of hydrogen peroxide (by 22 per cent) was noted at the rifampicin concentration of 1 microgram/ml.     

Quantitative analysis of rifampicin and 25-desacetylrifampicin in the plasma using high performance liquid chromatography

A procedure for determination of rifampicin and 25-desacetylrifampicin in plasma by HPLC was developed. The plasma proteins are precipitated by acetonitrile and the supernatant layer (50 microliters) is used for the assay under isocratic conditions on an analytical column 250 x 4.6 mm in size containing the reversed phase sorbent (C18). The size of the precolumn is 50 x 4.6 mm. An UV detector (at lambda 335 nm) is used. For preparing the mobile phase 630 ml of methanol and 370 ml of 0.058 M sodium nitrite solution are mixed. The flow rate of the mobile phase is 40.7 ml/min. The assay duration is about 10 min. The retention time is 9.6 min for rifampicin and 6.5 min for 25-desacetylrifampicin. The minimum detectable amount of the antibiotic and its metabolite is 0.10 micrograms/ml. The standard curves of rifampicin and 25-desacetylrifampicin are linear within the concentration ranges of 0.5-100 and 0.5-10 micrograms/ml respectively. The procedure is useful in studies on pharmacokinetics of rifampicin and 25-desacetylrifampicin.     

Effect of rifampicin and doxycycline on the chemiluminescence of leukocytes]

The effect of rifampicin and doxycycline on spontaneous and zymosan-induced chemiluminescence of polymorphonuclear leukocytes was studied on guinea pigs. The cells were incubated in the presence of the antibiotics, washed and stimulated by zymosan. Under such conditions rifampicin in therapeutic doses of 0.1 to 10 micrograms/kg and doxycycline in a dose of 100 micrograms/kg potentiated the leukocyte chemiluminescence. Investigation of the antibiotics effect on the cells without washing failed because of the direct interference of rifampicin and doxycycline with the cell-independent stage of the chemiluminescent reaction.     

Role of superoxide radicals in cytotoxic effects of Fe-NTA on cultured normal liver epithelial cells

We studied the cytotoxic effects of ferric nitrilotriacetate (Fe-NTA) on normal rat liver epithelial cells (RL34) cultured in medium containing 10% fetal calf serum. Marked cytolysis was present in cells exposed to greater than or equal to 25 micrograms/ml iron of Fe-NTA, but not all the cells exposed to 50 micrograms/ml iron were lethally injured. The remaining cells showed anomalous growth, namely cell pile-up and aggregation. Superoxide dismutase inhibited this iron-induced cytotoxicity, whereas catalase, mannitol, dimethyl sulfoxide, and 1,4-diazabicyclo-[2.2.2.] octane did not. RL34 cells exposed to Fe-NTA actually produced a large amount of superoxide radicals (O2-.), whereas unexposed control cells produced none. Allopurinol inhibited O2-. production and prevented cell injury by Fe-NTA. These results show that the injury to cells produced by Fe-NTA depends on the generation of O2-., the source of which may be xanthine oxidase.     

A new mutation in 16S rRNA of Escherichia coli conferring spectinomycin resistance.

We report a novel mutation, C1066U in 16S rRNA which was selected for resistance to spectinomycin, an antibiotic which inhibits ribosomal translocation. The minimal inhibitory concentration (MIC) of spectinomycin determined for this mutant (15 micrograms/ml) is greater than with the wild-type plasmid (5 micrograms/ml) but lower than with the well known C1192U mutation (> 80 micrograms/ml). The C1066U mutation also increases the cells sensitivity to fusidic acid, another antibiotic which inhibits translation at the translocation stage, whereas C1192U is unchanged relative to the wild type. We discuss why the acquisition of resistance to one of these drugs is often associated with hypersensitivity to the other.     

Involvement of multiple genetic determinants in high-level methicillin resistance in Staphylococcus aureus.

A methicillin-susceptible, novobiocin-resistant strain of Staphylococcus aureus (RN2677; methicillin MIC, 0.8 micrograms/ml) was transformed with DNA prepared from highly and homogeneously methicillin-resistant S. aureus strains (methicillin MIC, greater than or equal to 400 micrograms/ml) or from heterogeneous strains in which the majority of cells had a low level of resistance (methicillin MIC, 6.3 micrograms/ml). All methicillin-resistant transformants showed low and heterogeneous resistance (methicillin MIC, 3.1 micrograms/ml) irrespective of the resistance level of DNA donors. All transformants examined produced normal amounts of the low-affinity penicillin-binding protein (PBP) 2a, and methicillin resistance and the capacity to produce PBP 2a showed the same degree of genetic linkage to the novobiocin resistance marker with both homogeneous and heterogeneous DNA donors. Next, we isolated a methicillin-susceptible mutant from a highly and homogeneously resistant strain which had a Tn551 insertion near or within the PBP 2a gene and thus did not produce PBP 2a. With this mutant used as the recipient, genetic transformation of the methicillin resistance gene was repeated with DNA isolated either from highly and homogeneously resistant strains or from heterogeneous (low-resistance) strains. All transformants obtained expressed high and homogeneous resistance and produced PBP 2a irrespective of the resistance level of the DNA donors. Our findings suggest that (i) the methicillin resistance locus is identical to the structural gene for PBP 2a, (ii) although the ability to produce PBP 2a is essential for resistance, the MICs for the majority of cells are not related to the cellular concentration of PBP 2a, and (iii) high MICs and homogeneous expression of resistance require the products of other distinct genetic elements as well.     

Evaluation of anti-coccidial drugs' inhibition of Neospora caninum development in cell cultures

Eight anti-coccidial drugs were examined for their efficacies in preventing development of Neospora caninum in bovine monocyte cell cultures. Lasalocid sodium (0.05 microgram/ml), monensin sodium (0.05 microgram/ml), piritrexim (0.01 microgram/ml), pyrimethamine (0.05 microgram/ml), and trimethoprim (5.0 micrograms/ml) were effective in preventing development of intracellular N. caninum tachyzoites (P less than 0.05). No differences (P greater than 0.05) in mean numbers of infected cells compared to controls were observed in cultures treated with amprolium hydrochloride (10.0 micrograms/ml), sulfadiazine (200.0 micrograms/ml), and sulfamethoxazole (200.0 micrograms/ml).     

Amphotericin B susceptibility testing of Candida species by flow cytometry.

We have developed an 8 hr flow cytometry (FCM) method for assessing susceptibility of yeasts to amphotericin B (AmpB). The method detects both high-level and relative-resistance to the drug. Variables found to affect fluorescence of control and AmpB treated cells included pH, presence of glucose, incubation conditions, concentration and length of exposure to both AmpB and ethidium bromide (ETBR), and the degree of resistance to AmpB. The FCM method was optimized based on increased red fluorescence intensity (RF), decreased forward angle light scatter (FALS), and a negative gating technique. A dose response was seen between 0.1 and 10 micrograms AmpB/ml for the susceptible control strain. Greater than 50% of cells from all susceptible strains tested transfer into the negative gate when exposed to 2.5 micrograms Amp B/ml while fewer than 5% of cells of the highly resistant C. tropicalis (ATCC 28707) are affected at concentrations up to 20 micrograms/ml. This method may provide a more accurate assessment of Amp B susceptibility than conventional tube dilution methods.     

Genistein inhibition of the growth of human breast cancer cells: independence from estrogen receptors and the multi-drug resistance gene

The effect of isoflavones on the growth of the human breast carcinoma cell lines, MDA-468 (estrogen receptor negative), and MCF-7 and MCF-7-D-40 (estrogen receptor positive), has been examined. Genistein is a potent inhibitor of the growth of each cell line (IC50 values from 6.5 to 12.0 micrograms/ml), whereas biochanin A and daidzein are weaker growth inhibitors (IC50 values from 20 to 34 micrograms/ml). The isoflavone beta-glucosides, genistin and daidzin, have little effect on growth (IC50 values greater than 100 micrograms/ml). The presence of the estrogen receptor is not required for the isoflavones to inhibit tumor cell growth (MDA-468 vs MCF-7 cells). In addition, the effects of genistein and biochanin A are not attenuated by overexpression of the multi-drug resistance gene product (MCF-7-D40 vs MCF-7 cells).     

Protective activity of adult T cell leukemia-derived factor (ADF) against tumor necrosis factor-dependent cytotoxicity on U937 cells.

Adult T cell leukemia-derived factor (ADF) is a human homologue of thioredoxin with many biologic functions including IL-2R induction, growth promotion, thiol-dependent reducing activity, and radical scavenging activity. The regulatory effect of ADF on the cytotoxic activity of TNF was examined by using a human histiocytic lymphoma cell line, U937. When U937 cells were preincubated with recombinant ADF (rADF) (0.1-100 micrograms/ml) at 37 degrees C for 30 min, TNF-dependent cytotoxicity on U937 cells was markedly inhibited. This inhibitory effect was as high as 95% in the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay (rADF 100 micrograms/ml) and 85% in the 51Cr-releasing assay (rADF 10 micrograms/ml). After pretreatment of U937 cells with IFN-gamma to augment the sensitivity to TNF, an inhibitory effect of rADF was also found. When U937 cells were washed after preincubation with rADF, resistance to TNF-dependent cytotoxicity was still observed, indicating that rADF inhibited the sensitivity of U937 to TNF-dependent cytotoxicity rather than modifying TNF molecules. Scatchard analysis of TNF receptors on U937 cells using 125I-TNF showed that rADF modulated neither the density nor the affinity of the cell membrane significantly. rADF also reduced the cytotoxicity induced by anti-Fas IgM mAb which shows cytotoxicity quite similar to TNF. rADF (10 micrograms/ml) reduced 90% of the cytotoxicity by anti-Fas IgM mAb, without a detectable change either in Fas Ag expression (MFI 58.1 vs 53.3) or in the degradation of anti-Fas IgM mAb as determined by flow cytometric analysis. These findings indicated that the rADF-induced resistance to the cytotoxic effect of TNF and anti-Fas mAb was not related to the modulation of the TNF receptor or Fas Ag.     

Evaluation of genotoxicity of N-nitrosodibenzylamine in Chinese hamster V79 cells and in Salmonella

Health concerns have arisen due to the formation of N-nitrosodibenzylamine (NDBzA; CAS No. 5336-53-8) in pork processed in a new type of rubber netting. In view of the potent carcinogenicity of related nitrosamines (e.g. N-nitroso-n-dibutylamine and N-nitrosodiethylamine), NDBzA was evaluated for genotoxicity in vitro in both Chinese hamster V79 cells and in Salmonella. In V79 cells, concentrations up to 25 micrograms/ml were tested with and without activation by rat or hamster hepatocytes. Significant elevation of SCE frequency was seen only at 25 micrograms/ml in the presence of uninduced hamster hepatocytes. Mutation to 6-thioguanine resistance was observed at 25 micrograms/ml, in the absence of hepatocytes and in the presence of induced (Aroclor 1254) or uninduced hamster hepatocytes, but not with rat hepatocytes. With uninduced rat hepatocytes, a small but significant (p less than 0.05) increase in the mutation frequency was seen with 10 micrograms/ml NDBzA. In the Salmonella assay, using a pre-incubation protocol and concentrations up to 1000 micrograms/ml, NDBzA was negative in strain TA98, and in TA100 with rat S9, but was positive at the highest dose in TA100 with hamster S9, and more strongly with Aroclor 1254-induced hamster S9. When activated by uninduced rat or hamster hepatocytes, as opposed to S9, NDBzA was negative with all tester strains. Hamster hepatocytes activated more than rat in the V79 studies, and hamster S9 was more strongly activating in the Salmonella assay. These results indicate that NDBzA is weakly mutagenic to both Salmonella and V79 cells.     

Effect of antibiotics on Francisella tularensis]

The minimum inhibitory concentrations of rifampicin, doxycycline, sisomicin, ciprofloxacin and phosmidomycin for various strains of Francisella tularensis were 0.5 to 2.0, 0.5 to 2.0, 0.125 to 0.4, 0.625 to 0.125 and 2.0 to 12.5 micrograms/ml, respectively. Ciprofloxacin and sisomicin had a marked bactericidal effect. The bactericidal effect of rifampicin was insignificant. Doxycycline and phomidomycin had practically no such effect. All the antibiotics had a post effect. The level of the post-antibiotic effect was different and depended on the antibiotic concentration.     

Isolation of chloroquine-resistant Chinese hamster V79 cell variants that are also resistant to ammonium chloride

Chloroquine-resistant (CQr) clones (CQ-21 and CQ-22) have been isolated from mutagenized hamster lung V79 cells by exposing the cells to a high dose of chloroquine. CQ-21 and CQ-22 showed about 3-fold higher resistance to chloroquine than the parental V79 cells, and they showed specific cross-resistance to another amine, NH4Cl, which is also concentrated in lysosomes. CQr clone showed no cross-resistance to other unrelated agents. Chloroquine-induced inhibition of [125I]ricin internalization was observed in both cell lines at neutral pH, but the inhibition of uptake was less in the variant. Also, the degradation of endogenous protein was slowed in the mutant; further, treatment of cells with 30 micrograms/ml of chloroquine inhibited the degradation of endogenous proteins in the parental V79, but not in CQ-22 cells. Similar levels of acid phosphatase, beta-glucuronidase and cathepsin D were observed in V79 and CQ-22 cells, but the level of cathepsin B was lower in the mutant. Electron microscopy showed an increased number of electron-dense bodies, possibly autophagosomes/lysosomes, in the mutant cells grown for 4 days with 5 micrograms/ml of chloroquine. Similar aberrant structures were observed in the parental V79 cells treated for only 3 h with 5 micrograms/ml of chloroquine.     

Relative sensitivities of environmental legionellae to selective isolation procedures

A survey of water samples to determine the efficacy of standard procedures for the isolation of environmental legionellae was conducted. Marked variations in intraspecies resistance to selective agents and treatments were observed, and in experiments with one of the isolates, the response was modified by culture conditions. Five selective procedures incorporating acid (pH 2.2) and heat (50 degrees C, 30 min) treatments, with and without plating on buffered charcoal-yeast extract agar supplemented with vancomycin (5 micrograms/ml), polymyxin B (60 U/ml), and cycloheximide (80 micrograms/ml), caused 5 to 99% decreases in viable counts of pure cultures in water suspensions. The differences in the responses of the cultures to the five treatments were statistically significant. Cells in retained samples of naturally contaminated water from which the original cultures had been isolated were significantly less sensitive than artificially grown isolates. The sensitivities of the laboratory-grown cells to the treatments were affected by the length of incubation on buffered charcoal-yeast extract agar. Whereas acid resistance increased after 24 h of incubation, resistance to the antibiotic mixture decreased.     

Relative sensitivities of environmental legionellae to selective isolation procedures.

A survey of water samples to determine the efficacy of standard procedures for the isolation of environmental legionellae was conducted. Marked variations in intraspecies resistance to selective agents and treatments were observed, and in experiments with one of the isolates, the response was modified by culture conditions. Five selective procedures incorporating acid (pH 2.2) and heat (50 degrees C, 30 min) treatments, with and without plating on buffered charcoal-yeast extract agar supplemented with vancomycin (5 micrograms/ml), polymyxin B (60 U/ml), and cycloheximide (80 micrograms/ml), caused 5 to 99% decreases in viable counts of pure cultures in water suspensions. The differences in the responses of the cultures to the five treatments were statistically significant. Cells in retained samples of naturally contaminated water from which the original cultures had been isolated were significantly less sensitive than artificially grown isolates. The sensitivities of the laboratory-grown cells to the treatments were affected by the length of incubation on buffered charcoal-yeast extract agar. Whereas acid resistance increased after 24 h of incubation, resistance to the antibiotic mixture decreased.     

The drug sensitivity of meningococci and the characteristics of its determination

The results obtained in the study of antibiotic and sulfamide sensitivity of 197 Neisseria meningitidis strains of groups A, B and C, isolated from the spinal fluid and blood of patients with meningococcal infection hospitalized in the 2nd Clinico-Infectious Hospital, Moscow, in 1984-1989 and studied with the use of the disc diffusion method and the method of serial dilutions of antibiotics in solid culture media, are presented. As revealed in this study, N. meningitidis strains retained their high sensitivity to penicillin and ampicillin (MIC50 = 0.016 and 0.032 micrograms/ml respectively). Sensitivity to tetracycline decreased (MIC50 = 0.5 micrograms/ml) and to rifampicin increased (MIC50 = 0.063 micrograms/ml). 48.5% of strains were resistant to streptomycin. In recent years the proportion of N. meningitidis, resistant to sulfanilamide preparations, significantly decreased and MIC50 was equal to 2.5 micrograms/ml in comparison with 5-10 micrograms/ml in the preceding period. The results of testing sensitivity to antibiotics by both methods coincided. Still the disc diffusion method can be used in epidemiological surveillance on meningococcal infection, while for more exact differentiation of N. meningitidis strains the use of the method of serial dilutions is necessary.     

Comparison of in vitro susceptibilities of Rickettsia prowazekii, R. rickettsii, R. sibirica and R. tsutsugamushi to antimicrobial agents

The in vitro activities of 16 antimicrobial agents against Rickettsia prowazekii (Breinl strain), R. rickettsii (Bitterroot strain), R. sibirica (ATCC No. VR151) and R. tsutsugamushi (Gilliam, Karp, Kato, Shimokoshi, Kawasaki and Kuroki strains) were determined by the cell culture method. Tetracycline, demethylchlortetracycline, doxycycline, minocycline, chloramphenicol, kitasamycin and rifampicin were generally effective (MIC, 0.005-0.78 micrograms/ml) to all strains tested. Quinolones such as norfloxacin, ciprofloxacin and ofloxacin were moderately active, but they were less active against R. tsutsugamushi than other rickettsial species. Penicillins and cephems showed low activity against most of the strains tested, but high concentrations of benzylpenicillin (MIC, 25-50 micrograms/ml) inhibited R. prowazekii, R. rickettsii and R. sibirica. These findings may be applicable for differentiation of species of genus Rickettsia.