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.     

Diffusion gradient plates for herbicide toxicity tests on micro-algae and cyanobacteria

Single dimension agar gel diffusion gradient plates were used to test the tolerance of several micro-algae and cyanobacteria to five herbicides. Growth of most of the test organisms was prevented by terbutryn and diuron at concentrations between 1·1 and 2·8 μg/ml (minimum inhibitory concentration, MIC). Chlorpropham was intermediate in toxicity whilst monuron and atrazine generally gave higher MIC values (up to 17·1 μg/ml). The cyanobacteria were mostly more tolerant than the algae, but susceptibility to the herbicides was variable even between strains of an individual algal species. The gradient plate technique offers the advantages of simplicity and low cost in toxicity screening studies with herbicides or other biologically active chemicals and xenobiotics.     

Susceptibility of Brazilian Staphylococcal Strains to Glycopeptides Evaluated by Different Testing Methods

Reports of staphylococci with reduced susceptibility to glycopeptides are cause for concern. This study evaluated the susceptibility of 84 staphylococci clinical isolates to glycopeptides by the disk diffusion, agar dilution, E-test, and BHIA screening methods. Vancomycin agar dilution showed all strains presented minimum inhibitory concentration (MIC) ranging from 0.5 to 2 μg/ml, and the E-test showed similar results. Teicoplanin agar dilution test showed MICs ranging from ≤ 0.5 to 2 μg/ml for Staphylococcus aureus and MICs ranging from <0.25 to 32 μg/ml for coagulase-negative Staphylococcus (CNS). Ten CNS isolates presented MICs ranging from 8 to 32 μg/ml for agar dilution and/or E-test. All the staphylococci were susceptible to vancomycin by the disk diffusion test (DDT), but two CNS isolates presented intermediate resistance to teicoplanin by the DDT and MICs of susceptibility, with two other CNS strains, teicoplanin-susceptible by the DDT, presented MICs of intermediate resistance. On the vancomycin-containing agar, 20 CNS isolates were able to grow, but no S. aureus strain. All these isolates showed MICs to teicoplanin (4–32 μg/ml) higher than those isolates that did not grow on the agar screen plate. PFGE of chromosomal SmaI digests showed a wide diversity of these CNS strains, without any predominance of a single PFGE pattern. Received: 25 May 2001 / Accepted: 9 July 2001     

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.     

β-Lactam and aminoglycoside production from streptomycetes

Streptomyces cattleya, S. fradiae and S. griseus produced different amounts of growth when cultured sequentially through sporulation, vegetative and antibiotic production media. Only S. griseus grew well on all three types of medium. Streptomyces cattleya grew poorly on both sporulation and vegetative media. Growth was 1·6 and 8·0 mg/1/h respectively. For all three species, biomass yield in the final antibiotic production medium was dependent on amount of inoculum. Antibiotic yields were obtained only from production media. Under slow growth conditions l -cysteine and l -valine supplementation stimulated S. cattleya β-lactam production, giving 1000 μg/ml β-lactam equivalents compared with 45 μg/ml β-lactam equivalents for no supplementation. For aminoglycosides the agar well diffusion bioassay was more sensitive towards the hydrochloride than the neutral salt. Paper chromatography confirmed the main antibiotic classes. R _F values for replicate samples indicated aminoglycoside homogeneity and β-lactam heterogeneity.     

Comparative in vitro activity of moxalactam (LY127935), other beta-lactam antibiotics,and aminoglycosides

Moxalactam (LY127935), a novel beta-lactam antibiotic, was compared with semisynthetic penicillins, cephalosporins, and aminoglycosides by the agar dilution method against 5,317 recent clinical isolates of facultative and anaerobic bactria. At 0.5 μg/ml, moxalactam inhibited 90% of all Gram-negative bacilli tested except forPseudomonas aeruginosa (81% inhibited by 32 μg/ml) andAcinetobacter calcoaceticus (88% inhibited by 32 μg/ml). More than 90% ofBacteroides fragilis andStaphylococcus aureus were inhibited by 4 μg/ml and 8 μg/ml, respectively. Moxalactam was at least 16-fold more active by weight than cephalothin, cefamandole, and cefoxitin forEscherichia coli, Klebsiella pneumoniae, andEnterobacter species, and 2- to 4-fold more active than cefoxitin forB. fragilis. Moxalactam was 4-fold less active than cefamandole and cephalothin forS. aureus and 2- to 4-fold less active than piperacillin forP. aeruginosa. Moxalactam was as active or more active than the aminoglycosides for all facultative Gram-negative bacilli except forP. aeruginosa. Moxalactam was inhibitory (minimal inhibitory concentration <16 μg/ml) for 20/27 gentamicin-resistant isolates and 8/13 amikacin-resistant organisms. Moxalactam’s in vitro activity against Gram-negative bacilli is markedly superior to presently available cephalosporins and, except forP. aeruginosa, is comparable to the aminoglycosides.     

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.     

Co-existence of β-lactamase and penicillin acylase in bacteria; detection and quantitative determination of enzyme activities

Twenty-six bacteria were examined for the presence of penicillin acylase and β-lactamase. A copper reducing assay, which was sensitive in the analytical range 2–20 μg/ml, was used for determination of penicilloates and a fluorescamine assay was used to determine 6-aminopenicillanic acid concentrations when both substances were produced by the action of the enzymes on a single substrate. Seventeen bacteria contained β-lactamases, six contained penicillin acylases and four contained both enzymes. Two bacteria contained a Type 1 penicillin acylase and four bacteria contained a Type II enzyme. No ampicillin acylases were detected. All β-lactamases were constitutive enzymes in those organisms where both enzymes co-existed. Bacillus subtilis and B. cereus produced inducible and extracellular β-lactamases. Acinetobacter calcoaceticus ATCC 21288 produced a constitutive β-lactamase which was detected extracellularly.     

Response of Campylobacter jejuni to combinations of ferrous sulphate and cadmium chloride

On Mueller Hinton (MH) agar, Campylobacter jejuni showed 20.0 and 30.9mm zones of inhibition surrounding discs impregnated with 2.5 and 20 μg CdCl₂ respectively. The minimal inhibitory concentration (MIC) ranged from 0.64 to 3.2 μg CdCl₂/ml of MH agar for four C. jejuni strains. In the presence of 23 μg FeSO₄/ml of MH the MIC increased to a range of 1.5–5.4 μg CdCl₂/ml of MH. Moreover, the numbers of colonies present on MH supplemented with FeSO₄ were greater than on MH without iron. The growth response of C. jejuni in the presence of 0.025% (w/v) FeSO₄ in MH broth was increased about 10000 fold in three of four strains when compared with the growth in unsupplemented MH broth. Zones of inhibition surrounding 20 μg discs of CdCl₂ were 50.6 and 24.4 mm on MH and Campy-BAP media respectively, with cells grown on MH. These results suggest that the blood-containing medium 'neutralized' the biocidal influence of the CdCl₂. In comparison, C. jejuni inoculum from fluid thioglycollate (FT) medium showed smaller zones of inhibition. These decreased from 34.9 mm on MH agar to 19.6 mm on Campy-BAP agar, suggesting that components in the FT growth medium ameliorated the toxic influence of CdCl₂. Atomic absorption spectroscopy analysis indicated mean values (mg/100 g dry weight) of selected metals bound by C. jejuni as: Cu, 10.4; Mg, 146; Na, 2385; Fe, 45.1; Zn, 13.0; and K, 172.     

Biotransformation of mercury by bacteria isolated from a river collecting cinnabar mine waters

One hundred six strains of aerobic bacteria were isolated from the Fiora River which drains an area of cinnabar deposits in southern Tuscany, Italy. Thirty-seven of the strains grew on an agar medium containing 10g/ml Hg (as HgCl₂) with all of these strains producing elemental mercury. Seven of the 37 strains also degraded methylmercury. None of 106 sensitive and resistant strains produced detectable monomethylmercury although 15 strains produced a benzene-soluble mercury species. Two strains of alkylmercury (methyl-, ethyl- and phenylmercury) degrading bacteria were tested for the ability to degrade several other analogous organometals and organic compounds, but no activity was detected toward these compounds. Mercury methylation is not a mechanism of Hg resistance in aerobic bacteria from this environment. Growth of bacteria on the agar medium containing 10g/ml HgCl₂ was diagnostic for Hg detoxification based on reduction.     

Rapid methods for differentiating Gram-positive from Gram-negative aerobic and facultative anaerobic bacteria

M anafi , M. & K neifel , W. 1990. Rapid methods for differentiating Gram-positive from Gram-negative aerobic and facultative anaerobic bacteria. Journal of Applied Bacteriology 69 , 822–827.
Different tests based on lysis by KOH and on reaction with fluorogenic and chromogenic substrates, L-alanine-4-nitroanilide (LANA); L-alanine-4-methoxy-β-naphthylamide (MNA); 4-alanine-2-amidoacridone (AAA); L-alanine-7-amido-4-methylcoumarin (AAMC); 8-anilino-l-naphthalene-sulphonic acid (ANS) were compared for their suitability to distinguish Gram-positive from Gram-negative bacteria. A concentration of 100 μg/ml was chosen for incorporating LANA, AAA, AAMC and ANS into the growth medium, based on sensitivity tests. MNA did not show any detectable reaction over a concentration range from 50 to 200 μg/ml, and led to inhibition of all bacteria at 200 μ/ml. In the examination of a total of 146 bacterial strains, including Yersinia enterocoiitica, Bacillus cereus , and B. subtilis the KOH test was not comparable with the Gram staining. A good correlation with Gram staining was found between LANA, AAA and AAMC added to plate count agar on one hand, and LANA and AAMC impregnated paper strips on the other hand, thereby utilizing the aminopeptidase activity. Agar containing ANS showed detectable fluorescence with all Gram-negative strains, but with Staphylococcus aureus and Staph. epidermidis a weak reaction was also observed. AAMC was selected for a rapid paper strip test With this substrate a pronounced blue fluorescence was obtained with Gram-negative colonies.     

Rapid distinction between micrococci and staphylococci with furazolidone agars

Furazolidone agar proved to be a suitable medium for separating strains of the genera Micrococcus and Staphylococcus. 720 isolates (including 24 type strains) of gram- and catalase-positive cocci were tested for growth on tryptone soya and peptone agar with the addition of 50 g/ml furazolidone. The results were compared with the classification obtained by the standard-O/F-test and by the test system of Schleifer and Kloos.For routine identification and separation of staphylococci from micrococci a peptone agar with 20 g furazolidone/ml is recommended.     

Comparison of growth characteristics and roquefortin C production of<Emphasis Type="Italic">Penicillium roqueforti</Emphasis> from blue-veined cheese

The properties of 21 isolates ofPenicillium roqueforti from just as many commercial blue-veined cheeses, purchased from the Argentinean market (domestic and imported products) were comparatively examined. Isolates were investigated for their ability to grow at different temperatures, pH values and concentration of NaCl, as well as for their proteolytic and lipolytic activities, respectively. The potential of these strains to produce roquefortin in vitro, and the actual levels of roquefortin in 10 of these cheeses were analysed by TLC. All strains showed similar growth properties in aspects of salt concentration and pH-value of the medium, and all grew well at 10 °C. Only four strains showed proteolytic activity on casein agar, while all strains were lipolytic on trybutirin agar. After incubation at 25 °C for 16 days, all strains produced roquefortin in Yeast Extract Sucrose (25.6–426.7 μg/g) and in reconstituted (10%) sterile skim milk (26.9–488 μg/g). Roquefortin at >0.1 μg/g was also found in 9 out of 10 analysed samples of blue-veined cheeses (8 from Argentine, 1 from Spain), with a maximum value 3.6 μg/g. During the ripening process of blueveined cheese, production of roquefortin seems to be unavoidable. Care should be taken to select strains with low toxin production characteristics, to minimize potential health risks. Roquefortin C production byP. roqueforti in vitro was not correlated with roquefortin C levels found in cheese. Financial support: Research grants from the National University of Quilmes, Argentina     

Transformation of the entomopathogenic fungus Metarhizium anisopliae to benomyl resistance

Abstract Protoplasts of the entomopathogenic fungus Metarhizium anisopliae were transformed to benomyl resistance using cosmid pSV50 which harbours a β-tubulin gene cloned from a Neurospora crassa benomyl-resistant mutant. Transformant colonies, which appeared at a frequency of 4 per 50 μg DNA, grew and sporulated on 10 μg/ml benomyl, whereas the wild type was inhibited by 3 μg/ml. Southern blot hybridization of DNA from transformants showed that, in each case, tandem repeats of the cosmid had integrated at several chromosomal loci. The transformants were mitotically stable when subcultured on non-selective agar and retained the ability to infect and kill larvae of Manduca sexta . Two transformants were less virulent than the wild type and one of them showed slower in vitro spore germination. The benomyl-resistant phenotype persisted in reisolates from insect cadavers.     

Screening method for detecting staphylococci with reduced susceptibility to teicoplanin

The incidence of infections caused by staphylococci with decreased susceptibility to teicoplanin (MIC>/=8 microg/ml) is increasing, but the disk diffusion test has difficulty detecting this low level of resistance. In addition, detection is complicated because of the heterogeneous phenotypes for teicoplanin. In this study, we evaluated an agar screening method to detect staphylococci with decreased susceptibility to teicoplanin or heterogeneous resistance. First, to investigate the inoculum density and teicoplanin concentration of screening agar, we used 10(5) and 10(6) CFU/ml and Mueller-Hinton agars supplemented with 6 and 8 microg of teicoplanin/ml to test 39 genetically distinct staphylococcal strains (15 strains with teicoplanin MICs>/=8 microg/ml and 24 strains with teicoplanin MICs/=8 microg/ml or showing heteroresistance could be detected. These findings indicate that the method can be used as a reliable screening method for detecting staphylococci with reduced susceptibility to teicoplanin.     

Characterizing the production of a wild-type and benomyl-resistant Fusarium lateritium for biocontrol of Eutypa lata on grapevine

Benomyl-resistant (BR) and wild-type (WT) strains of Fusarium lateritium were examined for their tolerance to benomyl on potato dextrose agar (PDA) containing benomyl and control of the Eutypa lata in grapevine bioassays. The WT strain grew on PDA containing 1 μg/ml benomyl at 13, 26 and 29°C. The BR strain grew on PDA containing 10 μg/ml benomyl at 4°C, on PDA containing 100 μg/ml benomyl at 29°C, and on PDA containing 1000 μg/ml benomyl at 13 and 26°C. The BR strain was also able to colonize grapevine segments and control E. lata in the presence of 1000 μg/ml benomyl. Both strains were amenable to production via liquid fermentation and both achieved 100% control of E. lata in grapevine bioassays. Neither the duration of fermentation nor incubation temperature during grapevine bioassays influenced the efficacy of either strain against E. lata. The results suggest that application of BR F. lateritium alone or in combination with benomyl may provide good control of E. lata. Journal of Industrial Microbiology & Biotechnology (2001) 26, 151–155. Received 22 December 1999/ Accepted in revised form 20 October 2000     

SELECTIVE INHIBITION BY APHIDICOLIN OF THE ACTIVITY OF DNA POLYMERASE ALPHA LEADS TO BLOCKADE OF DNA SYNTHESIS AND CELL DIVISION IN SEA URCHIN EMBRYOS

Aphidicolin at 2 μg/ml caused 90% inhibition of mitotic cell division of sea urchin embryos at the I-cell stage. However, at 40 μg/ml it did not affect meiotic maturational divisions of starfish oocytes, which do not involve DNA replication. At 2 μg/ml it caused 90% inhibition of incorporation of tritiated thymidine into DNA of sea urchin embryos but did not affect protein or RNA synthesis even at a higher concentration. At 2 μg/ml it also caused 90% inhibition of the activity of DNA polymerase α, obtained from the nuclear fraction of sea urchin embryos, but did not affect the activity of DNA polymerase β or γ. These findings suggest that DNA polymerase α is responsible for replication of DNA in sea urchin embryos.     

Some observations on acquired resistance of tubercle bacteria to 4-acetylaminobenzaldehyde thiosemicarbazone

Summary Three strains of tubercle bacteria, two belonging to the human type and one of avian origin, were made resistant to 4-acetylaminobenzaldehyde thiosemicarbazone (Conteben) by being grown in progressively increasing concentrations of the drug using a Herrold agar medium. It could be shown that within four months the strains may be adapted to grow in the presence of 500g/ml of thiosemicarbazone, while at the beginning of treatment the bacteria were susceptible to an amount as low as 1g/ml of the substance. The impression was gained that resistance to streptomycin may be produced more readily than to thiosemicarbazone, and to the latter substance more easily than to P.A.S.     

Transfer of a benomyl resistance marker by heat-inactivated Trichoderma reesei protoplasts

Summary Protoplasts from a benomyl resistant Trichoderma reesei mutant were heat inactivated at 60°C for 8 min and fused with viable protoplasts from an osmosensitive, non-sporulating T. reesei strain. Fusants recovered on 50 g/ml benomyl containing potato dextrose agar plates grew and sporulated well. Cellulolytic enzyme activities produced in liquid culture by selected fusants were higher than those produced by parental strains.     

Ferritin-iron increases killing of Chinese hamster ovary cells by X-irradiation

Abstract. Stationary-phase Chinese hamster ovary cells were cultured in medium containing ferritin (-19% iron by weight) added at concentrations ranging from 0 to 128 μ g/ml. One set of cultures was unirradiated, and another set was exposed to 4.0 Gy of X-ray. Clonogenic cell survival was assessed in each set of cultures. In the absence of added ferritin, 4.0 Gy killed approximately 50% of the cells. In the absence of radiation, ferritin was not toxic at less than 48 μ g/ml; above 48 μ g/ml, toxicity increased with concentration. Apoferritin was not toxic at any concentration tested (up to 1000 μ g/ml). Although 32 μg/ ml ferritin, reflecting only a 3–6 fold increase in iron concentration over normal serum, was not toxic, it reduced the survival of X-irradiated cells by an additional 75%. These results indicate that a sublethal concentration of ferritin can be a potent radiosensitizer. This suggests the possibility that high body iron stores may increase susceptibility to radiation injury in humans.