In vitro susceptibility of Clostridium perfringens isolated from farm animals to growth-enhancing antibiotics

Minimal inhibitory concentration (MIC) determinations were carried out with seven growth-enhancing antibiotics against 95 Clostridium perfringens field isolates obtained during 1991 and 1992 from poultry, pigs and calves. All were resistant to 64 μg ml⁻¹ of the bambermycin antibiotic, flavomycin (flavophospholipol) and susceptible to avoparcin (MIC₉₀ 0.25 μg ml⁻¹), avilamycin (MIC₉₀ 0.5 μg ml⁻¹) and salinomycin (MIC₉₀≤ 0.12 μg ml⁻¹). Acquired resistance against bacitracin was detected in some isolates from poultry and bovines and resistance to tylosin and virginiamycin in some strains from all species investigated. Overall, the prevalence of resistance was comparable to the low levels recorded in 1979 in Cl. perfringens isolates from the same animal host species.     

Anti-bacterial activity of synthetic N-heterocyclic oxidizing compounds

Synthetic chlorochromate derivatives of pyridine and quinoline were active in vitro against type cultures of Escherichia coli (ATCC 128), Staphylococcus aureus (ATCC 14775), Pseudomonas aeruginosa (ATCC 10145) and Bacillus subtilis (NCTC 8236). The minimum inhibitory concentrations (MIC) were 125–250 μg ml⁻¹ and 250–500 μg ml⁻¹ for pyridinium chlorochromate and quinolinium chlorochromate, respectively. An established derivative of quinoline (Perfloxacin) had an MIC of 125–250 μg ml⁻¹. The extinction time for 10⁵ cfu in broth was 90 min for pyridinium chlorochromate and 120 min for quinolinium chlorochromate, except for B. subtilis which survived up to about 180 min and 360 min. A combination of the two compounds produced an antagonistic effect. The 50% lethal dose (LD₅₀ toxicity) in mice was estimated at 76 μg g⁻¹ and 33 μg g⁻¹ body weight for the quinolinium and pyridinium chlorochromates. The compounds also exhibited some potential for suppressing a simulated staphylococcal infection in mice at the dosage levels of ca 22 μg g⁻¹ for pyridinium chlorochromate and 45 μg g⁻¹ for quinolinium chlorochromate.     

Symbiotic effectiveness, rate of respiration and glutamine synthetase activity of sodium azide-resistant strains of Rhizobium leguminosarum biovar trifolii

Nitrogen fixing efficiency of sodium azide-resistant strains of Rhizobium leguminosarum bv. trifolii was studied in symbiosis with berseem clover plants in chillum jars. Rate of respiration and glutamine synthetase activity were tested in cultured cells and nodules, respectively. It was observed that shoot dry weight and percentage shoot nitrogen were maximum in plants inoculated with strains resistant to 15 μg ml⁻¹ sodium azide. Rate of respiration in cultured cells was lowest in strains resistant to 15 μg ml⁻¹ sodium azide and highest in strains resistant to 5 μg ml⁻¹ sodium azide. A negative correlation was observed between rate of respiration (in cultured cells) and shoot dry weight of host plants. Glutamine synthetase activity was maximum in nodule extracts of host plants inoculated with strains resistant to 5 and 10 μg ml⁻¹ sodium azide, whereas it was minimum for strains resistant to 15 μg ml⁻¹ sodium azide. Hence, resistance to low doses (15 μg ml⁻¹) of sodium azide, together with lower respiratory and glutamine synthetase activities, could be used as a potential method for isolating the symbiotically effective strains of Rh. leguminosarum bv. trifolii.     

WLIP, a lipodepsipeptide of Pseudomonas 'reactans', as inhibitor of the symptoms of the brown blotch disease of Agaricus bisporus

A cell-free crude extract containing the white line inducing principle (WLIP), a lipodepsipeptide produced by Pseudomonas 'reactans' , could inhibit browning of mushrooms caused by Pseudomonas tolaasii . Mushrooms inoculated with Ps. tolaasii at concentrations of 2·7 × 10⁶ cfu ml⁻¹ or higher showed the symptoms of the disease after 2 d of incubation. Mushroom caps treated with various concentrations of a crude WLIP preparation, and later inoculated with bacterial concentrations higher than the threshold value, did not develop the symptoms of the disease. One milligram of a crude WLIP preparation could block 50% of the symptoms caused by 1·2 × 10⁷ cfu. The inhibition of browning was effective when incubating at low temperatures for 4 d. A suspension containing 1·6 mg ml⁻¹ of pure WLIP was also able to inhibit the symptoms of brown blotch disease induced by 7·6 × 10⁶ cfu ml⁻¹ of Ps. tolaasii .     

Ecomycins, unique antimycotics from Pseudomonas viridiflava

A novel family of peptide antimycotics, termed ecomycins, is described from Pseudomonas viridiflava , a plant-associated bacterium. Ecomycins B and C have molecular masses of 1153 and 1181. They contain equimolar amounts of a β hydroxyaspartic acid, homoserine, threonine, serine, alanine, glycine and one unknown amino acid. Fatty acids were detectable after hydrolysis, methylation and gas chromatography and mass spectroscopy. The ecomycins have significant bioactivities against a wide range of human and plant pathogenic fungi. The minimum inhibitory concentration values for ecomycin B were 4·0 μg ml⁻¹ against Cryptococcus neoformans and 31 μg ml⁻¹ against Candida albicans. Pseudomonas viridiflava also produces what appears to be syringotoxin, an antifungal lipopeptide previously described from Ps. syringae.     

Antibacterial activity of bovine lactoferrin and its peptides against enterohaemorrhagic Escherichia coli O157:H7

The antimicrobial activities of bovine lactoferrin (bLF), its pepsin hydrolysate (bLFH) and the active peptide lactoferricin^® B (LFcinB) against four clinical isolates of enterohaemorrhagic Escherichia coli O157:H7 were studied. The MICs against these isolates were 3 mg ml⁻¹ for bLF, 0·1–0·2 mg ml⁻¹ for bLFH and 8–10 μg ml⁻¹ for LFcinB in 1% Bactopeptone broth. LFcinB killed these bacteria within 3 h at concentrations above 10 μg ml⁻¹. Transmission electron microscopy findings suggested that LFcinB acts on the bacterial surface and affects cytoplasmic contents. LFcinB was shown to influence the levels of verotoxins in the culture supernatant fluid of an E. coli 0157:H7 strain. These results demonstrate that E. coli O157:H7 strains are susceptible to the antimicrobial effects of bLF and its peptides.     

Antimicrobial action of essential oils : the effect of dimethylsulphoxide on the activity of cinnamon oil

Fifty-one essential oils extracted from plants of known origin were tested for their antimicrobial activity against three bacteria, Pseudomonas aeruginosa , Staphylococcus aureus , Escherichia coli and four yeasts, Torulopsis utilis , Schizosaccharomyces pombe , Candida albicans and Saccharomyces cerevisiae using the drop diffusion method. All showed antimicrobial activity against at least one of the micro-organisms. Following this preliminary screening, 13 essential oils showing antimicrobial activity against at least five of the micro-organisms were tested in the range 50 μg ml⁻¹ to 500 μg ml⁻¹ using broth micro dilution techniques with dimethylsulphoxide (DMSO) as a dispersing solvent. The concentration of most of the oils required for total inhibition of growth was >500 μg ml⁻¹. Further studies on the antimicrobial action of cinnamon oil in the range 10–150 μg ml⁻¹ showed that 50-fold higher activity was found when no dispersing solvent was used.     

Modulation of human polymorphonuclear leukocyte chemotaxis and superoxide anion production by Pseudomonas aeruginosa exoproducts, IL-1β and piroxicam

Abstract Whereas addition of 200 ng ml⁻¹ exotoxin A (exoA) did not modify PMNL chemotaxis, 20 U ml⁻¹ human recombinant interleukin-1β (hrIL-1β) primed polymorphonuclear leukocytes (PMNL) for migration towards Pseudomonas aeruginosa peptide chemotactins (PAPCs). Piroxicam (100 μg ml⁻¹), a non-steroidal anti-inflammatory agent (NSAIA), inhibited PMNL chemotaxis and abolished the priming effect of hrIL-1β. Both PAPCs and exoA induced PMNL superoxide anion production, but neither hrIL-1β nor piroxicam modified significantly PMNL superoxide anion production induced by PAPCs. The fact that hrIL-1β can prime PMNL for chemotaxis towards PAPCs and that piroxicam can abolish activation by primed PMNL are findings relevant to the pharmacological control of lung tissue damage during P. aeruginosa pneumonia.     

Effect of trifluoperazine on in vitro ATP synthesis by Mycobacterium leprae

The effect of trifluoperazine (TFP), a calmodulin antagonist, was investigated on in vitro ATP levels of human derived Mycobacterium leprae . M. leprae were obtained from biopsies from multi-bacillary forms of leprosy and were incubated in a modified Dubos medium system which supports limited in vitro synthesis of M. leprae . This incubation was carried out in the absence and presence of different concentrations of trifluoperazine. Samples for estimation of bacillary ATP levels were taken at day 0 and at 14 days of incubation. TFP inhibited ATP levels in M. leprae and this inhibitory effect was marginal at 2.5 μg ml⁻¹ (35% inhibition), highly significant at 5 μg ml⁻¹ (87% inhibition) and almost total at 10 μg ml⁻¹ (98.5% inhibition). This compound appears to have potential as an anti-leprotic drug and also as a broad spectrum anti-mycobacterial agent in view of its anti-tubercular activity reported earlier.     

Effect of high-temperature, short-time (HTST) pasteurization on milk containing low numbers of Mycobacterium paratuberculosis

The efficacy of high-temperature, short-time (HTST) pasteurization (72 °C/15 s) when low numbers (≤ 10³ cfu ml ⁻¹ ) of Mycobacterium paratuberculosis are present in milk was investigated. Raw cows' milk spiked with Myco. paratuberculosis (10³ cfu ml⁻¹, 10² cfu ml⁻¹, 10 cfu ml⁻¹, and 10 cfu 50 ml⁻¹) was subjected to HTST pasteurization using laboratory pasteurizing units. Ten bovine strains of Myco. paratuberculosis were tested in triplicate. Culture in BACTEC Middlebrook 12B radiometric medium detected acid-fast survivors in 14·8% and 10% of HTST-pasteurized milk samples at the 10³ and 10² cfu ml⁻¹ inoculum levels, respectively, whereas conventional culture on Herrold's egg yolk medium containing mycobactin J detected acid-fast survivors in only 3·7% and 6·7% of the same milk samples. IS900-based PCR confirmed that these acid-fast survivors were Myco. paratuberculosis . No viable Myco. paratuberculosis were isolated from HTST-pasteurized milk initially containing either 10 cfu ml⁻¹ or 10 cfu 50 ml⁻¹.     

Development of a microtitre ELISA to quantify development of Cryptosporidium parvum in vitro

Abstract An in situ enzyme-linked immunosorbent assay (ELISA) was developed to evaluate growth of Cryptosporidium parvum in vitro. Ninety-six-well tissue culture microtitre plates were each seeded with 4.0 X 104 human ileocecal adenocarcinoma (HCT-8) cells, then infected with CsCl-purified oocysts 24 h later. The growth medium consisted of RPMI 1640 supplemented with 10% fetal bovine serum, 15 mM HEPES (JV-2-hydroxyethylpiperazine N −2-ethanesulfonic acid), 50 mM glucose, 1 μg ml⁻¹ folic acid, 4 μg ml⁻¹ 4-aminobenzoic acid, 2 μg ml⁻¹ pantothenic acid and 35 μg ml⁻¹ ascorbic acid. Incubation conditions were at 37 ° C in a 5% CO₂/95% humidified air incubator. Oocysts were allowed to excyst in situ so that sporozoites could infect cells directly. Monolayers were then washed, new medium added, and infected cells re-incubated. Levels of infection were assessed 48 h later using a rat anti-C. parvum polyvalent antiserum directed against purified parasite membranes, followed by a goat anti-rat IgG conjugated to horseradish peroxidase and 3,3',5,5'-tetramethyl-benzidine as substrate. Using various parasite inoculating doses and incubation times, optimal results were obtained using a 90-min exposure of host cells to 2.5−3.0 × 10⁴ oocysts/well. Evaluation of various concentrations of four anti-microbials (monensin, lasalocid, paromomycin and sulfadimethoxine) in the system resulted in the acquisition of precise dose-response curves for each compound.     

Antibacterial spectrum of lactoferricin B, a potent bactericidal peptide derived from the N-terminal region of bovine lactoferrin

A physiologically diverse range of Gram-positive and Gram-negative bacteria was found to be susceptible to inhibition and inactivation by lactoferricin B, a peptide produced by gastric pepsin digestion of bovine lactoferrin. The list of susceptible organisms includes Escherichia coli, Salmonella enteritidis, Klebsiella pneumoniae, Proteus vulgaris, Yersinia enterocolitica, Pseudomonas aeruginosa, Campylobacter jejuni, Staphylococcus aureus, Streptococcus mutans, Corynebacterium diphtheriae, Listeria monocytogenes and Clostridium perfringens. Concentrations of lactoferricin B required to cause complete inhibition of growth varied within the range of 0.3 to 150 μg/ml, depending on the strain and the culture medium used. The peptide showed activity against E. coli O111 over the range of pH 5.5 to 7.5 and was most effective under slightly alkaline conditions. Its antibacterial effectiveness was reduced in the presence of Na⁺, K⁺, Mg²⁺ or Ca²⁺ ions, or in the presence of various buffer salts. Lactoferricin B was lethal, causing a rapid loss of colony-forming capability in most of the species tested. Pseudomonas fluorescens, Enterococcus faecalis and Bifidobacterium bifidum strains were highly resistant to this peptide.     

Selective growth inhibition of Porphyromonas gingivalis by bestatin

Abstract Recent work in our laboratory indicates that selected protease/peptidase inhibitors interfere with the growth of Porphyromonas gingivalis . The aim of the present study was to further investigate the inhibitory effect of bestatin on the growth of P. gingivalis . Complete growth inhibition of P. gingivalis (11 strains) was observed when bestatin was incorporated at 2.5 μg ml⁻¹ in a complex broth medium. Fifty percent inhibition was still obtained with bestatin at a final concentration of 0.5 μg ml⁻¹. The inhibitory effect of bestatin was highly specific as the growth of 20 different oral bacterial species, including Gram-positive and Gram-negative as well as saccharolytic and asacharolytic bacteria, was not affected even at bestatin concentrations up to 50 μg ml⁻¹. Bestatin did not significantly affect the viability of P. gingivalis indicating that it has a bacteriostatic rather than a bactericidal effect. Growth assays using other specific inhibitors suggested that the effect of bestatin on the growth of P. gingivalis was unlikely to be related to its aminopeptidase inhibitor activity. Cultivation of P. gingivalis with a subinhibitory concentration of bestatin did not modify the cell envelope protein profile, as determined by SDS-PAGE analysis, but significantly decreased the number of extracellular vesicles produced. The present study indicated that bestasin is a highly effective inhibitor of cell growth of P. gingivalis . Additional studies will indicate whether bestatin should be considered as a potential drug in the control of P. gingivalis , a suspected pathogen in adult chronic periodontitis.     

Electrotransformation of whole cells of Brevibacterium sp. R312 a nitrile hydratase producing strain: Construction of a cloning vector

Abstract: A rapid and effective method is described for electroporation of Brevibacterium sp. R312, a coryneform strain producing nitrile hydratase and amidase. The transformation efficiency of the method is 10⁸ transformants per μg of plasmid under optimal conditions. Parameters optimised included field strength (11.8 kV cm⁻¹), pulse length (2.4 ms), plasmid DNA concentration (0.25 μg ml⁻¹ and cell density (10¹⁰ cells ml⁻¹). Surprisingly, the transformation efficiency did not vary with the growth stage, in contrast to results in the literature. A shuttle vector was constructed containing several unique cloning sites down-stream of the SP6 RNA polymerase promoter.     

Bacteriological stability and growth kinetics of Pseudomonas aeruginosa in bottled water

Bacteriological stability of water bottled in plastic containers and the growth kinetics of Pseudomonas aeruginosa were determined. Samples of water from the source, water to be bottled, finished product and sterile water bottled in non-returnable and returnable containers were analysed for aerobic colony count, coliforms, Escherichia coli and Ps. aeruginosa. The samples were examined for up to 30 d storage. Aerobic colony count increased 6 d after bottling to between 10³ and 10⁵ cfu ml⁻¹. Coliforms and E. coli were not found in any sample. Pseudomonas aeruginosa was isolated from commercial products bottled in returnable plastic containers due to the contamination from the containers and the subsequent multiplication utilizing trace nutrients. The predominant Ps. aeruginosa strains showed high doubling time (26 h) due to competition from the accompanying flora. In the absence of competing flora Ps. aeruginosa reached higher density than the maximum reached by aerobic flora, with a doubling time of only 3·6 h. After 30 d storage, this micro-organism was predominant.     

Application of bioluminescence-based microbial biosensors to the ecotoxicity assessment of organotins

The toxicity of two common organotin pollutants and their initial breakdown products (tributyltin, dibutyltin, triphenyltin and diphenyltin) were assessed using two different bioluminescent microbial biosensors: Microtox and lux -modified Pseudomonas fluorescens pUCD 607. The organotins were made up as standards, and tested both in double-deionized water and in extracted soil solution, the latter representing a realistic matrix for terrestrial contamination. Microtox was especially sensitive to the organotins, with 50% effective concentration (EC₅₀) values (15 min) for tributyltin as low as 21·9 μg l⁻¹ in pure water, and 0·118 μg l⁻¹ in soil extract. The sensitivity of Microtox was increased by an order of magnitude in soil extract. The Ps. fluorescens was less sensitive, with EC₅₀ values (30 min) of around 800 μg l⁻¹ in pure water. The toxicity to Ps. fluorescens was decreased by around an order of magnitude in soil extract. The two biosensors showed different response patterns, with Microtox being more sensitive to the triorganotins and Ps. fluorescens being more sensitive to the diorganotins.     

Rapid, extensive and reversible vacuolation of Schizosaccharomyces pombe induced by amphotericin B

Abstract The fission yeast Schizosaccharomyces pombe has no large vacuoles under normal growth conditions, although budding yeasts usually have large central vacuoles. The minimum inhibitory concentration of amphotericin B to S. pombe was 0.5 μg ml⁻¹; treatment with 0.2 μg ml⁻¹ for 20 min induced rapid and extensive vacuolation in S. pombe exponential phase cells. Growth rate of the cells with 0.2 μg ml⁻¹ amphotericin B was much reduced for 6 h, showing extensive vacuolation. Vacuolation in itself was not fatal: on removal of the drug, most cells recovered gradually and eventually multiplied.     

The effect of cryptolepine on the morphology and survival of Escherichia coli, Candida albicans and Saccharomyces cerevisiae

The antimicrobial activity of the indoloquinoline alkaloid, cryptolepine, isolated from Cryptolepis sanguinolenta (Fam. Periplocaceae) was determined against selected micro-organisms. The minimum inhibitory concentration (MIC) ranges obtained, expressed as μg ml⁻¹, were: 5–10 for Saccharomyces cerevisiae NCPF 3139; 10–20 for S. cerevisiae NCPF 3178; 20–40 for Escherichia coli NCTC 10418; 40–80 for E. coli NCTC 11560, Candida albicans ATCC 10231 and C. tropicalis NCPF; and 80–160 for C. albicans NCPF 3242 and NCPF 3262.
Biocidal effects were noted at concentrations 2–4 times those of the MIC of the alkaloid following challenge with 10⁶ cfu ml⁻¹ of micro-organisms. Time-kill studies showed a reduction in viable count from 10⁶ to < 10 cfu ml⁻¹ in 4 h in C. albicans ATCC 10231 exposed to 320 μg ml⁻¹ of the agent; 3 log cycle reductions were recorded for the 6 h counts of E. coli NCTC 10418 and S. cerevisiae NCPF 3139 exposed to 40μg ml⁻¹ and 160 μg ml⁻¹ of the alkaloid respectively.
These results were consistent with findings using scanning electron microscopy. Exposure of cells to biocidal concentrations of cryptolepine produced filamentation prior to lysis in E. coli NCTC 10418 and extreme disturbance of surface structure, including partial and total collapse, followed by lysis in C. albicans ATCC 10231 and S. cerevisiae NCPF 3139.     

Development and characterization of a lux-modified 2,4-dichlorophenol-degrading Burkholderia sp. RASC

lux -marked biosensors for assessing the toxicity and bioremediation potential of polluted environments may complement traditional chemical techniques. lux CDABE genes were introduced into the chromosome of the 2,4-dichlorophenol (2,4-DCP)-mineralizing bacterium, Burkholderia sp. RASC c2, by biparental mating using the Tn 4431 system. Experiments revealed that light output was constitutive and related to cell biomass concentration during exponential growth. The transposon insertion was stable and did not interrupt 2,4-DCP-degradative genes, and expression of lux CDABE did not constitute a metabolic burden to the cell. A bioluminescence response was detectable at sublethal 2,4-DCP concentrations: at < 10.26 μg ml⁻¹, bioluminescence was stimulated (e.g. 218% of control), but at concentrations > 60 μg ml⁻¹ it declined to < 1%. Investigating the effect of [14C]-2,4-DCP concentration on the evolution of ¹⁴CO₂ revealed that, for initial concentrations of 2.5–25 μg ml⁻¹, ≈55% of the added ¹⁴C was mineralized after 24 h compared with < 1% at 50 and 100 μg ml⁻¹. Inhibition of 2,4-DCP mineralization between 25 and 50 μg ml⁻¹ corresponded well to the EC₅₀ value (33.83 μg ml⁻¹) obtained from bioluminescence inhibition studies. lux -marked RASC c2 may therefore be used as a functionally (i.e. 2,4-DCP degrader) and environmentally relevant biosensor of toxicity and biodegradation inhibition.     

Immunodot detection of nisin Z in milk and whey using enhanced chemiluminescence

A highly specific antisera was produced in New Zealand white rabbits against nisin Z, a 3400 Da bacteriocin produced by Lactococcus lactis ssp. lactis biovar. diacetylactis UL 719. A dot immunoblot assay was then developed to detect nisin Z in milk and whey. As few as 1·5 10⁻¹ international units per ml (IU ml⁻¹), corresponding to 0·003 μg ml⁻¹ of pure nisin Z, were detected in carbonate-bicarbonate buffer within 6 h using chemiluminescence. When milk and whey samples were tested, approximately 0·155 μg ml⁻¹ (7·9 IU ml⁻¹) of nisin Z was detected. The detection limit obtained was lower than that of traditional methods including microtitration and agar diffusion.