Screening of antagonistic bacterial strains against Meloidogyne incognita using protease activity

Twenty-six bacterial strains that had demonstrated antagonism to some fungal and bacterial pathogens were evaluated for their ability to inhibit Meloidogyne incognita Kofoid & White. The inhibition rates of egg-hatching and second-stage juveniles (J2) mortality of M. incognita by these strains ranged from ?16.5 to 87.4% and from 1.3 to 77.8%, respectively. The 12 strains causing J2 mortality over 40% were chosen for greenhouse experiments in which their biocontrol efficacy reached 33.3–65.6%. On the other hand, among the 26 strains, 20 demonstrated in vitro protease activity and 14 revealed chitinase activity. Significantly, strains Bacillus sp. AR156 and GJ24 in greenhouse tests showed the strongest protease activities. The analyses of the relationships of the efficacy of the 12 strains with their protease and chitinase activities, respectively, indicated that biocontrol efficacy was highly correlated with protease activity (r=0.92, P<0.001) but barely correlated with chitinase activity. The strong positive correlation between protease activity and efficacy suggests that in vitro protease activity could be used as a parameter for selecting biological control agents (BCAs) against root-knot nematodes. Consistently, the biocontrol efficacy of AR156, GJ24, abamectin reached 74.3, 73.4, and 40.9% in the field in Huai-an, Jiangsu; and 71, 69.9, and 37% in Zao-zhuang, Shandong, respectively. The fact that the strains with high protease activities also had significantly higher biocontrol efficacy than abamectin in the field implies that in vitro protease activity may be adopted as a reliable new parameter for speeding up the process of screening the biological control agents (BCAs).     

Germanium toxicity in selected bacterial and yeast strains

Summary The toxicity of germanium dioxide (GeO₂) to 21 bacterial and 13 yeast strains was investigated in liquid broth medium to obtain information on strains tolerant to high (1 to 2 mg/ml) GeO₂ concentrations.Arthrobacter sp. NRC 32005,enterobacter aerogenes NRC 2926,Klebsiella aerogenes NCTC 418 andPseudomonas putida NRC 5019 were tolerant to 1 mg/ml GeO₂.Bacillus sp. RC607 was able to grow in the presence of 2 mg/ml GeO₂ at pH 10 in broth culture. The yeastsCandida guilliermondii, Candida shehatae andPachysolen tannophilus were the most sensitive to GeO₂ as evidenced by their diminished growth rates at a GeO₂ concentration as low as 0.1 mg/ml. None of the yeast strains tested exhibited growth in the presence of 1 mg/ml GeO₂. The high pH of the medium containing germanium may be partially responsible for the growth inhibition of the yeast cultures. Select bacterial cultures previously exposed to 1 mg/ml GeO₂ could tolerate and grow better at 2 mg/ml GeO₂, suggesting the existence of very efficient adaptive mechanisms. The pH of the medium could modulate GeO₂ tolerance and this effect was found to be strain-dependent.     

Evaluation of antibacterial activity of crude protein extracts from seeds of six different medical plants against standard bacterial strains

A huge group of natural antimicrobial compounds are active against a large spectrum of bacterial strains causing infectious threat. The present study was conducted to investigate the crude extracts of antimicrobial protein and peptide efficacy from six medicinal plant seeds. Extraction was carried out in Sodium phosphate citrate buffer, and Sodium acetate buffer using different pH. Antimicrobial activities of these plants were determined by the microbiological technique using Agar well diffusion Assay. Extremely strong activity was observed in the seed extracts of Allium ascolinicum extracted in sodium phosphate citrate buffer at pH (5.8) against Proteus vulgaris, Escherichia coli and Staphylococcus aureus with zone of inhibition 17 mm, 17 mm and 15 mm and Rumex vesicarius at pH (7.6), Ammi majus at pH (6.8), Cichorium intybus at pH (7.4) and Cucumis sativus at pH (7.8) also showed better sensitivity against the bacterial strains with zone of inhibition ranges 16–10 mm and some of the strains were found to be resistant. Antibacterial activity pattern of different plant extracts prepared in sodium acetate buffer pH (6.5), among all the plant seed extracts used Foeniculum vulgare had shown good inhibition in all the bacterial strains used, with zone of inhibition ranges 11–12.5 mm, The extracts of C. intybus and C. sativus were found to be effective with zone of inhibition 11–6 mm and some of the strains were found to be resistant. Most of the strains found to have shown better sensitivity compared with the standard antibiotic Chloramphenicol (25 mcg). Our results showed that the plants used for our study are the richest source for antimicrobial proteins and peptides and they may be used for industrial extraction and isolation of antimicrobial compounds which may find a place in medicine industry as constituents of antibiotics.     

Antimicrobial activity of some plant extracts against bacterial strains causing food poisoning diseases

Prevention of food spoilage and food poisoning pathogens is usually achieved by use of chemical preservatives which have negative impacts including: human health hazards of the chemical applications, chemical residues in food & feed chains and acquisition of microbial resistance to the used chemicals. Because of such concerns, the necessity to find a potentially effective, healthy safer and natural alternative preservatives is increased. Within these texts, Plant extracts have been used to control food poisoning diseases and preserve foodstuff. Antimicrobial activity of five plant extracts were investigated against Bacillus cereus, Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa and Salmonella typhi using agar disc diffusion technique. Ethanolic extracts of Punica granatum, Syzygium aromaticum, Zingiber officinales and Thymus vulgaris were potentially effective with variable efficiency against the tested bacterial strains at concentration of 10 mg/ml while extract of Cuminum cyminum was only effective against S. aureus respectively. P. granatum and S. aromaticum ethanolic extracts were the most effective plant extracts and showed bacteriostatic and bactericidal activities against the highly susceptible strains of food borne pathogenic bacteria (S. aureus and P. aeruginosa) with MIC's ranged from 2.5 to 5.0 mg/ml and MBC of 5.0 and 10 mg/ml except P. aeruginosa which was less sensitive and its MBC reached to 12.5 mg/ml of S. aromaticum respectively. These plant extracts which proved to be potentially effective can be used as natural alternative preventives to control food poisoning diseases and preserve food stuff avoiding healthy hazards of chemically antimicrobial agent applications.     

In vitro assay of the antimicrobial activity of kephir against bacterial and fungal strains

Kephir is a fermented carbonated refreshing milk, with a slightly acidic aromatic taste and creamy foam composition which contains lactobacilli, leuconostocci, acetic acid bacteria, lactostreptococci and yeasts. Recent studies have demonstrated its antibacterial, immunostimulating, antitumoral and cholesterol-lowering activities.

Purpose

The purpose of this study was to investigate the antimicrobial activity of kephir against Bacillus subtilis spp. spizizenii ATCC 6633, Staphylococcus aureus ATCC 6538, Enterococcus faecalis ATCC 29212, Escherichia coli ATCC 8739, Salmonella enteritidis ATCC 13076, Pseudomonas aeruginosa ATCC 9027 and Candida albicans ATCC 10231. The kephir fermented for 24 h and 48 h, as well and after 7 days preservation at 4–8 °C was tested by in vitro disk diffusion method. The intensity of the antimicrobial activity was interpreted by comparison with two antibiotics, i.e. ampicillin and neomycin.

Results

The antimicrobial activity of 24 h as well as 48 fermented kephir, fresh or after 7 days preservation at 4–8 °C was similar and observed against B. subtilis, S. aureus, E. coli, E. faecalis and S. enteritidis. For E. coli, E. faecalis and S. enteritidis the antimicrobial activity was superior to both tested antibiotics and for B. subtilis and S. aureus to one antibiotic. The tested products exhibited no activity against P. aeruginosa and C. albicans.

Conclusion

Kephir is exhibiting large spectrum and strong antibacterial activity probably due to the complex viable probiotic strains association producing antimicrobial substances.     

Interaction of neomycin with other antibiotics on selected bacterial strains

Antimicrobial combinations are used most frequently to provide broad-spectrum empirical coverage in the treatment of bacterial infections. However, combination of two antibiotics may not influence their activity, may lead to synergy or antagonism in the activity. Neomycin may be combined with one of the following antibiotics: ampicillin, procaine penicillin, gramicidin, bacitracin, polymyxin B, lincomycin, oxytetracycline, and erythromycin in some human and veterinary multiantibiotic drugs distributed in Poland. The checkerboard method has been one of the traditional assays for the measurement of antibiotic interactions. The aim of this study was to analyse the activity interaction of neomycin with second antibiotic in multiantibiotic drugs distributed in Poland on standards and clinical bacterial strains. Checkerboard results for all strains demonstrated synergism for 2.5% of combinations, only for standards strains. In one case Salmonella Enteritidis, in combination of neomycin with bacitracin, inhibition effect was observed. Additive effects were predominant--49%. In 18% neutral effects were shown, but in 26% of combinations FIC indexes were not possible to calculate, because of the resistance of clinical strains to the highest concentration of at least one antibiotic. In combination of aminoglycoside (neomycin) with beta-lactams antibiotics (ampicillin, procaine penicillin) in vitro, no synergy was observed for all examined strains. The best results were achieved for combinations of neomycin with peptide antibiotics (polymyxin, gramicidin and bacitracin)--5 for all 6 synergy effect observed.     

In vitro activity of cefepime against ESBL-positive clinical strains of gram-negative rods

The aim of this study was to determine an in vitro activity of cefepime against ESBL-positive clinical strains of Gram-negative rods isolated from hospitalized patients. Experiments were performed with 100 ESBL-positive strains of Gram-negative rods isolated from clinical samples in 2004. Strains were identified with the use of automatic ATB Expression system and biochemical ID 32 GN tests (bioMdrieux sa). Extended-spectrum beta-lactamases (ESBLs) were detected by means of disc diffusion methods: the double-disc synergy test (DDST) and the diagnostic disc test (DD, Oxoid Ltd, UK). Susceptibility in vitro of ESBL producers to 4th generation--cefepime was determined with gradient diffusion method Etest (AB Biodisk, Solna, Sweden). MIC value of cefepime was assessed for each strain. Among 100 ESBL-producing strains, 94--belonged to enteric rods and 6--to nonfermentative rods. The greatest number of strains belonged to the species Serratia marcescens (27% of all strains) and next--to the species Enterobacter cloacae (21%). Fourteen strains were susceptible (S) in vitro to cefepime, 12--intermediately susceptible (I) and 74--resistant (R). Application of cefepime in a therapy of infections caused by ESBL-positive strains of Gram-negative rods highly susceptible in vitro to this antibiotic, should be considered.     

The in vitro antibiofilm activity of selected marine bacterial culture supernatants against Vibrio spp.

The aim of the work is to investigate the effect of marine bacterial culture supernatants on biofilm formation of Vibrio spp., a major menace in aquaculture industries. Vibrio spp. biofilm cause life-threatening infections in humans and animals. Forty-three marine bacterial culture supernatants were screened against the hydrophobicity index, initial attachment and biofilm formation in Vibrio spp. Twelve culture supernatants showed antibiofilm activity. The bacterial culture supernatants S8-07 (Bacillus pumilus) and S6-01 (B. indicus) inhibited the initial attachment, biofilm formation and dispersed the mature biofilm at 5% v/v concentration without inhibiting the growth. Analysis by light microscopy and confocal laser scanning microscopy showed that the architecture of the biofilm was destroyed by bacterial supernatants when compared to the control. The bacterial supernatants also reduce the surface hydrophobicity of Vibrio spp. which is one of the important requirements for biofilm formation. Further characterization of antibiofilm activity in S8-07 culture supernatant confirmed that it is an enzymatic activity and the size is more than 10 kDa and in S6-01, it is a heat-stable, non-protein compound. Furthermore, both the supernatants failed to show any biosurfactant activity. The culture supernatants of S8-07 and S6-01 with promising antibiofilm property have potential for application in medicine and marine aquaculture.     

Efficacy of multiple metals against copper-resistant bacterial strains

Aims: The antibacterial efficacy of zeolites containing copper (Cu) or silver (Ag) ions or a combination was assessed against several reported copper‐resistant (Cu^R) bacterial strains. Methods and Results: Comparison strains were obtained from the American Type Culture Collection that had no documented metal resistance. Reductions in bacterial populations were determined after exposure time intervals of 3, 6 and 24 h. All three Cu^R strains of Salmonella enterica exhibited resistance to Cu, Ag and Cu/Ag after three and 6 h of exposure. Both the Cu^R and comparison strain of Enterococcus faecium were resistant to both metals and the metal combination. Cu^RPseudomonas putida was significantly reduced by all zeolites within 3 h. The Cu^REscherichia coli strain was more sensitive to Cu, but more resistant to Ag than the comparison strain; however, significant reductions were achieved within 3 h with both Cu and Cu/Ag, and within 24 h with Ag. Conclusions: Some strains with reported resistance to Cu were also resistant to Ag, suggestive of a shared resistance mechanism such as an indiscriminate Cu efflux pump. Ent. faecium appears to have innate resistance to both metals. In general, Ent. faecium was the most resistant species to the individual metals and the combination of metals, Ps. putida the least resistant, and the Salmonella strains were more resistant than E. coli. Significance and Impact of the Study: Several of the comparison strains with no reported copper resistance were resistant to one or both metals. This may call into question the methods for determining bacterial metal resistance, which typically use nutrient‐rich media containing metals to assess the ability of the bacteria to grow in comparison with a wild‐type strain. Nevertheless, all the Cu^R strains evaluated in this study, with the exception of Ent. faecium, were reduced using the Cu and Ag zeolite combination.     

Evaluation of Lactobacillus strains for selected probiotic properties

Eleven strains of Lactobacillus collected in the Culture Collection of Dairy Microorganisms (CCDM) were evaluated for selected probiotic properties such as survival in gastrointestinal fluids, antimicrobial activity, and competition with non-toxigenic Escherichia coli O157:H7 for adhesion on Caco-2 cells. The viable count of lactobacilli was reduced during 3-h incubation in gastric fluid followed by 3-h incubation in intestinal fluid. All strains showed antimicrobial activity and the three most effective strains inhibited the growth of at least 16 indicator strains. Antimicrobial metabolites of seven strains active against Lactobacillus and Clostridium indicator strains were found to be sensitive to proteinase K and trypsin, which indicates their proteinaceous nature. The degree of competitive inhibition of non-toxigenic E. coli O157:H7 adhesion on the surface of Caco-2 cells was strain-dependent. A significant decrease (P?<?0.05) in the number of non-toxigenic E. coli O157:H7 adhering to Caco-2 cells was observed with all lactobacilli. Three strains were selected for additional studies of antimicrobial activity, i.e., Lactobacillus gasseri CCDM 215, Lactobacillus acidophilus CCDM 149, and Lactobacillus helveticus CCDM 82.     

Evaluation of antimicrobial activity of selected plant extracts by rapid XTT colorimetry and bacterial enumeration

The aim of this study was to screen and evaluate the antimicrobial activity of indigenous Jordanian plant extracts, dissolved in dimethylsulfoxide, using the rapid XTT assay and viable count methods. XTT rapid assay was used for the initial screening of antimicrobial activity for the plant extracts. Antimicrobial activity of potentially active plant extracts was further assessed using the "viable plate count" method. Four degrees of antimicrobial activity (high, moderate, weak and inactive) against Bacillus subtilis, Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa, respectively, were recorded. The plant extracts of Hypericum triquetrifolium, Ballota undulata, Ruta chalepensis, Ononis natrix, Paronychia argentea and Marrubium vulgare had shown promising antimicrobial activity. This study showed that while both XTT and viable count methods are comparable when estimating the overall antimicrobial activity of experimental substances, there is no strong linear correlation between the two methods.     

Interaction of aminoglycosides and the other antibiotic on selected bacterial strains

The aim of this study was to analyse the activity interaction of aminoglycosides (gentamicin, kanamycin, streptomycin and dihydrostreptomycin) when combined with other antibiotics (lincomycin, benzylpenicillin, amoxicillin, cephalexin, spectinomycin and erythromycin), on selected clinical bacterial strains. The checkerboard method has been selected from the traditional assays for the measurement of antibiotic interaction. Checkerboard results for all strains demonstrated synergism for nine cases (9/112--8%). Additive effects were predominant--73/112--65.2%. In 12.5% neutral effects were shown, but in 11.6% of combinations FIC indexes were not possible to calculate, because of the resistance of clinical strains to the highest concentration of at least one antibiotic. The best results were achieved for combinations of dihydrostreptomycin with procaine penicillin because of higher number of cases synergy effect was observed. Antagonism of aminoglycosides and beta-lactams in case of gentamicin and amoxicillin for E. coli and E. cloacea strains were shown. Potential activity for combination of streptomycin and erythromycin was shown.