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PubMed1997

Oxygen scavenging, NADH oxidase and metronidazole resistance in Helicobacter pylori.

Failure of triple-therapy regimes to eradicate Helicobacter pylori from the stomach is thought to be due to the occurrence of a metronidazole-resistant bacterial population. Exposure of metronidazole-resistant (MtzR) strains of H. pylori to an anaerobic environment causes the activation of metronidazole and the loss of resistance. Using metronidazole-sensitive (MtzS) clinical isolates, we selected mutants conferring resistance to metronidazole, which were used to investigate the effect of bacterial cell density upon the activation of metronidazole. The addition of metronidazole, at a final concentration of 10 mg/L, to MtzR cultures of a bacterial cell density >1 x 10(6) cfu/mL, caused a loss in viability. No loss in viability, however, occurred upon addition of metronidazole to MtzR cultures of a cell density of <1 x 10(6) cfu/mL. MtzS cultures lost viability irrespective of the initial cell density, indicating that oxygen scavenging at the site of metronidazole reduction may occur in these cultures. The ability of MtzS wild types, MtzR isogenic mutants and MtzR wild types to scavenge oxygen from the intracellular environment was investigated. H. pylori cultures contained NADH and NADPH oxidase activity. NADPH oxidase activity was always more than double the NADH oxidase activity. MtzR mutants possessed approximately one-third the NADH oxidase activity found in their respective MtzS parent wild types. MtzR wild types possessed a low NADH oxidase level similar to that found in the MtzR mutants. We propose that metronidazole resistance may be mediated through an inability of MtzR strains to remove oxygen from the site of metronidazole reduction, thereby preventing metronidazole activation.

Anti-Bacterial Agents↗

PubMed1994

Activities of the benzoxazinorifamycin KRM 1648 and ethambutol against Mycobacterium avium complex in vitro and in macrophages.

KRM 1648 is a 4-aminobenzoxazine derivative of rifamycin S with potent in vitro activity against the Mycobacterium avium complex (MAC); the MIC for 90% of 24 MAC isolates from AIDS patients was 0.25 microgram/ml as determined by a radiometric broth macrodilution assay. KRM 1648 was bactericidal for MAC isolates in Middlebrook 7H9 broth, with a reduction in viability of 1 to 4 orders of magnitude over 72 h. In human macrophages, KRM 1648 also was bactericidal, with a reduction of 3 to 4 orders of magnitude in CFU per ml of macrophage lysate at a concentration of 1 microgram/ml; however, the bactericidal activity varied approximately 10-fold among the three MAC serovars tested. In growth medium, ethambutol potentiated the effect of KRM 1648, but this potentiation was modest when tested against MAC in macrophages and also varied between MAC strains. KRM 1648 has potential as an antimycobacterial agent for MAC disease, perhaps in combination with other agents so that the use of lower dosages of KRM 1648 than are needed with other rifamycins may be possible.

Antibiotics, Antitubercular↗

PubMed2000

Persistence of Chlamydia trachomatis is induced by ciprofloxacin and ofloxacin in vitro.

An in vitro cell culture model was used to investigate the long-term effect of ciprofloxacin and ofloxacin on infection with Chlamydia trachomatis. Standard in vitro susceptibility testing clearly indicated successful suppression of chlamydial growth. To mimic better in vivo infection conditions, extended treatment with the drugs was started after infection in vitro had been well established. Incubation of such established chlamydial cultures with ciprofloxacin and ofloxacin not only failed to eradicate the organism from host cells, but rather induced a state of chlamydial persistence. This state was characterized by the presence of nonculturable, but fully viable, bacteria and the development of aberrant inclusions. In addition chlamydia exhibited altered steady-state levels of key chlamydial antigens, with significantly reduced major outer membrane protein and near constant hsp60 levels. Resumption of overt chlamydial growth occurred after withdrawal of ciprofloxacin, confirming the viability of persisting chlamydia. In vitro ciprofloxacin results are consistent with clinical data, thereby providing an explanation for treatment failures of ciprofloxacin. Parallel in vitro studies with ofloxacin suggest a better correlation between clinical and laboratory-defined efficacy, although the clinical studies on which this assessment is based did not include monitoring of chlamydial persistence. The data presented here clearly demonstrate that under at least some circumstances, standard determination of MICs and minimal bactericidal concentrations for C. trachomatis allows no more than a simple definition of whether an antibiotic has some anti chlamydial activity; however, such testing is not always sufficient to verify that the antibiotic will eliminate the organism in vivo.

Anti-Infective Agents↗

PubMed2004

Functional genomic analysis of fluconazole susceptibility in the pathogenic yeast Candida glabrata: roles of calcium signaling and mitochondria.

The pathogenic yeast Candida glabrata exhibits innate resistance to fluconazole, the most commonly used antifungal agent. By screening a library of 9,216 random insertion mutants, we identified a set of 27 genes which upon mutation, confer altered fluconazole susceptibility in C. glabrata. Homologues of three of these genes have been implicated in azole and/or drug resistance in Saccharomyces cerevisiae: two of these belong to the family of ABC transporters (PDR5 and PDR16), and one is involved in retrograde signaling from mitochondria to nucleus (RTG2). The remaining 24 genes are involved in diverse cellular functions, including ribosomal biogenesis and mitochondrial function, activation of RNA polymerase II transcription, nuclear ubiquitin ligase function, cell wall biosynthesis, and calcium homeostasis. We characterized two sets of mutants in more detail. Strains defective in a putative plasma membrane calcium channel (Cch1-Mid1) were modestly more susceptible to fluconazole but showed a significant loss of viability upon prolonged fluconazole exposure, suggesting that calcium signaling is required for survival of azole stress in C. glabrata. These mutants were defective in calcium uptake in response to fluconazole exposure. The combined results suggest that, in the absence of Ca(2+) signaling, fluconazole has a fungicidal rather than a fungistatic effect on C. glabrata. The second set of mutants characterized in detail were defective in mitochondrial assembly and organization, and these exhibited very high levels of fluconazole resistance. Further analysis of these mutants indicated that in C. glabrata a mechanism exists for reversible loss of mitochondrial function that does not involve loss of mitochondrial genome and that C. glabrata can switch between states of mitochondrial competence and incompetence in response to fluconazole exposure.

Antifungal Agents↗

PubMed1975

Protozoa as agents responsible for the decline of Xanthomonas campestris in soil.

A streptomycin-resistant mutant of Xanthomonas campestris was used to assess the persistence of the plant pathogen in soil and the changes in populations that might be important for its survival. In soil into which large numbers of the organism were introduced, a marked decline in its abundance occurred, but after about 1 week its population density reached a level of about 105 and did not continue to fall during the test period. No such marked decline was evident in sterile soil inoculated with X. campestris. The bacterium did not lose viability if starved for carbon or inorganic nitrogen. Although abundant in soil, the numbers of propagules capable of producing antibiotics or lytic enzymes active against X. campestris did not increase coincident with the pathogen's decline, and no increase in tartrate-extractable toxins was observed. Neither bdellovibrios nor bacteriophages active against the xanthomonad were found in the soil, but a marked increase in the frequency of protozoa paralleled the phase of rapid diminution in the X. campestris population. In actidione-treated soil, in which protozoan activity was severly limited, the high cell density of the pathogen was maintained. On the basis of these data, it is concluded that predation by protozoa is responsible for the abrupt fall in frequency of the bacterium in natural soil.

Animals↗

PubMed1975

Effect of dichlorodifluoromethane on the appearance, viability, and integrity of Escherichia coli.

Cultures of Escherichia coli H52 were treated with liquid dichlorodifluoromethane (fluorocarbon-12 [f-12]) for 2 h at 22 C and then examined microscopically. Treated cells tended to clump, and their cytoplasms were generally less dense and less uniform in appearance than those of control cells. E. coli ML30 was exposed to f-12 at a concentration of 1.25 X saturation for times up to 1,200 min at 22 C. Cells were examined for changes in viability (plate count), permeability (as measured by exit of alpha-[14-C]methylglucoside or uptake of omicron-nitrophenyl-beta-D-galactopyranoside), release of compounds absorbing at 260 nm, and lysis (changes in absorbance at 420 nm). Large losses of alpha-methylglucoside and of percentage of viability occurred after brief exposure to f-12. Release of compounds absorbing at 260 nm occurred more slowly than the aforementioned events, possibly because these molecules are larger than alpha-methylglucoside. During 1,200-min exposure to f-12, the number of survivors decreased from 10-9 to 10-4 organisms/ml, the loss of compounds absorbing at 260 nm amounted to 50 percent, and 32 percent lysis occurred. Most of these changes occurred during the first 300 min of treatment. Loss of alpha-methylglucoside was almost complete after 1-min exposure to f-12. These results suggest that death of the cell involves several stages, with a change of permeability, occurring first, followed by leakage of compounds of increasing size and, finally, lysis.

Bacteriolysis↗

PubMed1989

[Effect of storage conditions on population variability and activity of Streptomyces recifensis var. lyticus].

The results of the study on survival and variation of Streptomyces recifensis var. lyticus 2435 producing lytic enzymes are presented. The culture was maintained for 2.5 years under a layer of vaseline oil, at a temperature of -20 degrees C and in lyophilized state. It was shown that irrespective of the storage method strain 2435 preserved its viability. However, the most intensive growth was observed in the lyophilized cultures. During the storage the content of the productive colonies characteristic of the morphological type culture in the population decreased while the number of the low active variants increased. Lyophilization of the strain spores in the sucrose-gelatine medium provided insignificant morphological variation of the culture and preservation of the initial level of its lytic activity against a number of test-microbes except S. aureus and M. lysodeikticus. Storage of the culture under vaseline oil and at a temperature of -20 degrees C resulted in lowering of its lytic activity against all the test-microbes used. For long-term maintenance of Streptomyces recifensis var. lyticus 2435 the method of lyophilization in the sucrose-gelatine medium is recommended.

Bacteriolysis↗

PubMed2006

Yeast cell inactivation related to local heating induced by low-intensity electric fields with long-duration pulses.

The effects of electric field (EF) treatments on Saccharomyces cerevisiae viability were investigated using a PG200 electroporator (Hoefer Scientific Instrument, San Fransisco, CA, USA) with specific attention to induced thermal effects on cell death. Lethal electric fields (1.5 kV cm(-1) for 5 s) were shown to cause heat variations in the cell suspension medium (water+glycerol), while corresponding classical thermal treatments at equivalent temperatures had no effect on the cells viability. Variations of the electrical conductivity of the intra- and extracellular matrix caused by ions and solutes transfer across the membrane were shown to be involved in the observed heating. The results permitted to build a theoretical model for the temperature variations induced by electric fields. Using this model and the electrical conductivity of the different media, a plausible explanation of the cell death induced by low-intensity electric fields with long-duration pulses has been proposed. Indeed, cell mortality could in part be caused by direct and indirect effects of electric fields. Direct effects are related to well known electromechanical phenomena, whereas indirect effects are related to secondary thermal stress caused by plasma membrane thermoporation. This thermoporation was attributed to electrical conductivity variations and the corresponding intracellular heating.

Colony Count, Microbial↗

PubMed2006

Potential use of copper surfaces to reduce survival of epidemic meticillin-resistant Staphylococcus aureus in the healthcare environment.

Epidemic meticillin-resistant Staphylococcus aureus (EMRSA) emerged in the early 1980s with EMRSA-15 and -16 being the most prevalent strains within the UK. MRSA transmission between patients is largely via the hands of healthcare workers, and contamination of the hospital environment may occur. The objective of this study was to evaluate the effectiveness of copper and brass to reduce the viability of air-dried deposits of three MRSA strains [MRSA (NCTC 10442), EMRSA-1 (NCTC 11939) and EMRSA-16 (NCTC 13143)] compared with stainless steel. MRSA and EMRSA [10(7)colony-forming units (CFU)] were inoculated on to coupons (1 cm x 1 cm) of copper, brass or stainless steel and incubated at either 22 degrees C or 4 degrees C for various time periods. Viability was determined by resuspending removed CFUs and plating out on tryptone soy agar plates in addition to staining with the respiratory indicator fluorochrome 5-cyano-2,3-ditolyl tetrazolium. On pure copper surfaces, 10(7) MRSA, EMRSA-1 and EMRSA-16 were completely killed after 45, 60 and 90 min, respectively, at 22 degrees C. In contrast, viable organisms for all three strains were detected on stainless steel (grade 304) after 72 h at 22 degrees C. At 4 degrees C, complete kill was achieved on copper for all three strains within 6 h. The results demonstrate an antimicrobial effect of copper on MRSA, EMRSA-1 and -16 in contrast to stainless steel. Consequently, the contemporary application of stainless steel in hospital environments for work surfaces and door furniture is not recommended.

Colony Count, Microbial↗

PubMed2000

Exploring the frontier between life and death in Escherichia coli: evaluation of different viability markers in live and heat- or UV-killed cells.

A number of methods have been proposed to assess the viability of cells without culture. Each method is based on criteria that reflect different levels of cellular integrity or functionality. As a consequence, the interpretation of viability is often ambiguous. The purposes of this work were to evaluate the capacity of current viability markers to distinguish between live and dead Escherichia coli K-12 cells. Methods that assess 'viability' by the demonstration of metabolic activities (esterase activity, active electron transport chain, transport of glucose), cellular integrity (membrane integrity, presence of nucleic acids) or the building up of cellular material (cell elongation) have been evaluated in live and UV- or heat-killed cells. With live cells, viability markers detected cells in counts similar to the colony count. However, these so-called viability markers could stain dead cells for some time after the lethal treatment. For the UV-killed cells, residual activities were detected even after 48 h of storage at 20 degrees C. However, for heat-treated cells, these activities disappeared within hours after heat treatment. Only a combination of fluorescence in situ hybridization with rRNA probes and cell elongation in response to nutrients (in the presence of an inhibitor of cell division) had the ability to differentiate live from dead cells. Problems in the definition of a viable but nonculturable state are in part due to the lack of a clear definition of bacterial death. We consider death as an irreversible state where no growth, cell elongation or protein synthesis may occur.

Bacterial Proteins↗

PubMed1989

Intracellular activity of cefamandole and aztreonam against phagocytosed Escherichia coli and Staphylococcus aureus.

The intracellular activity of cefamandole and aztreonam against phagocytosed Escherichia coli and cefamandole against phagocytosed Staphylococcus aureus was studied using a sensitive and standardized method of murine peritoneal macrophages. Cefamandole and aztreonam exerted an intracellular antibacterial activity against E. coli which was greater than their extracellular one. With concentrations of both antibiotics up to 16 x MBC a dose-dependent decrease of the initial number of intracellular E. coli which ranged from 32% to 90% was observed. However, similar antibiotic concentrations above the MBC affected the viability of extracellular E. coli by only 20% to 30%. The intracellular antibacterial activity of both antibiotics against E. coli was further enhanced by immune serum. Cefamandole at 4 x the MBC did not affect the survival of intracellular S. aureus, but killed 41% of extracellular bacteria by 1 h and 99% after 3 h. The intracellular activity of both antibiotics against E. coli was also maintained in NaF-pulsed macrophages which have an impaired oxidative metabolism. The data suggest that both cefamandole and aztreonam possess an intracellular antibacterial activity against E. coli that seems at least in part due to a positive cooperation of antibiotics with the O2-independent microbicidal system of macrophages.

Animals↗

PubMed2003

Garlic extract and two diallyl sulphides inhibit methicillin-resistant Staphylococcus aureus infection in BALB/cA mice.

OBJECTIVES: The inhibitory effect of garlic extract, diallyl sulphide and diallyl disulphide against methicillin-resistant Staphylococcus aureus (MRSA) infection in BALB/cA mice was studied. The influence of these agents upon the levels of fibronectin, interleukin-6 and lipid oxidation in MRSA-infected mice was examined. METHODS: Garlic extract at 100% and 50%; diallyl sulphide (DAS) at 10% and 5%; diallyl disulphide (DADS) at 1% and 0.5% were used in this study. Sixteen clinical MRSA isolates obtained from infected patients were used in this study (n=16). Mice were infected by injecting 200 microL MRSA-PBS solution, which contained 10(7) cfu, via the tail vein. At 16 h post-infection (p.i.), garlic extract, DAS or DADS at 200 microL was administrated orally. At 24 h p.i., mice were killed and blood, liver, kidney and spleen of each mouse were collected. Plasma and the filtrate from each organ and serial dilutions were used to determine colony count. Plasma fibronectin level was determined by rabbit anti-rat fibronectin antibody and quantified by ELISA. Interleukin-6 levels were determined by commercial kit. Lipid oxidation was determined by measuring malondialdehyde levels. RESULTS: The oral administration of these agents significantly decreased the viability of MRSA, in plasma, liver, kidney and spleen (P<0.05). MRSA infection significantly increased fibronectin and interleukin-6 levels in plasma of MRSA-infected mice (P<0.05); however, the oral administration of garlic extract and two diallyl sulphides significantly reduced both fibronectin and interleukin-6 levels (P<0.05). MRSA infection also significantly enhanced lipid oxidation in plasma and three organs (P<0.05). The treatments of garlic extract and two diallyl sulphides significantly decreased the malondialdehyde level and showed antioxidant protection (P<0.05). CONCLUSIONS: These data strongly supported the conclusion that garlic extract, diallyl sulphide and diallyl disulphide possessed multiple protective functions against MRSA infection, in which diallyl sulphide and diallyl disulphide could be considered as novel therapeutic agents for the treatment of MRSA infection.

Allyl Compounds↗

PubMed1988

Truncation of the carboxy-terminal domain of yeast beta-tubulin causes temperature-sensitive growth and hypersensitivity to antimitotic drugs.

beta-tubulin of budding yeast Saccharomyces cerevisiae is a polypeptide of 457 amino acids encoded by the unique gene TUB2. We investigated the function of the carboxy-terminal part of yeast beta-tubulin corresponding to the carboxy-terminal variable domain of mammalian and avian beta-tubulins. The GAA codon for Glu-431 of TUB2 was altered to TAA termination codon by using in vitro site-directed mutagenesis so that the 27-amino acid residues of the carboxyl terminus was truncated when expressed. The mutagenized TUB2 gene (tub2(T430)) was introduced into a haploid strain in which the original TUB2 gene had been disrupted. The tub2(T430) haploid strain grows normally less than 30 but not at 37 degrees C. The truncation of the carboxyl terminus caused hypersensitivity to antimitotic drugs and low spore viability at the permissive temperature for vegetative growth. Immunofluorescence labeling with antitubulin antibody and DNA staining with 4',6'-diamidino-2-phenylindole showed that in these cells at 37 degrees C, formation of spindle microtubules and nuclear division was inhibited and cytoplasmic microtubule distribution was aberrant. These results suggest that functions of the carboxy-terminal domain of yeast beta-tubulin are necessary for cells growing under suboptimal growth conditions although it is not essential for growth under the optimal growth conditions. Cells bearing tub2(411), a tub2 gene in which the GAA codon for Glu-412 was altered to TAA were no more viable at any temperature. In addition, a haploid strain carrying two functional beta-tubulin genes is not viable.

Antineoplastic Agents↗

PubMed2000

Evaluation of a human monocytic cell line THP-1 model for assay of the intracellular activities of antimicrobial agents against Legionella pneumophila.

We examined the intracellular activities of 11 antimicrobial agents against Legionella pneumophila using a human monocyte-derived cell line, THP-1. Colony counting and microscopic examination of L. pneumophila co-incubated with THP-1 cells (5 x 105 cells/well) were performed. Both extra- and intra-cellular multiplication of L. pneumophila were observed and were dependent on the inoculum of L. pneumophila in the culture; L. pneumophila did not grow in the cell culture medium alone. Light microscopic examination confirmed that extracellular L. pneumophila originated from THP-1 cells disrupted by bacterial multiplication. L. pneumophila multiplied by 3-4 logs after 24 h incubation with THP-1 cells and their number remained stable at 106-107 cfu/mL until 72 h. The results of viability studies using four antimicrobial agents-ciprofloxacin, erythromycin, minocycline and rifampicin-demonstrated that our system was suitable for the intracellular activity assay. We used a concept of 'minimum extracellular concentration inhibiting intracellular multiplication' (MIEC) to evaluate the intracellular activity of antimicrobial agents. The MIECs of three beta-lactams were markedly higher than their conventional MICs while those of macrolides, quinolones, rifampicin and minocycline were similar to their MICs. Our results suggest that evaluation of the clinical efficacy of drugs against L. pneumophila should include determination of their intracellular activity against the bacteria, which could be measured using our assay system in THP-1 cells.

Anti-Bacterial Agents↗

PubMed2003

RAM2: an essential gene in the prenylation pathway of Candida albicans.

Prenylation is a post-translational protein modification process that results in correct protein localization to membranes in the cell. It is mediated by at least three prenyltransferases (PTFs) in eukaryotic cells. The RAM2 gene encodes the common alpha-subunit of two PTFs, farnesyltransferase (FTase) and geranylgeranyltransferase I (GGTase I). In this study, the RAM2 gene of the prenylation pathway in the opportunistic pathogen Candida albicans was analysed. The heterozygote trisomy test was used to demonstrate that RAM2 is essential to the viability of C. albicans. RAM2 mRNA in the yeast and hyphae growth states was not detected by Northern blot analysis, but was detected by RT-PCR. Drugs that inhibit mammalian PTFs do not alter Candida cell growth, but they do inhibit FTase and GGTase I activities in cell-free enzyme assays. The data from genetic studies and cell-free enzyme assays suggest that the drugs may not have access to the Candida cytoplasm. The regulation of PTF subunits was also examined in a strain in which RAM2 was under the control of a regulable promoter. Overall, this study demonstrated that RAM2 is essential to C. albicans, indicating that protein prenylation is an indispensable cellular process in this yeast.

Alkyl and Aryl Transferases↗

PubMed1999

Viability of Lactobacillus gasseri and its cholesterol-binding and antimutagenic activities during subsequent refrigerated storage in nonfermented milk.

The effect storage at 4 degrees C on the viability of Lactobacillus gasseri and its sodium taurocholate-deconjugating and cholesterol-binding abilities as well as desmutagenic activity was investigated. Unfermented milks containing L. gasseri strains SBT0274 and SBT0270 at 10(9) cfu/ml were prepared using 10% skim milk. Total and bile-tolerant lactobacilli for strains SBT0274 and SBT0270 generally decreased after 14 d of storage at 4 degrees C; however, viable cells of these strains were still at 10(8) cfu/ml after 28 d of storage. The amounts of cholic acid released and of cholesterol bound by strains SBT0274 and SBT0270 declined over time, especially at 21 d of storage. Antimutagenic activity of unfermented milk made from both strains was attributed to the bacterial cells, and the activity was stable during storage at 4 degrees C for 28 d.

Animals↗

PubMedDate not supplied

Effect of metal mixtures on Salmonella enteritidis viability in synthetic sewage.

We sought to determine the effect of 26 multiple metals solutions, containing Cd, Pb, As and/or Ni, on the viability of Salmonella enteritidis. We used the direct viable count effected by immunofluorescence technique (IFA-DVC) in comparison with the culturable cell count. The metals concentrations were prepared considering the single as well as the overall limits provided by Italian regulation on the water pollution. The mixtures containing nickel showed always toxicity more elevated than other metal solutions tested. Thus we consider too high the nickel law limit. The interactions among the elements, causing generally toxicity decrease, are very well assessable by IFA-DVC test. This method permits to calculate the lethal concentrations of toxicants on bacteria that play a pivotal role on the ecosystem components.

Area Under Curve↗

PubMed2004

The viable but nonculturable state of Kanagawa positive and negative strains of Vibrio parahaemolyticus.

Ingestion of shellfish-associated Vibrio parahaemolyticus is the primary cause of potentially severe gastroenteritis in many countries. However, only Kanagawa phenomenon (hemolysin) positive (KP+) strains of V. parahaemolyticus are isolated from patients, whereas >99% of strains isolated from the environment do not produce this hemolysin (i.e. are KP-). The reasons for these differences are not known. Following a temperature downshift, Vibrio parahaemolyticus enters the viable but nonculturable (VBNC) state wherein cells maintain viability but cannot be cultured on routine microbiological media We speculated that KP+ and KP- strains may respond differently to the temperature and salinity conditions of seawater by entering into this state which might account for the low numbers of culturable KP+ strains isolated from estuarine waters. The response of eleven KP+ and KP- strains of V. parahaemolyticus following exposure to a nutrient and temperature downshift in different salinities, similar to conditions encountered in their environment, was examined. The strains included those from which the KP+ genes had been selectively removed or added. Our results indicated that the ability to produce hemolysin did not affect entrance into the VBNC state. Further, VBNC cells of both biotypes could be restored to the culturable state following an overnight temperature upshift.

Adaptation, Physiological↗