Improvement of Zn (II) and Cd (II) Biosorption by Priestia megaterium PRJNA526404 Isolated from Agricultural Waste Water.

Heavy metals are considered as dangerous pollutants even in relatively low concentrations. Biosorption is an ecofriendly technology that uses microbial biomasses for adsorbing heavy metals from wastewater on their surfaces based on physicochemical pathways. Ten agricultural wastewater samples were collected from different sites in Sohag Governorate, Egypt. One hundred and nineteen zinc and cadmium-resistant bacterial isolates were recovered from the water samples. Interestingly, the isolate R1 was selected as the most resistant to Zn2+ and Cd2+. This isolate was morphologically and biochemically characterized and identified by sequencing of 16S rRNA gene as Priestia megaterium, and then deposited in the GenBank database under the accession number PRJNA526404. Studying the effects of pH and contact time on the biosorption process revealed that the maximum biosorption was achieved within 50 min at pH 7 and 8 for Zn2+ and Cd2+, respectively, by the living and lyophelized biomass of Priestia megaterium PRJNA526404. The preliminary characterization of the main chemical groups present on the cell wall, which are responsible for heavy metal biosorption, was performed by Infrared analysis (IR). Kinetics studies revealed that data were fitted towards the models hypothesized by Langmuir and Freundlich isotherm equations. The maximum capacity values (qmax) for biosorption of zinc and cadmium reached by using living and lyophelized biomass were 196.08; 227.27 and 178.57; 212.777 mg/g, respectively, and it was indicated that lyophilization improved efficiency of the biomass to heavy metals compared to living cells. The results indicated that Priestia megaterium PRJNA526404 had good application prospect in cadmium and zinc water remediation.

Prevalence and Molecular Characterization of Methicillin-Resistant Staphylococcus aureus from Nasal Specimens: Overcoming MRSA with Silver Nanoparticles and Their Applications.

Staphylococcus aureus is a cause of high mortality in humans and therefore it is necessary to prevent its transmission and reduce infections. Our goals in this research were to investigate the frequency of methicillin-resistant S. aureus (MRSA) in Taif, Saudi Arabia, and assess the relationship between the phenotypic antimicrobial sensitivity patterns and the genes responsible for resistance. In addition, we examined the antimicrobial efficiency and application of silver nanoparticles (AgNPs) against MRSA isolates. Seventy-two nasal swabs were taken from patients; MRSA was cultivated on Mannitol Salt Agar supplemented with methicillin, and 16S rRNA sequencing was conducted in addition to morphological and biochemical identification. Specific resistance genes such as ermAC, aacA-aphD, tetKM, vatABC and mecA were PCR-amplified and resistance plasmids were also investigated. The MRSA incidence was ~49 % among the 72 S. aureus isolates and all MRSA strains were resistant to oxacillin, penicillin, and cefoxitin. However, vancomycin, linezolid, teicoplanin, mupirocin, and rifampicin were effective against 100% of MRSA strains. About 61% of MRSA strains exhibited multidrug resistance and were resistant to 3-12 antimicrobial medications (MDR). Methicillin resistance gene mecA was presented in all MDR-MRSA strains. Most MDR-MRSA contained a plasmid of > 10 kb. To overcome bacterial resistance, AgNPs were applied and displayed high antimicrobial activity and synergistic effect with penicillin. Our findings may help establish programs to control bacterial spread in communities as AgNPs appeared to exert a synergistic effect with penicillin to control bacterial resistance.

Biogenic Synthesis of ZnO Nanoparticles and Its Potential Use as Antimicrobial Agent Against Multidrug-Resistant Pathogens.

In case of Escherichia coli and Klebsiella pneumoniae infection, the increased prominence of multidrug-resistance strains has become the greatest challenge in the urinary tract disease treatment. Therefore, the 16S rRNA sequencing of multidrug-resistant strains was performed, in addition to those of plasmids and genes responsible for multidrug resistance. These strains showed containing responsible genes Sulfonamides sul1, Tetracycline Tet(A), Tetracycline Tet(B), chloramphenicol catA1, ß-lactams blaSHV, and cmlA. Also, the strains demonstrated resistance to at least 10 types of antibiotics or more due to carrying various plasmids. For increasing the level of public health in daily life and treatment of multidrug-resistant bacteria, the nanomedicine was employed. Consequently, ZnO nanoparticles (ZnONPs-E) were synthesized by employing supernatant of Escherichia hermannii strain isolated from raw milk source. The E. hermannii strain produces high concentration of ZnONPs-E compared to other strains so we used it in this study. This ZnONPs-E has a minimal inhibitory concentration (MIC) ranged from the concentration 10 µg/ml to 40 µg/ml against E. coli and K. pneumoniae, respectively. The antimicrobial efficiency of ZnONPs-E was 40 µg/ml and it was superior to the reported values in literature. Moreover, SEM results evident for distorted membrane morphology, blebbing of membrane, cell elongation, and leakage of cellular contents due to ZnONPs-E activity against tested bacteria. These results indicated that the ZnONPs-E exhibited interesting antimicrobial activity against pathogenic extended-spectrum ß-lactamases (ESBLs) strains. The present study revealed that the active components entered in biosynthesis of ZnONPs-E pave the way to lead its effective nano-medical and drug delivery applications.

Isolation and antimicrobial resistance of Escherichia coli isolated from farm chickens in Taif, Saudi Arabia.

OBJECTIVES: Poultry is one of the main sources of food in the world. Antimicrobial-resistant Escherichia coli can be transmitted to humans by contact with poultry waste or by contaminated poultry products, contributing to the increasing crisis of antimicrobial resistance. This study aimed to determine the incidence of antimicrobial resistance in E. coli isolated from chickens in Taif province, Saudi Arabia, and to identify the genes responsible for any resistance observed. METHODS: A total of 150 cloacal swabs were aseptically obtained from chickens from different farms, from which 180 colonies of E. coli were identified using standard microbiology procedures. Antimicrobial susceptibility testing was performed by the Kirby-Bauer disk diffusion method. The genes blaSHV, aac(3)-IV, tet(A), tet(B), aadA1, catA1, cmlA, ere(A) and sul1 were detected by PCR. RESULTS: Most of the E. coli isolates showed resistance to oxacillin (99%), lincomycin (98%) and oxytetracycline (97%). The prevalence of resistance to chloramphenicol (73%), ciprofloxacin (59%) and ampicillin (51%) was lower. Genes conferring resistance to ß-lactams (blaSHV) and tetracyclines [tet(A) and tet(B)] were observed at prevalences of 96% and 95%, respectively, among the E. coli isolates. Chloramphenicol (catA1 and cmlA) and erythromycin [ere(A)] resistance genes showed prevalences of 72% and 15%, respectively, whereas gentamicin [aac(3)-IV], streptomycin (aadA1) and sulfonamide (sul1) resistance genes were detected in 20%, 20% and 10% of the studied isolates, respectively. CONCLUSION: A significant prevalence of antimicrobial resistance genes was observed among E. coli isolates from farm chickens, supporting strict regulatory procedures for the use of antimicrobial agents.

Role of wild birds as carriers of multi-drug resistant Escherichia coli and Escherichia vulneris

Emergence and distribution of multi-drug resistant (MDR) bacteria in environments pose a risk to human and animal health. A total of 82 isolates of Escherichia spp. were recovered from cloacal swabs of migrating and non-migrating wild birds. All bacterial isolates were identified and characterized morphologically and biochemically. 72% and 50% of isolates recovered from non-migrating and migrating birds, respectively, showed positive congo red dye binding (a virulence factor). Also, hemolysin production (a virulence factor) was showed in 8% of isolates recovered from non-migrating birds and 75% of isolates recovered from migrating birds. All isolates recovered from non-migrating birds were found resistant to Oxacillin while all isolates recovered from migrating birds demonstrated resistance to Oxacillin, Chloramphenicol, Oxytetracycline and Lincomycin. Some bacterial isolates recovered from non-migrating birds and migrating birds exhibited MDR phenotype. The MDR isolates were further characterized by API 20E and 16S rRNA as E. coli and E. vulneris. MDR Escherichia isolates contain ~1-5 plasmids of high-molecular weights. Accordingly, wild birds could create a potential threat to human and animal health by transmitting MDR bacteria to water streams and other environmental sources through their faecal residues, and to remote regions by migration.

Role of wild birds as carriers of multi-drug resistant Escherichia coli and Escherichia vulneris.

Emergence and distribution of multi-drug resistant (MDR) bacteria in environments pose a risk to human and animal health. A total of 82 isolates of Escherichia spp. were recovered from cloacal swabs of migrating and non-migrating wild birds. All bacterial isolates were identified and characterized morphologically and biochemically. 72% and 50% of isolates recovered from non-migrating and migrating birds, respectively, showed positive congo red dye binding (a virulence factor). Also, hemolysin production (a virulence factor) was showed in 8% of isolates recovered from non-migrating birds and 75% of isolates recovered from migrating birds. All isolates recovered from non-migrating birds were found resistant to Oxacillin while all isolates recovered from migrating birds demonstrated resistance to Oxacillin, Chloramphenicol, Oxytetracycline and Lincomycin. Some bacterial isolates recovered from non-migrating birds and migrating birds exhibited MDR phenotype. The MDR isolates were further characterized by API 20E and 16S rRNA as E. coli and E. vulneris. MDR Escherichia isolates contain ~1-5 plasmids of high-molecular weights. Accordingly, wild birds could create a potential threat to human and animal health by transmitting MDR bacteria to water streams and other environmental sources through their faecal residues, and to remote regions by migration.

Biosorption of aluminum, cobalt, and copper ions by Providencia rettgeri isolated from wastewater.

Twenty-three bacterial isolates from polluted water and soil were screened for heavy metals resistance (i.e., Al(3+), Co(2+), and Cu(2+)). The most potent isolate was identified by morphological characteristics, biochemical tests and confirmed by API20E kits as Providencia rettgeri MAM-4. Removal of Al(3+) from aqueous solution by P. rettgeri is more efficient (∼fourfold) than that by B. cereus ATCC 11778 (a comparison strain) at concentration of 200 mg L(-1) Al(3+). P. rettgeri was able to remove Co(2+) more than B. cereus ATCC 11778 at concentration of 50 mg L(-1) Co(2+). Inoculation of P. rettgeri into clay enhanced significantly the removal of Al(3+), Co(2+), and Cu(2+). P. rettegri MI (mutant strain) was able to tolerate more Al(3+) than that of the parent strain. P. rettgeri was resistant to 7 out of 15 antibiotics tested. P. rettgeri MAM-4 isolated from wastewater had ability to remove Al(3+), Co(2+), and Cu(2+) efficiently from aqueous media; and enhanced significantly metal biosporption by clay. This study has revealed that P. rettgeri could be employed as an effective and economic technology for the removal such metal elements from polluted environment.

Characterization of a strain of Pseudomonas putida isolated from agricultural soil that degrades cadusafos (an organophosphorus pesticide).

Bacteria capable of degrading the pesticide, cadusafos, were isolated from agricultural soil using an enrichment method. In this way, five distinct cadusafos-degrading strains of Pseudomonas putidia were isolated, and were characterized using morphological and biochemical analysis, as well as 16S rRNA sequencing. Strain PC1 exhibited the greatest cadusafos degradation rate and was consequently selected for further investigation. Degradation of cadusafos by strain PC1 was rapid at 20 and 37°C, but was greatly reduced (~1.5-fold) by the presence of carbon sources. Strain PC1 was able to effectively degrade cadusafos in sterilized soil using low inoculum levels. The maximum degradation rate of cadusafos (V ( max )) was calculated as 1.1 mg l(-1 )day(-1), and its saturation constant (K ( s )) was determined as 2.5 mg l(-1). Bacteria such as strain PC1, that use cadusafos as a carbon source, could be employed for the bioremediation of sites contaminated with pesticides.

Isolation and characterization of heavy-metal resistant microbes from roadside soil and phylloplane.

Contamination by heavy metals is one of the major environmental problems in many countries and these contaminants reach from various sources such as traffic cars and other activities. Soil and phylloplane samples were collected from eight traffic and two non-traffic sites in Sohag city, Egypt. Heavy metal contents of Cd²âº, Zn²âº and Pb²âº of soil and phylloplane samples were determined and revealed high levels of Zn²âº and Pb²âº in traffic samples. A total of 112 bacterial and 62 fungal isolates were obtained from soil and phylloplane. Bacterial isolates were characterized on the basis of morphological, physicochemical and biochemical characteristics; and 16S rRNA gene sequences. Fungal isolates were identified according to morphological characterization. Minimal inhibitory concentrations (MICs) of Cd²âº, Zn²âº and Pb²âº for each isolate were detected. All bacterial and fungal isolates demonstrated resistance to lead with MICs >0.528 mM and >0.211, respectively. Moreover, the maximum MICs of cadmium and zinc for bacteria were 0.821 mM and 1.471 mM, respectively, where as, MICs for fungi were 0.328 mM and 0.588 mM, respectively. The most resistant bacterial and fungal isolates were Pseudomonas aeruginosa RA65 and Penicillium corylophyllum, respectively. Therefore, P. aeruginosa RA65 was selected for further investigations. Growth curve study showed that 0.264 mM lead had no efficiently effect on the growth of P. aeruginosa RA65. Plasmid isolation evidenced by transformation studies indicated that P. aeruginosa RA65 harbored a single plasmid (~9.5 kb) which mediated heavy meal resistance. Consequently, these microbial isolates could be potentially used in bioremediation of heavy metal-contaminated environment.

Molecular approach and bacterial quality of drinking water of urban and rural communities in Egypt.

Water is necessary to life so when supplied as drinking water to consumers, a satisfactory quality must be maintained. In Egypt, infectious intestinal diseases are the major cause of hospitalization in almost all regions. The purpose of this study was to evaluate the microbiological quality of treated and untreated water samples from urban and rural communities. Thirty-five samples of treated (chlorinated) water from taps, 25 samples of bottled water and 15 samples of hand pump (untreated) water collected from different cities alongside the River Nile during the winter of 2007 were bacteriologically tested for safety as drinking water. This study indicated good quality of tap water and bottled water. The untreated water samples (hand pumps) were, however, slightly contaminated by faecal coliforms, faecal enterococci, Clostridium perfringens, Salmonella and Shigella. Consequently, the consumers in the villages receiving water through hand pumps are often exposed to the risk of water-borne diseases due to inadequate treatment of the raw water. Therefore, there are guidelines necessary to protect groundwater quality. Moreover, PCR-amplified by some functional gene fragments such as dctA, dcuB, frdA, dcuS and dcuR genes of the E. coli was adapted for use as a non-cultivation-based molecular approach for detection of E. coli populations from water samples without the need for pure and identified cultures.

Optimization of bacteriocin production by Lactobacillus plantarum AA135.

Growth of Lactobacillus plantarum AA135 in MRS broth at 30 degrees C yielded maximal plantaricin AA13 activity of 10000 AU/ml at pH 3.8 after 16 hours. Maximal activity (10000 AU/ml) of plantaricin AA13 was recorded in MRS broth at initial pH of 5 or 5.5. Optimal production (10000 AU/ml) was recorded in the presence of tryptone (20 g/l), a combination of tryptone plus meat extract (1:0.6) or tryptone with yeast extract (1:0.6) as sole nitrogen source. Growth of Lactobacillus plantarum AA135 in the presence of 20 g/l glucose yielded bacteriocin activity of 10000 AU/ml. Moreover, when 40 g/l glucose was added, the plantaricin AA13 levels doubled to 20000 AU/ml. Concentrations of 5, 10 and 20 g/l K2HPO4 lowered bacteriocin activity by 60%. Supplementing MRS with 1 g/l or more glycerol repressed bacteriocin production. Maximal activity of 20000 AU/ml was recorded in MRS supplemented with Vitamin DL-6,8-thioctic acid.

Molecular characterization of antimicrobial compound produced by Lactobacillus acidophilus AA11.

Approximately 63 strains of Lactobacillus acidophilus were isolated from Egyptian home-made cheese and examined for production of antagonism. Only eight strains demonstrated inhibitory activity against spoilage microorganisms (i.e. Staphylococcus aureus and Bacillus cereus) and pathogens (i.e. E. coli, Salmonella sp. and Shigella sp.). Lactobacillus acidophilus AA11 produced higher antimicrobial activity with a wide range of inhibition. The agent AA11 was sensitive to proteolytic enzymes and retained full activity after 30 min at 100 degrees C. Activity against sensitive cells was bactericidal but not bacteriolytic. The compound was produced during growth phase and could be extracted from the culture supernatant fluids with n-butanol. 12% SDS-PAGE analysis of 40% ammonium sulphate precipitated agent showed two peptides with molecular weights of approximately 36 kDa and approximately 29 kDa. No plasmid was identified in Lactobacillus acidophilus AA11 indicating that the genes encoding the inhibitory agent were located on the chromosome. These characteristics identify the inhibitory substance as a bacteriocin, designated acidocin AA11 and confer the agent an application potential as a biopreservative.

Involvement of chromosomally-encoded genes in malathion utilization by Pseudomonas aeruginosa AA112.

Malathion is an organophosphate insecticide that has been widely used for both domestic and commercial agricultural purposes. However, malathion has the potential to produce toxic effects in mammalian systems. In this study, Pseudomonas aeruginosa AA 112 which was isolated from soil using enrichment technique could utilize the malathion as a sole carbon source and a source of energy. Pseudomonas aeruginosa AA112 was able to grow in MSMPY medium containing 42.75 mg/ml malathion. However, the optimum concentration of malathion which supported the maximum bacterial growth was found to be 22. 8 mg/ml. Malathion was used as an initial source of energy and carbon when it was found without additional carbon sources (in MSM medium) while it was utilized as second source of energy and carbon in a nutrient-supplemented medium (in MSMPY medium). Moreover, lead acetate test indicated that malathion was first attacked at a sulphur site 1-2 hours after the start of incubation. TLC and IR analysis indicated that malathion was completely degraded into diethyl succinate, hydrogen sulphide and phosphates. Therefore a malathion degradation pathway was proporsed. The degradation of malathion is attributed to the genes located on the chromosome and at least three proteins of high molecular size might be involved in malathion utilization. Bacteria able to use malathion as a food source or metabolize its residues in the environment to inactive, less toxic, and harmless compounds, could be used in bioremediation of an environmental pollution caused by the pesticide.

Chromosomal-gene-mediated inhibition of intestinal and foodborne pathogens by Lactobacillus acidophilus AA11.

Approximately 63 strains of Lactobacillus acidophilus were isolated from Egyptian home-made cheese and examined for production of antagonism. Only eight strains demonstrated inhibitory activity against spoilage microorganisms (i.e. Staphylococcus aureus and Bacillus cereus) and pathogens (i.e. E. coli, Salmonella sp. and Shigella sp.). Lactobacillus acidophilus AA11 produced a more antimicrobial activity with a wide range of inhibition. The agent AA11 was sensitive to proteolytic enzymes and retained full activity after 30 min at 100 degrees C. Activity against sensitive cells was bactericidal but not bacteriolytic. The compound was produced during growth phase and can be extracted from the culture supernatant fluids with n-Butanol. 12 % SDS-PAGE analysis of 40% ammonium sulphate precipitated agent showed two peptides with molecular weights of approximately 36 kDa and approximately 29 kDa. No plasmid was identified in Lactobacillus acidophilus AA11 indicating that the genes encoding the inhibitory agent located on the chromosome. These characteristics identify the inhibitory substance as a bacteriocin, designated acidocin AA11 and confer the agent an application potential as a biopreservative.

Heavy metals resistant plasmid-mediated utilization of solar by Pseudomonas aeruginosa AA301.

Solar-degrading bacteria, Pseudomonas aeruginosa strains, were isolated from Egyptian soil by Mineral Salt Medium (MSM) supplemented with Solar (motor fuel) from different oil-contaminated sites in Sohag province. The strain AA301 of Pseudomonas aeruginosa showed appreciable growth in MSM medium containing high concentrations of Solar ranging from 0.5 to 3% (v/v), with optimum concentration at 1.5%. Solar was used as a sole carbon source and a source of energy by the bacterium. The ability to degrade Solar was found to be associated with a single 60-kb plasmid designated pSOL15. The plasmid-cured variant, which was obtained by culturing in LB broth with kanamycin, lost the plasmid indicative the ability to degrade Solar must depend on this plasmid. The wild type isolate, Pseudomonas aeruginosa AA301 and transformant strain, have maximum growth (OD600 = approximately 2) on Solar, however the plasmid-cured variant did not have any significant growth on Solar. Moreover, resistance to a wide range of heavy metals such as Mn2+, Hg2+, Mg2+, Cd2+, Zn2+, and Ni2+ was also 60-kb plasmid-mediated. Therefore, the strain AA301 could be good candidate for remediation of some heavy metals and oil hydrocarbons in heavily polluted sites.

DNA interaction and phosphotransfer of the C4-dicarboxylate-responsive DcuS-DcuR two-component regulatory system from Escherichia coli.

The DcuS-DcuR system of Escherichia coli is a two-component sensor-regulator that controls gene expression in response to external C(4)-dicarboxylates and citrate. The DcuS protein is particularly interesting since it contains two PAS domains, namely a periplasmic C(4)-dicarboxylate-sensing PAS domain (PASp) and a cytosolic PAS domain (PASc) of uncertain function. For a study of the role of the PASc domain, three different fragments of DcuS were overproduced and examined: they were PASc-kinase, PASc, and kinase. The two kinase-domain-containing fragments were autophosphorylated by [gamma-(32)P]ATP. The rate was not affected by fumarate or succinate, supporting the role of the PASp domain in C(4)-dicarboxylate sensing. Both of the phosphorylated DcuS constructs were able to rapidly pass their phosphoryl groups to DcuR, and after phosphorylation, DcuR dephosphorylated rapidly. No prosthetic group or significant quantity of metal was found associated with either of the PASc-containing proteins. The DNA-binding specificity of DcuR was studied by use of the pure protein. It was found to be converted from a monomer to a dimer upon acetylphosphate treatment, and native polyacrylamide gel electrophoresis suggested that it can oligomerize. DcuR specifically bound to the promoters of the three known DcuSR-regulated genes (dctA, dcuB, and frdA), with apparent K(D)s of 6 to 32 micro M for untreated DcuR and < or =1 to 2 microM for the acetylphosphate-treated form. The binding sites were located by DNase I footprinting, allowing a putative DcuR-binding motif [tandemly repeated (T/A)(A/T)(T/C)(A/T)AA sequences] to be identified. The DcuR-binding sites of the dcuB, dctA, and frdA genes were located 27, 94, and 86 bp, respectively, upstream of the corresponding +1 sites, and a new promoter was identified for dcuB that responds to DcuR.