Differential effects of catecholamines on in vitro growth of pathogenic bacteria

Supplementation of minimal medium inoculated with bacterial cultures with norepinephrine, epinephrine, dopamine, or isoproterenol resulted in marked increases in growth compared to controls. Norepinephrine and dopamine had the greatest enhancing effects on growth of cultures of Pseudomonas aeruginosa and Klebsiella pneumoniae, while epinephrine and isoproterenol also enhanced growth to a lesser extent. The growth of Escherichia coli in the presence of norepinephrine was greater than growth in the presence of the three other neurochemicals used in the study. Growth of Staphylococcus aureus was also enhanced in the presence of norepinephrine, but not to the same degree as was the growth of gram negative bacteria. Addition of culture supernatants from E. coli cultures that had been grown in the presence of norepinephrine was able to enhance the growth of K. pneumoniae. Addition of the culture supernatant fluid culture from E. coli cultures that had been grown in the presence of norepinephrine did not enhance growth of P. aeruginosa or S. aureus. Culture supernatant fluids from bacteria other than E. coli grown in the presence of norepinephrine were not able to enhance the growth of any bacteria tested. The results suggest that catecholamines can enhance growth of pathogenic bacteria, which may contribute to development of pathogenesis; however, there is no uniform effect of catecholamines on bacterial growth.     

Detection and validation of volatile metabolic patterns over different strains of two human pathogenic bacteria during their growth in a complex medium using multi-capillary column-ion mobility spectrometry (MCC-IMS)

Headspace analyses over microbial cultures using multi-capillary column-ion mobility spectrometry (MCC-IMS) could lead to a faster, safe and cost-effective method for the identification of pathogens. Recent studies have shown that MCC-IMS allows identification of bacteria and fungi, but no information is available from when on during their growth a differentiation between bacteria is possible. Therefore, we analysed the headspace over human pathogenic reference strains of Escherichia coli and Pseudomonas aeruginosa at four time points during their growth in a complex fluid medium. In order to validate our findings and to answer the question if the results of one bacterial strain can be transferred to other strains of the same species, we also analysed the headspace over cultures from isolates of random clinical origin. We detected 19 different volatile organic compounds (VOCs) that appeared or changed their signal intensity during bacterial growth. These included six VOCs exclusively changing over E. coli cultures and seven exclusively changing over P. aeruginosa cultures. Most changes occurred in the late logarithmic or static growth phases. We did not find differences in timing or trends in signal intensity between VOC patterns of different strains of one species. Our results show that differentiation of human pathogenic bacteria by headspace analyses using MCC-IMS technology is best possible during the late phases of bacterial growth. Our findings also show that VOC patterns of a bacterial strain can be transferred to other strains of the same species.     

The production of ginsenosides in hairy root cultures of American Ginseng, Panax quinquefolium L. and their antimicrobial activity

Panax quinquefolium, American ginseng, is valued for its triterpene saponins, known as ginsenosides. These constituents possess a number of pharmacological properties and hairy root cultures can synthesize similar saponins to those of field-cultivated roots. The antibacterial activity of extracts from three hairy root clones of P. quinquefolium L. was tested against a range of standard bacterial and yeast strains. The agar diffusion method was used to evaluate inhibition of microbial growth at various extract concentrations. Commercial antibiotics were used as positive reference standards to determine the sensitivity of the strains. Susceptibility testing to antibiotics was also tested using the disk diffusion method. The minimal inhibitory concentration values of the extracts, obtained by agar diffusion, ranged from 0.8 to 1.4 mg/ml. The results showed that extracts from hairy root cultures inhibited the growth of bacteria and yeast strains and suggest that they may be useful in the treatment of infections caused by pathogenic microorganisms.     

From Grazing Resistance to Pathogenesis: The Coincidental Evolution of Virulence Factors

To many pathogenic bacteria, human hosts are an evolutionary dead end. This begs the question what evolutionary forces have shaped their virulence traits. Why are these bacteria so virulent? The coincidental evolution hypothesis suggests that such virulence factors result from adaptation to other ecological niches. In particular, virulence traits in bacteria might result from selective pressure exerted by protozoan predator. Thus, grazing resistance may be an evolutionarily exaptation for bacterial pathogenicity. This hypothesis was tested by subjecting a well characterized collection of 31 Escherichia coli strains (human commensal or extra-intestinal pathogenic) to grazing by the social haploid amoeba Dictyostelium discoideum. We then assessed how resistance to grazing correlates with some bacterial traits, such as the presence of virulence genes. Whatever the relative population size (bacteria/amoeba) for a non-pathogenic bacteria strain, D. discoideum was able to phagocytise, digest and grow. In contrast, a pathogenic bacterium strain killed D. discoideum above a certain bacteria/amoeba population size. A plating assay was then carried out using the E. coli collection faced to the grazing of D. discoideum. E. coli strains carrying virulence genes such as iroN, irp2, fyuA involved in iron uptake, belonging to the B2 phylogenetic group and being virulent in a mouse model of septicaemia were resistant to the grazing from D. discoideum. Experimental proof of the key role of the irp gene in the grazing resistance was evidenced with a mutant strain lacking this gene. Such determinant of virulence may well be originally selected and (or) further maintained for their role in natural habitat: resistance to digestion by free-living protozoa, rather than for virulence per se.     

A strategy for mitigating avian colibacillosis disease using plant growth promoting rhizobacteria and green synthesized zinc oxide nanoparticles

Avian colibacillosis caused by the zoonotic pathogen Escherichia coli is a common bacterial infection that causes major losses in the poultry sector. Extracts of different medicinal plants and antibiotics have been used against poultry bacterial pathogens. However, overuse of antibiotics and extracts against pathogenic strains leads to the proliferation of multi-drug resistant bacteria. Due to their environmentally friendly nature, nanotechnology and beneficial bacterial strains can be used as effective strategies against poultry infections. Green synthesis of zinc oxide nanoparticles (ZnO-NPs) from Eucalyptus globulus leaves was carried out in this study. Their characterization was done by UV–vis spectroscopy, X-ray diffraction (XRD), and Fourier transmission infrared spectroscopy (FT-IR) which confirmed their synthesis, structure, and size. In vitro, antimicrobial activities of plant leaf extract, ZnO-NPs, and plant growth-promoting rhizobacteria (PGPR) were checked against E. coli using well diffusion as well as disc diffusion method. Results proved that the antimicrobial activity of ZnO-NPs and PGPR strains was more enhanced when compared to eucalyptus leaf extract at 36 h. The maximum relative inhibition shown by ZnO-NPs, PGPR strains and eucalyptus leaf extracts was 88%, 67% and 58%, respectively. The effectiveness of ZnO-NPs was also increased with an increase in particle dose and treatment time. The 90 mg/ml of ZnO-NPs was more effective. PGPR strains from all over the tested strains, Pseudomonas sp. (HY8N) exhibited a strong antagonism against the E. coli strain as compared to other PGPR strains used in this study. However, combined application of PGPR (Pseudomonas sp. (HY8N)) and ZnO-NPs augment antagonistic effects and showed maximum 69% antagonism. The study intends to investigate the binding affinity of ZnO-NPs with the suitable receptor of the bacterial pathogen by in silico methods. The binding site conformations showed that the ligand ZnO binds with conserved binding site of penicillin-binding protein 6 (PBP 6) receptor. According to the interactions, ZnO-NPs form the same interaction pattern with respect to other reported ligands, hence it can play a significant role in the inhibition of PBP 6. This research also found that combining ZnO-NPs with Pseudomonas sp. (HY8N) was a novel and effective technique for treating pathogenic bacteria, including multidrug-resistant bacteria.     

Administration of non-pathogenic isolates of Escherichia coli and Clostridium perfringens type A to piglets in a herd affected with a high incidence of neonatal diarrhoea

A bacterial cocktail of living strains of Clostridium perfringens type A (CPA) without β2-toxin gene and non-pathogenic Escherichia coli was administered orally to newborn piglets before first colostrum intake and on 2 consecutive days on a farm with a high incidence of diarrhoea and antibiotic treatment in suckling piglets associated with E. coli and CPA. This clinical field study was driven by the hypothetic principle of competitive exclusion of pathogenic bacteria due to prior colonization of the gut mucosal surface by non-pathogenic strains of the same bacterial species with the aim of preventing disease. Although CPA strains used in this study did not produce toxins in vitro, their lack of pathogenicity cannot be conclusively confirmed. The health status of the herd was impaired by a high incidence of postpartum dysgalactia syndrome in sows (70%) and a high incidence of neonatal diarrhoea caused by enterotoxigenic E. coli and CPA during the study. No obvious adverse effect of the bacterial treatment occurred. On average, more piglets were weaned in litters treated (P=0.009). Visual pathological alterations in the small intestinal wall were more frequent in dead piglets of the control group (P=0.004) and necrotizing enteritis was only found in that group. A higher average daily weight gain of piglets in the control group (P<0.001) may be due to an increased milk uptake due to less competition in the smaller litters. The bacterial cocktail was tested under field conditions for its potential to stabilize gut health status in suckling piglets before disease development due to colibacillosis and clostridial infections; however, the gut flora stabilizing effect of the bacterial cocktail was not clearly discernible in this study. Further basic research is needed to confirm the positive effects of the bacterial treatment used and to identify additional potential bacterial candidates for competitive exclusion.     

Small Heat-Shock Proteins,IbpAB, Protect Non-Pathogenic Escherichia coli from Killing by Macrophage-Derived Reactive Oxygen Species

Many intracellular bacterial pathogens possess virulence factors that prevent detection and killing by macrophages. However, similar virulence factors in non-pathogenic bacteria are less well-characterized and may contribute to the pathogenesis of chronic inflammatory conditions such as Crohn’s disease. We hypothesize that the small heat shock proteins IbpAB, which have previously been shown to reduce oxidative damage to proteins in vitro and be upregulated in luminal non-pathogenic Escherichia strain NC101 during experimental colitis in vivo, protect commensal E. coli from killing by macrophage-derived reactive oxygen species (ROS). Using real-time PCR, we measured ibpAB expression in commensal E. coli NC101 within wild-type (wt) and ROS-deficient (gp91phox^-/-) macrophages and in NC101 treated with the ROS generator paraquat. We also quantified survival of NC101 and isogenic mutants in wt and gp91phox^-/- macrophages using gentamicin protection assays. Similar assays were performed using a pathogenic E. coli strain O157:H7. We show that non-pathogenic E. coli NC101inside macrophages upregulate ibpAB within 2 hrs of phagocytosis in a ROS-dependent manner and that ibpAB protect E. coli from killing by macrophage-derived ROS. Moreover, we demonstrate that ROS-induced ibpAB expression is mediated by the small E. coli regulatory RNA, oxyS. IbpAB are not upregulated in pathogenic E. coli O157:H7 and do not affect its survival within macrophages. Together, these findings indicate that ibpAB may be novel virulence factors for certain non-pathogenic E. coli strains.     

A New Zebrafish Model of Oro-Intestinal Pathogen Colonization Reveals a Key Role for Adhesion in Protection by Probiotic Bacteria

The beneficial contribution of commensal bacteria to host health and homeostasis led to the concept that exogenous non-pathogenic bacteria called probiotics could be used to limit disease caused by pathogens. However, despite recent progress using gnotobiotic mammal and invertebrate models, mechanisms underlying protection afforded by commensal and probiotic bacteria against pathogens remain poorly understood. Here we developed a zebrafish model of controlled co-infection in which germ-free zebrafish raised on axenic living protozoa enabled the study of interactions between host and commensal and pathogenic bacteria. We screened enteric fish pathogens and identified Edwardsiella ictaluri as a virulent strain inducing a strong inflammatory response and rapid mortality in zebrafish larvae infected by the natural oro-intestinal route. Using mortality induced by infection as a phenotypic read-out, we pre-colonized zebrafish larvae with 37 potential probiotic bacterial strains and screened for survival upon E. ictaluri infection. We identified 3 robustly protective strains, including Vibrio parahaemolyticus and 2 Escherichia coli strains. We showed that the observed protective effect of E. coli was not correlated with a reduced host inflammatory response, nor with the release of biocidal molecules by protective bacteria, but rather with the presence of specific adhesion factors such as F pili that promote the emergence of probiotic bacteria in zebrafish larvae. Our study therefore provides new insights into the molecular events underlying the probiotic effect and constitutes a potentially high-throughput in vivo approach to the study of the molecular basis of pathogen exclusion in a relevant model of vertebrate oro-intestinal infection.     

Response of bacteria to earthworm surface excreta

Response of bacteria to the surface excreta of the Aporrectodea caliginosa earthworm was studied. The excreta were obtained by a 1 h incubation of the earthworms in petri dishes with subsequent collection of the slime. Both inhibition and stimulation of growth were revealed, as well as suppression of the respiratory activity of some bacterial species treated with A. caliginosa surface excreta. The organisms studied included various taxa of soil bacteria (19 strains), bacteria isolated from A. caliginosa intestine and excrements (82 strain), and 48 Bacillus thuringiensis strains. For the cultures of soil bacteria, the respiratory activity was determined using the formazan color reaction due to the activity of the respiratory cycle enzymes. Earthworm excreta caused a consistent 30–50% decrease of dehydrogenase activity in 13 out of the 19 cultures. Determination of the growth rates (derived from OD₆₂₀ of cell suspensions) after 10 h of incubation revealed growth stimulation in 48 out of the 82 strains isolated from intestines and excrement. Other strains exhibited no reaction to the excreta. For 29 out of 45 B. thuringiensis strains, growth stimulation was observed, while growth of two strains was suppressed; other strains exhibited no reaction to the excreta. No relation was found between bacterial reaction to the excreta and their taxonomic position. These results correlate with the research, demonstrating antibacterial and antifungal activity of the extracts from the earthworm body and digestive tract. Thus, earthworms, apart from their medium-forming function, affect the formation of soil microbial communities by direct stimulation or suppression of specific microbial populations.     

Analysis of anti-bacterial and anti oxidative activity of Azadirachta indica bark using various solvents extracts

Herbal medications have been used for relief of symptoms of disease. Regardless of the great advances observed in current medicine in recent decades, plants still make a significant contribution to health care. An alarming increase in bacterial strains resistant to a number of antimicrobial agents demands that a renewed effort be made to seek antibacterial agents effective against pathogenic bacteria resistant to or less sensitive to current antibiotics. Anti-bacterial activity of Azadirachta indica stem bark was tested against pathogenic Salmonella paratyphi and Salmonella typhi using various solvent extracts. The in vitro anti-bacterial activity was performed by agar well diffusion method and the results were expressed as the average diameter of zone of inhibition of bacterial growth around the well. The ethanol and methanol extracts showed better anti-bacterial activity with zone of inhibition (20–25 mm) when compared with other tested extracts and standard antibiotic Erythromycin (15 mcg) with zone of inhibition (13–14 mm). Using Fisher’s exact test of significance difference was found between two Salmonella strains sensitivity patterns against tested extracts (P ⩽ 0.035). Extracts of A. indica stem bark also exhibited significant antioxidant activity, thus establishing the extracts as an antioxidant. The results obtained in this study give some scientific support to the A. indica stem bark for further investigation of compounds and in future could be used as drug.     

Complete Genome Sequence and Comparative Analysis of the Wild-type Commensal Escherichia coli Strain SE11 Isolated from a Healthy Adult

We sequenced and analyzed the genome of a commensal Escherichia coli (E. coli) strain SE11 (O152:H28) recently isolated from feces of a healthy adult and classified into E. coli phylogenetic group B1. SE11 harbored a 4.8 Mb chromosome encoding 4679 protein-coding genes and six plasmids encoding 323 protein-coding genes. None of the SE11 genes had sequence similarity to known genes encoding phage- and plasmid-borne virulence factors found in pathogenic E. coli strains. The comparative genome analysis with the laboratory strain K-12 MG1655 identified 62 poorly conserved genes between these two non-pathogenic strains and 1186 genes absent in MG1655. These genes in SE11 were mostly encoded in large insertion regions on the chromosome or in the plasmids, and were notably abundant in genes of fimbriae and autotransporters, which are cell surface appendages that largely contribute to the adherence ability of bacteria to host cells and bacterial conjugation. These data suggest that SE11 may have evolved to acquire and accumulate the functions advantageous for stable colonization of intestinal cells, and that the adhesion-associated functions are important for the commensality of E. coli in human gut habitat.Key words: Escherichia coli, commensal, human gut, genome sequencing     

Relationship of periodontal clinical parameters with bacterial composition in human dental plaque

More than 600 bacterial species have been identified in the oral cavity, but only a limited number of species show a strong association with periodontitis. The purpose of the present study was to provide a comprehensive outline of the microbiota in dental plaque related to periodontal status. Dental plaque from 90 subjects was sampled, and the subjects were clustered based on bacterial composition using the terminal restriction fragment length polymorphism of 16S rRNA genes. Here, we evaluated (1) periodontal clinical parameters between clusters; (2) the correlation of subgingival bacterial composition with supragingival bacterial composition; and (3) the association between bacterial interspecies in dental plaque using a graphical Gaussian model. Cluster 1 (C1) having high prevalence of pathogenic bacteria in subgingival plaque showed increasing values of the parameters. The values of the parameters in Cluster 2a (C2a) having high prevalence of non-pathogenic bacteria were markedly lower than those in C1. A cluster having low prevalence of non-pathogenic bacteria in supragingival plaque showed increasing values of the parameters. The bacterial patterns between subgingival plaque and supragingival plaque were significantly correlated. Chief pathogens, such as Porphyromonas gingivalis, formed a network with other pathogenic species in C1, whereas a network of non-pathogenic species, such as Rothia sp. and Lautropia sp., tended to compete with a network of pathogenic species in C2a. Periodontal status relates to non-pathogenic species as well as to pathogenic species, suggesting that the bacterial interspecies connection affects dental plaque virulence.     

Catecholamine induced growth of gram negative bacteria.

The addition of various catecholamines to cultures of gram negative bacteria resulted in dramatic increases in growth. The ability of norepinephrine, epinephrine, dopamine and dopa to enhance the growth of Escherichia coli, Yersinia enterocolitica and Pseudomonas aeruginosa was observed to be dependent on the bacterium employed with each strain showing marked preference for one or more of the catecholamines. Catecholamine induced increases in growth were confirmed by one or more of the following methods: uptake of tritiated thymidine into newly synthesized DNA, changes in optical density or pour plate analysis. None of the catecholamine metabolites resulting from either oxidative deamination or catechol-O-methylation were able to effect similar increases in bacterial growth as compared to either norepinephrine, epinephrine or dopamine. Norepinephrine was consistently observed to effect the greatest increase in bacterial growth for all strains tested.     

An evaluation of the antimicrobial properties of the eggs of 11 species of scleractinian corals

Potential sources of mortality of marine invertebrate larvae are numerous and include predation and diseases caused by marine microorganisms. Extracts from the eggs of 11 coral species were evaluated for their ability to deter surface attachment and inhibit the growth of two marine tolerant laboratory bacteria and 92 bacterial strains isolated from seawater and the surface of coral colonies on the Great Barrier Reef (GBR). Extracts of the eggs of Montipora digitata inhibited the growth of the two laboratory bacteria, Vibrio harveyii and Bacillus subtilis, and one bacterial isolate from the mucus of the coral Favia pallida in disc diffusion and liquid culture assays. No other microbial strains (n=91) from the surface of corals and the reef environment were inhibited by M. digitata extracts. No antibacterial activity was found in the egg extracts of the remaining ten coral species and none of the extracts inhibited surface attachment of various bacteria. Extrapolation of estimated surface concentrations of the biologically active extract of M. digitata suggests that the level of the growth inhibitory compounds may be sufficient to deter microbial growth in situ.     

Significance of Fecal Coliform-Positive Klebsiella

A total of 191 Klebsiella pneumoniae isolates of human clinical, bovine mastitis, and a wide variety of environmental sources were tested for fecal coliform (FC) response with the membrane filtration and most probable number techniques. Twenty-seven Escherichia coli cultures of human clinical and environmental origins were also tested. Eighty-five percent (49/58) of known pathogenic K. pneumoniae were FC positive, compared with 16% (19/120) of the environmental strains. E. coli results indicated 93% (13/14) of the clinical and 85% (11/13) of the environmental strains as FC positive. There was no significant difference in the incidence of FC-positive cultures between pathogenic Klebsiella and E. coli. pH measurements of K. pneumoniae and E. coli cultures growing in m-FC broth at 44.5°C revealed three distinct pH ranges correlating with colony morphology. β-Galactosidase assays of Klebsiella and E. coli cultures at 44.5°C indicated all were able to hydrolyze lactose, even if they were FC negative by the membrane filtration or most probable number techniques. The FC response pattern appears stable in K. pneumoniae. Three pathogenic cultures showed no change in FC responses after 270 generations of growth in sterile pulp mill effluent. Since K. pneumoniae is carried in the gastrointestinal tract of humans and animals and 85% of the tested pathogenic strains were FC positive, the isolation of FC-positive Klebsiella organisms from the environment would indicate their fecal or clinical origin or both. The added fact that K. pneumoniae is an opportunistic pathogen of increasing importance makes the occurrence of FC-positive environmental Klebsiella, particularly in large numbers, a potential human and animal health hazard.     

Retention in Treated Wastewater Affects Survival and Deposition of Staphylococcus aureus and Escherichia coli in Sand Columns

The fate and transport of pathogenic bacteria from wastewater treatment facilities in the Earth''s subsurface have attracted extensive concern over recent decades, while the impact of treated-wastewater chemistry on bacterial viability and transport behavior remains unclear. The influence of retention time in effluent from a full-scale municipal wastewater treatment plant on the survival and deposition of Staphylococcus aureus and Escherichia coli strains in sand columns was investigated in this paper. In comparison to the bacteria cultivated in nutrient-rich growth media, retention in treated wastewater significantly reduced the viability of all strains. Bacterial surface properties, e.g., zeta potential, hydrophobicity, and surface charges, varied dramatically in treated wastewater, though no universal trend was found for different strains. Retention in treated wastewater effluent resulted in changes in bacterial deposition in sand columns. Longer retention periods in treated wastewater decreased bacterial deposition rates for the strains evaluated and elevated the transport potential in sand columns. We suggest that the wastewater quality should be taken into account in estimating the fate of pathogenic bacteria discharged from wastewater treatment facilities and the risks they pose in the aquatic environment.     

2,4-Dihydroxy-7-methoxy-2H-1,4-benzoxazin-3(4H)-one, an Inhibitor from Zea mays with Differential Activity against Soft Rotting Erwinia Species

[2,4-Dihydroxy-7-methoxy-2H-1,4-benzoxazin-3(4H)-one] DIMBOA was extracted with ethyl acetate from acidified water homogenates of corn (Zea mays L.) seedlings. Pure DIMBOA or ethyl acetate extracts of corn tissue were added to bacterial growth medium at five concentrations (measured as hydroxamates). DIMBOA and corn extracts were more inhibitory to soft rot bacteria (Erwinia spp.) that are nonpathogenic to corn than to soft rot bacteria that are corn pathogens. The inhibitory activity of DIMBOA was similar to that of the ethyl acetate extracts. Both corn extracts and DIMBOA prolonged the lag phase of bacterial growth without significantly changing log phase growth rates. At various concentrations of the inhibitor, 50 to 100% of the activity of corn extracts inhibitory to different bacterial isolates was attributable to DIMBOA. Extracts of DIMBOA-deficient plants (genotype bxbx) were not inhibitory to Erwinia spp. It was concluded that DIMBOA is the major active component in those corn extracts which are inhibitory to soft rot Erwinia species.     

Immune reactions of Chironomus larvae (Insecta: Diptera) against bacteria

Humoral encapsulation (“melanization”) represents the predominant defence reaction of Chironomus larvae against injected bacteria. Only low levels of phagocytic activity were observed; cellular encapsulation and nodule formation were completely missing due to low numbers of haemocytes. No other humoral antibacterial activity was detected in normal Chironomus haemolymph and even haemolymph of preinjected (“immunized”) Chironomus larvae showed little inhibition of bacterial growth on agar test plates.Low cellular and lytic activity of Chironomus haemolymph against bacteria is well compensated for by its fast and efficient capacity of humoral encapsulation. Within 5–10 min, even high numbers of injected bacteria (up to 10⁵ per larva) were surrounded by capsular material. Within this range of injection dose, the fates of pathogenic and non-pathogenic strains were identical, and bacteria which are highly pathogenic for many other insects, e.g. larvae of Galleria mellonella, proved to be harmless to Chironomus larvae. The rapidity of humoral encapsulation may prevent the release of toxins or enzymes by which pathogenic bacteria normally damage its host and weaken its immune system.     

Utilization of agro-industrial orange peel and sugar beet pulp wastes for fungal endo- polygalacturonase production

The pectinase enzymes are involved in several industrial applications, and industrial waste is one of the largest environmental pollutants, so this study aims to Endo-polygalacturonase (endo-PG) producing using Aspergillus niger AUMC 4156, Penicillium oxalicum AUMC 4153 and P. variotii AUMC 4149 by using some agro-industrial wastes (dried orange peel and sugar beet pulp) as a sole raw carbon source for degradation these waste in the process of urban wastes disposal. The fermentation process was carried out as a submerged culture technique under both shaken and static culture conditions. A. niger AUMC 4156 was the most promising producer of endo-PG under static conditions while P. oxalicum AUMC 4153 was the highest producer of endo-PG under shaken conditions. Sugar beet pulp proved to be the most preferable to orange peel as the only source of carbon in both shaken and static cultures. The medium that encompassing orange peel as a single carbon source afforded the highest protein content with all tested fungal strains in stirred and static cultures in comparison with sugar beet pulp. The highest activity of endo-polygalacuronase that produced using A. niger AUMC 4156 and P. oxalicum AUMC 4153 was achieved by using sugar beet pulp at 3% concentration under static cultures, meanwhile maximal enzyme activity produced by both fungal strains required 2% sugar beet pulp under shaken cultures. Sugar beet pulp showed promised potential as a good inducer for endo-polygalacturoase production, and enzymes production depended on fungal strains, culture medium, and submerged fermentation conditions.     

Prevalence of antibiotic resistant mastitis pathogens in dairy cows in Egypt and potential biological control agents produced from plant endophytic actinobacteria

Dairy production is threatened by antibiotic resistant pathogens worldwide, and alternative solutions to treat mastitis are not available. The prevalence of antibiotic resistant strains is not well known in less developed countries. The prevalence of pathogenic bacteria and their resistance to 21 commercial antibiotics were studied in milk samples taken from 122 dairy cows suffering from the symptoms of mastitis in Egypt. The bacterial species were identified with molecular methods, and antibiotic resistance was studied with disc diffusion method. The prevalence of Streptococcus aureus, Escherichia coli and Pseudomonas aeruginosa were 30%, 17% and 3.5%, respectively. Most (90%) of the S. aureus strains showed resistance to penicillin whereas only 10% of the strains were resistant to oxacillin. Nearly half (40%) of E. coli strains showed resistance to streptomycin. Six P. aeruginosa strains showed resistance to several antibiotics, including ceftriaxone, enrofloxacin and levofloxacin. This points out that despite P. aeruginosa was not common, it should be followed up carefully. Potential biocontrol agents against antibiotic resistant mastitis bacteria were searched among 30 endophytic actinobacterial strains derived from wild medicinal plants. Three plants, namely Mentha longifolia, Malva parviflora and Pulicaria undulata were chosen for a more detailed study; their endophytic actinobacteria were used to prepare metabolic extracts. The crude metabolites of the actinobacteria were extracted with ethyl acetate. All metabolic extracts inhibited the growth of S. aureus, methicillin-resistant Staphylococcus aureus (MRSA), E. coli and P. aeruginosa in vitro. The 16S rRNA sequence analysis revealed that the most efficient actinobacterial strains were two Micromonospora sp. and one Actinobacteria bacterium. We conclude that the combination of the metabolites of several endophytic actinobacteria derived from several medicinal plants would be the most efficient against pathogens. Different metabolite cocktails should be studied further in order to develop novel biocontrol agents to treat antibiotic resistant mastitis bacteria in dairy cows.