Scaling up production of cephalosporin C by Acremonium chrysogenum W42-I in a fermenter using submerged fermentation.

Cephalosporins presently stand as the most extensively utilized antibiotic in clinical settings. Acremonium (A.) chrysogenum is the main strain used in the manufacturing of cephalosporin C (CPC), which offers distinct advantages, including a wide-ranging antibacterial spectrum and powerful antibacterial efficacy. Our study aimed to determine the optimal conditions for scaling up the production of CPC from A. chrysogenum W42-I starting with the optimized conditions on the shake flask level obtained from our previous study and utilizing the optimized media (CPC2). The results indicated that an inoculum size equivalent to 1% v/v, aeration at 1 vvm, and an agitation rate of 400 rpm, with controlled pH at 4, were the most favorable conditions for the CPC production using a laboratory fermentor (14 L). The concentration of generated CPC was assessed using two standard curves obtained from agar well diffusion and high-performance liquid chromatography (HPLC). These optimized conditions resulted in a production of 399.52 µg/mL showing a significant increase of approximately 3.4 folds when compared to the unoptimized fermentation run. In conclusion, our findings demonstrated a more favorable time course for CPC production in the fermentor compared to that in the shake flask. Notably, there was a two-fold increase in production within the first three days. Fortunately, the fermentor achieved a noteworthy increase in output, generating 1.598 gm of the CPC within 4 L.

Statistical optimization and gamma irradiation on cephalosporin C production by Acremonium chrysogenum W42-I.

Most antibiotics now used in clinical practice are cephalosporins. Acremonium (A.) chrysogenum W42-I is an intermediate strain out of W42 strain improvement program whose productivity is above that of the wild-type strain to produce the broad-spectrum antibacterial cephalosporin C (CPC). As a result, fermentation process optimization is considered because it offers the ideal environment for strains to reach their full potential. Our research aimed to combine a rational design to regulate the fermentation process environment and culture media as well as to develop mutants with high productivity. Different media were tested to obtain maximum CPC production. To maximize the production of CPC, some environmental parameters were experimentally optimized via the Box-Behnken design used for response surface methodology (RSM). There were 17 tests conducted, and each experiment's reaction was recorded. Improvement of the CPC production was further achieved via mutagenesis using gamma radiation. Results revealed that a pH of 4, an incubation period of 4 days, and an inoculum size of 1% v/v using the optimized media (CPC2) were the optimum conditions for enhancing the CPC production by 4.43-fold. In addition, gamma irradiation further enhanced production to reach 3.46-fold using an optimum dose of 2 KGy. In conclusion, in comparison to initial production levels, CPC production increased 4.43-fold because of nutritional and environmental optimization. The mutant AC8 demonstrated a roughly 3.46-fold increase in activity against its parent type. Moreover, subsequent AC8 mutant culture demonstrated excellent genetic stability.

Characterization and anti-biofilm activity of bacteriophages against urinary tract Enterococcus faecalis isolates.

Strong biofilm-forming Enterococcus feacalis urinary tract pathogens (n = 35) were used to determine the lytic spectrum of six bacteriophages isolated from sewage samples. Only 17 Enterococcus feacalis isolates gave lytic zones with the tested bacteriophages from which five isolates were susceptible to all of them. The isolated enterococcal phages are characterized by wide range of thermal (30-90 °C) and pH (3-10) stability. They belong to order Caudovirales, from which four bacteriophages (EPA, EPB, EPD, EPF) belong to family Myoviridae and two (EPC, EPE) belong to family Siphoviridae. In addition, they have promising antibiofilm activity against the tested strong-forming biofilm E. faecalis isolates. The enterococcal phages reduced the formed and preformed biofilms to a range of 38.02-45.7% and 71.0-80.0%, respectively, as compared to the control. The same promising activities were obtained on studying the anti-adherent effect of the tested bacteriophages on the adherence of bacterial cells to the surface of urinary catheter segments. They reduced the number of adherent cells to a range of 30.8-43.8% and eradicated the pre-adherent cells to a range of 48.2-71.1%, as compared to the control. Overall, the obtained promising antibiofilm activity makes these phages good candidates for application in preventing and treating biofilm associated Enterococcus faecalis infections.

In vitro Anti SARS-CoV-2 Activity and Docking Analysis of Pleurotus ostreatus, Lentinula edodes and Agaricus bisporus Edible Mushrooms.

Background: Fungi are rich source of biologically active metabolites aimed for the improvement of human health through the prevention of various diseases, including infections and inflammatory disorders. Aim: We aimed to in vitro examine the anti-SARS CoV-2 activity of the aqueous extract of each Pleurotus (P.) ostreatus, Lentinula (L.) edodes and Agaricus (A.) bisporus edible mushroom followed by docking analysis of certain metabolites against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-main protease (protease Mpro). Methods: Antiviral and cytotoxic effects were tested on hCoV-19/Egypt/NRC-3/2020/Vero-E6 cells and analyzed via (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide Assay (MTT) assay. Ligand-protein and protein-protein docking studies were performed to explore the interaction of different mushroom extracts at the binding site of protease Mpro. Molecular dynamics (MD) simulations were performed on the most promising ligand-target complexes to investigate their dynamic properties and confirm docking results. Results: Substantial antiviral activities with an IC50 of 39.19, 26.17, and 10.3.3 µg/mL and a selectivity index (SI) of 4.34, 3.44, and 1.5 for P. ostreatus, L. edodes and A. bisporus, were observed, respectively. Docking analysis revealed that, catechin from three mushroom isolates, chlorogenic acid from A. bisporus, kamperferol of P. ostreatus and quercetin from L. edodes, with a C-DOCKER interaction energy in the range of 22.8-37.61 (Kcal/mol) with protease compared to boceprevir ligand of 41.6 (Kcal/mol). Docking of superoxide dismutase, catalase from the three mushrooms, tyrosinase from A. bisporus showed ligand contact surface area with the protein as 252.74 Å2 while receptor contact surface area was 267.23 Å2. Conclusion: P. ostreatus, L. edodes and A. bisporus have potential and remarkable in vitro antiviral activities against SARS-CoV-2. Quercetin from L. edodes, Kaempferol from P. ostreatus, chlorogenic acid and ascorbic acid, catechin, superoxide dismutase and catalase of the three mushrooms extracts were effectively bounded to Mpro of SARS-CoV-2 as conferred by docking analysis.

Immunomodulatory activity of extracts from five edible basidiomycetes mushrooms in Wistar albino rats.

Mushrooms are nutritious foods that are widely cultivated all over the world. They are rich in a range of compounds linked to improving functions of the immune system including carotenoids, alkaloids, lectins, enzymes, folates, fats, organic acids, minerals, polysaccharides, phenolics, proteins, tocopherols, terpenoids, and volatile compounds. In this study we investigated, the immunomodulatory activity in rats of the aqueous extracts of five of the most common edible mushrooms belonging to Family Basidiomycota-white-rot fungi including, Lentinula edodes, Agaricus bisporus, Pleurotus ostreatus, Pleurotus columbinus, and Pleurotus sajor-caju. Male Wistar albino rats were assigned to thirteen groups and Immunosuppression was induced by oral administration of dexamethasone (0.1 mg/kg), followed by oral administration of the mushroom extracts at low (200 mg/kg) and high (400 mg/kg) doses. A positive control group received the immune stimulant Echinacea extract Immulant® at (30 mg/kg), while the negative control group received only saline. From each animal, in each group, blood samples were collected after 15 days for complete blood counts and for measurement of immunologic parameters, including lysozyme activity, nitric oxide (NO) production and serum cytokines including tumor necrosis factor-alpha (TNF-α), interferon-gamma (IFN-γ) and interleukin 1 beta (IL-1ß) levels. Results have shown that white blood cells (WBCs) and lymphocytic counts were significantly boosted by high doses of each of the five mushroom extracts (207-289% increase for WBC and 153-175% for lymphocytes) with a significant increase in lysozyme activity (110-136% increase), NO concentration (159-232% increase) and cytokines as compared to the negative control group. Histopathological examination of the rats' spleen and thymus tissues has shown marked lymphocytic proliferation that was more obvious at the higher doses. In conclusion, our results showed that the five edible mushroom extracts revealed significant immunostimulatory effects preclinically particularly, at the higher doses (400 mg/kg) which can be considered the effective dose.

Antibiofilm activity of green synthesized silver nanoparticles against biofilm associated enterococcal urinary pathogens.

Biofilm-formed enterococcal urinary tract clinical isolates (n = 92) were used for studying the antibiofilm activity of cinnamon, ginger, and chemical AgNPs. The average particle sizes of cinnamon, ginger, and chemical AgNPs were 8.7, 41.98, and 55.7 nm, respectively. The results of Fourier transform infrared analysis revealed that phytocompounds, such as cinnamaldehyde and gingerol, were the main compounds incorporated in the synthesis of cinnamon and ginger AgNPs, respectively. The purity and crystalline nature of the AgNPs have been confirmed by energy dispersive X-ray and X-ray Diffraction analysis. The results of antimicrobial activity showed that MIC of ginger, cinnamon, and chemical AgNPs were 37.64, 725.7, and 61.08 µg/ml, respectively. On studying the antibiofilm activity of AgNPs at sub-MIC values (1/2, 1/4, and 1/8 MIC), the results revealed that it was concentration dependent. Therefore, further studies were carried out to evaluate the antibiofilm activity of AgNPs at a concentration of 18 µg/ml. The results showed that ginger and chemical AgNPs reduced the formed biofilm to 39.14% and 65.32% and the number of adherent cells on the urinary catheter surface to 42.73% and 69.84%, respectively, as compared to that of the control, while cinnamon AgNPs showed no significant activity. Accordingly, ginger AgNPs had the most potent antibacterial and antiadherent activity against biofilm-associated enterococcal isolates.

Association of Macrolide Resistance Genotypes and Synergistic Antibiotic Combinations for Combating Macrolide-Resistant MRSA Recovered from Hospitalized Patients.

Macrolide-resistant methicillin-resistant Staphylococcus aureus (MAC-MRSA) is one of the most clinically relevant pathogens due to its significant ability of resistance acquisition to different antimicrobial agents. This study aimed to evaluate antimicrobial susceptibility and the use of different combinations of azithromycin with other antibiotics for combating MAC resistance. Seventy-two Staphylococci (38.5%) (n = 187), showed resistance to MACs; of these, 53 isolates (73.6%, n = 72) were S. aureus and 19 (26.4%, n = 72) were coagulase-negative staphylococci (CoNS). Out of the 53 S. aureus and 19 CoNS isolates, 38 (71.7%, n = 53) and 9 (47.4%, n = 19) were MRSA and methicillin-resistant CoNS, respectively. The constitutive MACs, lincosamides and streptogramin-B (cMLS) comprised the predominant phenotype among S. aureus isolates (54.7%) and CoNS isolates (78.9%). The PCR analysis showed that the ermC gene was the most prevalent (79.2%), followed by msrA (48.6%), and ermA (31.9%). Azithromycin combinations with either linezolid, ceftriaxone, gentamicin, or cefotaxime provided synergy in 42.1%, 44.7%, 31.6% and 7.9% of the 38 MAC-MRSA isolates, respectively. Statistical analysis showed significant association between certain MAC resistance genotypes and the synergistic effect of certain azithromycin combinations (p value < 0.05). In conclusion, azithromycin combinations with either linezolid, or ceftriaxone showed synergism in most of the MAC-resistant MRSA clinical isolates.

Antiviral, Cytotoxic, and Antioxidant Activities of Three Edible Agaricomycetes Mushrooms: Pleurotus columbinus, Pleurotus sajor-caju, and Agaricus bisporus.

In this study, we investigated aqueous extracts of three edible mushrooms: Agaricus bisporus (white button mushroom), Pleurotus columbinus (oyster mushroom), and Pleurotus sajor-caju (grey oyster mushroom). The extracts were biochemically characterized for total carbohydrate, phenolic, flavonoid, vitamin, and protein contents besides amino acid analysis. Triple TOF proteome analysis showed 30.1% similarity between proteomes of the two Pleurotus spp. All three extracts showed promising antiviral activities. While Pleurotus columbinus extract showed potent activity against adenovirus (Ad7, selectivity index (SI) = 4.2), Agaricus bisporus showed strong activity against herpes simplex II (HSV-2; SI = 3.7). The extracts showed low cytotoxicity against normal human peripheral blood mononuclear cells (PBMCs) and moderate cytotoxicity against prostate (PC3, DU-145); colorectal (Colo-205); cecum carcinoma (LS-513); liver carcinoma (HepG2); cervical cancer (HeLa); breast adenocarcinoma (MDA-MB-231 and MCF-7) as well as leukemia (CCRF-CEM); acute monocytic leukemia (THP1); acute promyelocytic leukemia (NB4); and lymphoma (U937) cell lines. Antioxidant activity was evaluated using 2,2-diphenyl-1-picryl-hydrazyl-hydrate (DPPH) radical scavenging, 2,2'-Azinobis-(3-Ethylbenzthiazolin-6-Sulfonic Acid) ABTS radical cation scavenging, and oxygen radical absorbance capacity (ORAC) assays. The three extracts showed potential antioxidant activities with the maximum activity recorded for Pleurotus columbinus (IC50 µg/mL) = 35.13 ± 3.27 for DPPH, 13.97 ± 4.91 for ABTS, and 29.42 ± 3.21 for ORAC assays.

Streptomyces griseus KJ623766: A Natural Producer of Two Anthracycline Cytotoxic Metabolites ß- and γ-Rhodomycinone.

BACKGROUND: This study aimed to produce, purify, structurally elucidate, and explore the biological activities of metabolites produced by Streptomyces (S.) griseus isolate KJ623766, a recovered soil bacterium previously screened in our lab that showed promising cytotoxic activities against various cancer cell lines. METHODS: Production of cytotoxic metabolites from S. griseus isolate KJ623766 was carried out in a 14L laboratory fermenter under specified optimum conditions. Using a 3-(4,5-dimethylthazol-2-yl)-2,5-diphenyl tetrazolium-bromide assay, the cytotoxic activity of the ethyl acetate extract against Caco2 and Hela cancer cell lines was determined. Bioassay-guided fractionation of the ethyl acetate extract using different chromatographic techniques was used for cytotoxic metabolite purification. Chemical structures of the purified metabolites were identified using mass, 1D, and 2D NMR spectroscopic analysis. RESULTS: Bioassay-guided fractionation of the ethyl acetate extract led to the purification of two cytotoxic metabolites, R1 and R2, of reproducible amounts of 5 and 1.5 mg/L, respectively. The structures of R1 and R2 metabolites were identified as ß- and γ-rhodomycinone with CD50 of 6.3, 9.45, 64.8 and 9.11, 9.35, 67.3 µg/mL against Caco2, Hela and Vero cell lines, respectively. Values were comparable to those of the positive control doxorubicin. CONCLUSIONS: This is the first report about the production of ß- and γ-rhodomycinone, two important scaffolds for synthesis of anticancer drugs, from S. griseus.

Cloning and sequencing of lsaE efflux pump gene from MDR Enterococci and its role in erythromycin resistance.

Enterococci are opportunistic members of intestinal microbiota with notable ability to transmit antimicrobial resistance genes. Among the different resistance mechanisms, multidrug efflux is evolving as a huge problem in conferring multidrug resistance to bacterial cells because these pumps extrude a broad range of antimicrobials. Therefore, the aim of this work was to evaluate role of efflux pumps in the development of multi-drug resistance in Enterococci through studying the antimicrobial resistance profiles of Enterococci isolates, phenotypically and genotypically investigating the role of active efflux pumps in development of resistance, in addition to characterizing the most common efflux pump genes. The study involved the recovery of 149 Enterococci isolates from specimens of patients suffering infections in some hospitals in Egypt. Antimicrobial resistance profiles of isolates showed that only 1.3% of the isolates were resistant to each of linezolid, daptomycin, and fosfomycin. The highest resistance was to ampicillin (60.4%) while 47 of the isolates (31.54%) were found to be multidrug-resistant. Efflux pumps have shown to have a significant role in erythromycin resistance in 11 isolates (23.4% of MDR isolates) as indicated by an 8 or more fold decrease in minimum inhibitory concentration in the presence of the efflux pump inhibitor, carbonyl cyanide m- chlorophenylhydrazone (CCCP). End point PCR was used to detect efflux pump genes lsaE, msrC, and mefA in the 11 isolates at which efflux pumps were found to play a significant role in resistance. Nine out of the 11 isolates (81.8%) were found to carry lsaE gene. This gene was inserted into pUC21 vector and cloned into DH5α E. coli resulting in successful transformation and expression of erythromycin resistance in this host. Finally, sequencing of the lsaE gene was carried out. To the best of our knowledge, this is the first report on the cloning of lsaE gene from MDR Enterococcus isolates.

In vivo evaluation of a recombinant N-acylhomoserine lactonase formulated in a hydrogel using a murine model infected with MDR Pseudomonas aeruginosa clinical isolate, CCASUP2.

Failure in the treatment of P. aeruginosa, due to its broad spectrum of resistance, has been associated with increased patient mortality. One alternative approach for infection control is quorum quenching which was found to decrease virulence of such pathogen. In this study, the efficiency of a recombinant Ahl-1 lactonase formulated as a hydrogel was investigated to control the infection of multidrug resistant (MDR) P. aeruginosa infected burn using a murine model. The recombinant N-acylhomoserine lactonase (Ahl-1) was formulated as a hydrogel. To test its ability to control the infection of MDR P. aeruginosa, a thermal injury model was used. Survival rate, and systemic spread of the infection were evaluated. Histopathological examination of the animal dorsal skin was also done for monitoring the healing and cellular changes at the site of infection. Survival rate in the treated group was 100% relative to 40% in the control group. A decrease of up to 3 logs of bacterial count in the blood samples of the treated animals relative to the control group and a decrease of up to 4 logs and 2.3 logs of bacteria in lung and liver samples, respectively were observed. Histopathological examination revealed more enhanced healing process in the treated group. Accordingly, by promoting healing of infected MDR P. aeruginosa burn and by reducing systemic spread of the infection as well as decreasing mortality rate, Ahl-1 hydrogel application is a promising strategy that can be used to combat and control P. aeruginosa burn infections.

Correlation between the Antibiotic Resistance Genes and Susceptibility to Antibiotics among the Carbapenem-Resistant Gram-Negative Pathogens.

In this study, the correlation between the antibiotic resistance genes and antibiotic susceptibility among the carbapenem-resistant Gram-negative pathogens (CRGNPs) recovered from patients diagnosed with acute pneumonia in Egypt was found. A total of 194 isolates including Klebsiella pneumoniae (89; 46%), Escherichia coli (47; 24%) and Pseudomonas aeruginosa (58; 30%) were recovered. Of these, 34 (18%) isolates were multiple drug resistant (MDR) and carbapenem resistant. For the K. pneumoniae MDR isolates (n = 22), blaNDM (14; 64%) was the most prevalent carbapenemase, followed by blaOXA-48 (11; 50%) and blaVIM (4; 18%). A significant association (p value < 0.05) was observed between the multidrug efflux pump (AcrA) and resistance to ß-lactams and the aminoglycoside acetyl transferase gene (aac-6'-Ib) gene and resistance to ciprofloxacin, azithromycin and ß-lactams (except for aztreonam). For P. aeruginosa, a significant association was noticed between the presence of the blaSHV gene and the multidrug efflux pump (MexA) and resistance to fluoroquinolones, amikacin, tobramycin, co-trimoxazole and ß-lactams and between the aac-6'-Ib gene and resistance to aminoglycosides. All P. aeruginosa isolates (100%) harbored the MexAB-OprM multidrug efflux pump while 86% of the K. pneumoniae isolates harbored the AcrAB-TolC pump. Our results are of great medical importance for the guidance of healthcare practitioners for effective antibiotic prescription.

Production and statistical optimization of Paromomycin by Streptomyces rimosus NRRL 2455 in solid state fermentation.

BACKGROUND: Paromomycin is a 2-deoxystreptamine aminocyclitol aminoglycoside antibiotic with broad spectrum activity against Gram-negative, Gram-positive bacteria and many protozoa. This study introduces a strategy for paromomycin production through solid-state fermentation using Streptomyces rimosus subsp. paromomycinus NRRL 2455. Solid state fermentation has gained enormous attention in the development of several products because of their numerous advantages over submerged liquid fermentation. After selecting the best solid substrate, a time course study of paromomycin production was carried out followed by optimization of environmental conditions using response surface methodology. Paromomycin yields obtained using this technique were also compared to those obtained using submerged liquid fermentation. RESULTS: Upon screening of 6 different substrates, maximum paromomycin concentration (0.51 mg/g initial dry solids) was obtained with the cost-effective agro-industrial byproduct, corn bran, impregnated with aminoglycoside production media. Optimization of environmental conditions using D-optimal design yielded a 4.3-fold enhancement in paromomycin concentration reaching 2.21 mg/g initial dry solids at a pH of 8.5, inoculum size of 5% v/w and a temperature of 30 °C. CONCLUSION: Compared to submerged liquid fermentation, solid state fermentation resulted in comparable paromomycin concentrations, cost reduction of raw materials, less energy consumption and waste water discharge, which have major implications in industrial fermentation. Therefore, solid state fermentation is a promising alternative to submerged liquid fermentation for paromomycin production. To the best of our knowledge, this is the first report on the optimized paromomycin production through solid state fermentation process.

Common childhood vaccines do not elicit a cross-reactive antibody response against SARS-CoV-2.

Anecdotal evidence showed a negative correlation between Bacille Calmette-Guérin (BCG) vaccination and incidence of COVID-19. Incidence of the disease in children is much lower than in adults. It is hypothesized that BCG and other childhood vaccinations may provide some protection against SARS-CoV-2 infection through trained or adaptive immune responses. Here, we tested whether BCG, Pneumococcal, Rotavirus, Diphtheria, Tetanus, Pertussis, Hepatitis B, Haemophilus influenzae, Hepatitis B, Meningococcal, Measles, Mumps, and Rubella vaccines provide cross-reactive neutralizing antibodies against SARS-CoV-2 in BALB/c mice. Results indicated that none of these vaccines provided antibodies capable of neutralizing SARS-CoV-2 up to seven weeks post vaccination. We conclude that if such vaccines have any role in COVID-19 immunity, this role is not antibody-mediated.

Prevalence of plasmid-mediated resistance genes among multidrug-resistant uropathogens in Egypt.

BACKGROUND: The emergence of multidrug-resistant (MDR) uropathogens has become a public health threat and current knowledge of the genotypic basis of bacterial resistance is essential for selecting appropriate treatment options. OBJECTIVES: To determine the prevalence of antimicrobial resistance among MDR uropathogens and to elucidate the molecular bases of plasmid-mediated resistance. METHODS: Bacterial isolates were recovered from urine specimens of 150 out-patients with signs and symptoms of urinary tract infections (UTIs) at El-Demerdash Hospital, Cairo, Egypt. Standard methods were used for identification, antimicrobial susceptibility testing was performed according to CLSI guidelines. RESULTS: Among the recovered isolates, 22.7% and 77.3% were Gram-positive, and negative, respectively. Of which; 43.3% were MDR with 60% harboring plasmids. Extended spectrum ß-lactamase (ESBL) genes bla CTX-M, bla SHV, and bla TEM were detected on plasmids of 89.7%, 41%, and 84.6% of the tested isolates, respectively. The aminoglycoside resistance gene aac6'-Ib/aac-6'-Ib-cr was found on plasmids of 92.3% of the tested isolates followed by qnrS (92.3%), qnrB (46.2%), and qnrA (7.7%). The most prevalent quinolone efflux pump gene was oqxB (38.5%), followed by oqxA (20.5%), then qepA (10.3%). CONCLUSION: High levels of resistance to nitrofurans, ß-lactam/ß-lactamase inhibitor, cephalosporins, aminoglycosides, and fluoroquinolones were detected, and their use as empirical treatment for UTIs has become questionable.

New insight into poly (3-hydroxybutyrate) production by Azomonas macrocytogenes isolate KC685000: large scale production, kinetic modeling, recovery and characterization.

About 24 h incubation of Azomonas (A.) macrocytogenes isolate KC685000 in 14L fermenter produced 22% poly (3-hydroxybutyrate) (PHB) per cell dry weight (CDW) biopolymer using 1 vvm aeration, 10% inoculum size, and initial pH of 7.2. To control the fermentation process, Logistic and Leudeking-Piret models were used to describe the cell growth and PHB production, respectively. These two models were in good agreement with the experimental data confirming the growth associated nature of PHB production. The best method for recovery of PHB was chemical digestion using sodium hypochlorite alone. The characterization of the produced polymer was carried out using FT-IR, 1HNMR spectroscopy, gel permeation chromatography and transmission electron microscope. The analysis of the nucleotide sequences of PHA synthase enzyme revealed class III identity. The putative tertiary structure of PHA synthase enzyme was analyzed using Modular Approach to Structural class prediction software, Tied Mixture Hidden Markov Model server, and Swiss model software. It was deduced that PHA synthases' structural class was multidomain protein (α/ß) containing a conserved cysteine residue and lipase box as characteristic features of α/ß hydrolase super family. Taken together, all the results of molecular characterization and transmission electron microscope images supported that the PHB formation was attained by the micelle model. To the best of our knowledge, this is the first report on production of growth associated PHB polymer using A. macrocytogenes isolate KC685000, and its class III PHA synthase.

Paromomycin production from Streptomyces rimosus NRRL 2455: statistical optimization and new synergistic antibiotic combinations against multidrug resistant pathogens.

BACKGROUND: Response surface methodology (RSM) employing Box-Behnken design was used to optimize the environmental factors for the production of paromomycin, a 2 deoxystreptamine aminocyclitol aminoglycoside antibiotic, (2DOS-ACAGA) from Streptomyces (S.) rimosus NRRL 2455. Emergence of bacterial resistance caught our attention to consider the combination of antimicrobial agents. The effect of paromomycin combination with other antimicrobial agents was tested on some multiple drug resistant isolates. To the best of our knowledge, this is the first report on optimization of paromomycin production from S. rimosus NRRL 2455. A Quadratic model and response surface method were used by choosing three model factors; pH, incubation time and inoculum size. A total of 17 experiments were done and the response of each experiment was recorded. Concerning the effect of combining paromomycin with different antimicrobial agents, it was tested using the checkerboard assay against six multidrug resistant (MDR) pathogens including; Pseudomonas (P.) aeruginosa (2 isolates), Klebsiella (K.) pneumoniae, Escherichia (E.) coli, methicillin sensitive Staphylococcus aureus (MSSA) and methicillin resistant Staphylococcus aureus (MRSA). Paromomycin was tested in combination with ceftriaxone, ciprofloxacin, ampicillin/sulbactam, azithromycin, clindamycin and doxycycline. RESULTS: The optimum conditions for paromomycin production were a pH of 6, an incubation time of 8.5 days and an inoculum size of 5.5% v/v using the optimized media (soybean meal 30 g/L, NH4CL 4 g/L, CaCO3 5 g/L and glycerol 40 ml/L), 28 °C incubation temperature, and 200 rpm agitation rate that resulted in 14 fold increase in paromomycin production as compared to preliminary fermentation level using the basal medium. The tested antibiotic combinations showed either synergistic effect on paromomycin activity on most of the tested MDR pathogens (45.83%), additive effect in 41.67% or indifferent effect in 12.5%. CONCLUSION: RSM using multifactorial design was a helpful and a reliable method for paromomycin production. Paromomycin combination with ceftriaxone, ciprofloxacin, ampicillin/sulbactam, azithromycin, clindamycin or doxycycline showed mostly synergistic effect on certain selected clinically important MDR pathogens.

Overexpressed recombinant quorum quenching lactonase reduces the virulence, motility and biofilm formation of multidrug-resistant Pseudomonas aeruginosa clinical isolates.

The increasing occurrence of resistance among Pseudomonas aeruginosa clinical isolates necessitates finding alternatives to antibiotics for controlling the infection of such pathogenic bacteria. In this study, lactonase gene ahl-1 from Bacillus weihenstephanensis isolate-P65 was successfully cloned and expressed in Escherichia coli BL21 (DE3) under the control of T7 promoter for utilizing its quorum quenching activity against three multidrug-resistant (MDR) P. aeruginosa clinical isolates. The biological activity of the overexpressed lactonase enzyme (Ahl-1), tested using a synthetic signal and Chromobacterium violaceum CV026 as a biosensor, displayed good catalytic activity using hexanoyl homoserine lactone (HHL) as a substrate and Chromobacterium violaceum (CV026) as a biosensor (77.2 and 133 nm min-1 for the crude and the purified Ahl-lactonase enzymes, respectively). Upon challenging its ability to inhibit the virulence of three MDR P. aeruginosa clinical isolates, recombinant Ahl-1 successfully prevented the accumulation of acylhomoserine lactone signals resulting in a significant reduction in the investigated virulence determinants; protease (from 40 up to 75.5%), pyocyanin (48-75.9%), and rhamnolipids (52.7-63.4%) (P value < 0.05). Ahl-1 also displayed significant inhibitory activities on the swarming motility and biofilm formation of the three tested MDR P. aeruginosa clinical isolates (P value < 0.05). Consequently, Ahl-1 lactonase enzyme in this study is considered a promising therapeutic agent to inhibit P. aeruginosa pathogenicity with no fear of emergence of resistance.

Kinetic modeling, recovery, and molecular characterization of poly-beta-hydroxybutyrate polymer in Acinetobacter baumannii isolate P39.

To control the poly-ß-hydroxybutyrate (PHB) biopolymer production by Acinetobacter baumannii isolate P39 kinetic modeling of the fermentation process, polymer downstream processing, enzymological analysis, and molecular characterization of PHA synthase, key biosynthetic enzyme, should be addressed. A. baumannii isolate P39 produced 0.15 g/L PHB after 24 h of incubation with a polymer content of 28% per dry weight. Logistic and Leudeking-Piret models were used for describing cell growth and PHB production, respectively. They showed good agreement with the experimental data describing both cell growth and PHB production (average regression coefficient r2:0.999). The growth-associated production of PHB biopolymer as an electron acceptor was confirmed using Leudeking-Piret model and victim substrate experiment. The best method of recovery of PHB biopolymer was chemical digestion using sodium hypochlorite, since it produced the largest amount of polymer and highest molecular weight (16,000 g/mole) in comparison to other recovery methods. DTNB assay showed high activity of PHA synthase enzyme, 600 U activity, and 153.8 U/mg specific activity. Molecular analysis of PHA synthase enzyme confirmed class III identity. Taken together, micelle model was proposed for polyhydroxybutyrate formation in A. baumannii isolate P39.

Anti-tuberculous activity of treponemycin produced by a streptomyces strain MS-6-6 isolated from Saudi Arabia.

A Streptomyces strain MS-6-6 with promising anti-tuberculous activity was isolated from soil samples in Saudi Arabia. The nucleotide sequence of its 16S rRNA gene (1426 bp) evidenced a 100% similarity to Streptomyces mutabilis. Through an anti-tuberculous activity-guided approach, a polyketide macrolide was isolated and identified as treponemycin (TP). The structure of the isolated compound was determined by comprehensive analyses of its 1D and 2D NMR as well as HRESI-MS. In addition to the promising anti-tuberculous activity (MIC = 13.3 µg/mL), TP showed broad spectrum of activity against the Gram positive, Gram negative strains, and Candida albicans. Improvement of TP productivity (150%) was achieved through modification in liquid starch nitrate medium by replacing KNO3 with corn steep liquor and yeast extract or tryptone, and removing CaCO3 and K2HPO4. The follow up of TP percentage as well as its metabolites profile for each media was assessed by LC/DAD/MS.