Sequencing analysis and efficient biodiesel production by lipase from Pseudomonas aeruginosa.

BACKGROUND: Recently, lipase processing for biodiesel production has shown a global increase as it is considered a potential alternative clean-fuel source. The current study's objective is to investigate of lipolytic activity of lipase produced from different strains of Pseudomonas aeruginosa (P. aeruginosa) in biodiesel production using edible plant oils. The goal is to develop an efficient and cost-effective method for producing inexpensive and environmentally friendly biodiesel. METHODS AND RESULTS: Four P. aeruginosa isolates were obtained from different environmental sources (soil), phenotypically identified, and it was confirmed by the PCR detection of the 16SrRNA gene. The isolated P. aeruginosa strains were screened for lipase production, and the recovered lipase was purified. Besides, the lipase (lip) gene was detected by PCR, and the purified PCR products were sequenced and analyzed. The production of biofuel was conducted using gas chromatography among tested oils. It was found that castor oil was the best one that enhances lipase production in-vitro.

The secrets of environmental Pseudomonas aeruginosa in slaughterhouses: Antibiogram profile, virulence, and antibiotic resistance genes.

Environmental pollution is a serious problem that can cause sicknesses, fatality, and biological contaminants such as bacteria, which can trigger allergic reactions and infectious illnesses. There is also evidence that environmental pollutants can have an impact on the gut microbiome and contribute to the development of various mental health and metabolic disorders. This study aimed to study the antibiotic resistance and virulence potential of environmental Pseudomonas aeruginosa (P. aeruginosa) isolates in slaughterhouses. A total of 100 samples were collected from different slaughterhouse tools. The samples were identified by cultural and biochemical tests and confirmed by the VITEK 2 system. P. aeruginosa isolates were further confirmed by CHROMagar™ Pseudomonas and genetically by rpsL gene analysis. Molecular screening of virulence genes (fimH, papC, lasB, rhlI, lasI, csgA, toxA, and hly) and antibiotic resistance genes (blaCTX-M, blaAmpC, blaSHV, blaNDM, IMP-1, aac(6')-Ib-, ant(4')IIb, mexY, TEM, tetA, and qnrB) by PCR and testing the antibiotic sensitivity, biofilm formation, and production of pigments, and hemolysin were carried out in all isolated strains. A total of 62 isolates were identified as P. aeruginosa. All P. aeruginosa isolates were multidrug-resistant and most of them have multiple resistant genes. blaCTX-M gene was detected in all strains; 23 (37.1%) strains have the ability for biofilm formation, 33 strains had virulence genes, and 26 isolates from them have more than one virulence genes. There should be probably 60 (96.8%) P. aeruginosa strains that produce pyocyanin pigment. Slaughterhouse tools are sources for multidrug-resistant and virulent pathogenic microorganisms which are a serious health problem. Low-hygienic slaughterhouses could be a reservoir for resistance and virulence genes which could then be transferred to other pathogens.

Anti-capsular activity of CuO nanoparticles against Acinetobacter baumannii produce efflux pump.

Copper oxide nanoparticles are modern kinds of antimicrobials, which may get a lot of interest in the clinical application. This study aimed to detect the anti-capsular activity of CuO nanoparticles against Acinetobacter baumannii produce efflux pump. Thirty-four different clinical A. baumannii isolates were collected and identified by the phenotypic and genetic methods by the recA gene as housekeeping. Antibiotic sensitivity and biofilm-forming ability, capsular formation were carried out. The effect of CuO nanoparticles on capsular isolates was detected, the synergistic effects of a combination CuO nanoparticles and gentamicin against A. baumannii were determined by micro broth checkerboard method, and the effect of CuO nanoparticles on the expression of ptk, espA and mexX genes was analyzed. Results demonstrated that CuO nanoparticles with gentamicin revealed a synergistic effect. Gene expression results show reducing the expression of these capsular genes by CuO nanoparticles is major conduct over reducing A. baumannii capsular action. Furthermore, results proved that there was a relationship between the capsule-forming ability and the absence of biofilm-forming ability. As bacterial isolates which were negative biofilm formation were positive in capsule formation and vice versa. In conclusion, CuO nanoparticles have the potential to be used as an anti-capsular agent against A. baumannii, and their combination with gentamicin can enhance their antimicrobial effect. The study also suggests that the absence of biofilm formation may be associated with the presence of capsule formation in A. baumannii. These findings provide a basis for further research on the use of CuO nanoparticles as a novel antimicrobial agent against A. baumannii and other bacterial pathogens, also to investigate the potential of CuO nanoparticles to inhibit the production of efflux pumps in A. baumannii, which are a major mechanism of antibiotic resistance.

Binary CuO\CoO nanoparticles inhibit biofilm formation and reduce the expression of papC and fimH genes in multidrug-resistant Klebsiella oxytoca.

BACKGROUND AND AIM: Binary copper-cobalt oxide nanoparticles (CuO\CoO NPs) are modern kinds of antimicrobials, which may get a lot of interest in clinical application. This study aimed to detect the effect of the binary CuO\CoO NPs on the expression of papC and fimH genes in multidrug-resistant (MDR) isolates of Klebsiella oxytoca to reduce medication time and improve outcomes. METHODS: Ten isolates of K. oxytoca were collected and identified by different conventional tests besides PCR. Antibiotic sensitivity and biofilm-forming ability were carried out. The harboring of papC and fimH genes was also detected. The effect of binary CuO\CoO nanoparticles on the expression of papC and fimH genes was investigated. RESULTS: Bacterial resistance against cefotaxime and gentamicin was the highest (100%), while the lowest percentage of resistance was to amikacin (30%). Nine of the ten bacterial isolates had the ability to form a biofilm with different capacities. MIC for binary CuO\CoO NPs was 2.5 µg/mL. Gene expression of papC and fimH was 8.5- and 9-fold lower using the NPs. CONCLUSION: Binary CuO\CoO NPs have a potential therapeutic effect against infections triggered by MDR K. oxytoca strains due to the NPs-related downregulation ability on the virulence genes of K. oxytoca.

Spread of ESßL-producing Escherichia coli and the anti-virulence effect of graphene nano-sheets.

Despite the studies worldwide, the prevalence of ESßL E. coli in the Iraq is still unknown. Realization of the demographic characterization of ESßL E. coli infections will assist the prevention efforts. This study aimed to isolate clinical E. coli, determine their antimicrobial susceptibility, phenotypic and genotypic detection of ESßL-producing ability, detection of some virulence-related genes, estimate the impact of graphene nano-sheets as antibacterial, and study the adherence-related gene expressions in E. coli isolates. Graphene nano-sheets were synthesized and characterized using XRD, UV, TEM, and SEM. E. coli isolates were identified using 16S rRNA. Antibiotic resistance was detected, virulence genes (blaTEM, blaSHV, BlaCTX-M-15, papC, and fimH) were screened using PCR. The antibacterial activity of graphene nano-sheets was screened using well-diffusion assay and MIC. The gene expression of adherence genes after treatment with graphene nano-sheets was evaluated using QRT-PCR. From a total of 512 identified using 16S rRNA, 359 (69.9%) were ESßL-producing E. coli. The ESßL genotypes positive were 83.56% (300/359) of E. coli isolates with the frequencies: 85% for blaCTX-M gene, 26% for blaSHV gene, and 28% for blaTEM gene. Graphene nano-sheets showed effective antibacterial activity with MIC 25 µg/ml. Furthermore, graphene nano-sheets reduced the expression of papC, and fimH genes. This study has helped us to better understand the characteristics of ESßL E. coli, their adherence gene harboring, and the potential ability of graphene nano-sheets to reduce bacterial growth, and the expression of adherence genes. Furthermore, the current study showed further step to understand the mechanisms by which graphene nano-sheets can conflict bacterial virulence and resistance.

Multidrug-resistant Acinetobacter baumannii as an emerging concern in hospitals.

Acinetobacter baumannii has become a major concern for scientific attention due to extensive antimicrobial resistance. This resistance causes an increase in mortality rate because strains resistant to antimicrobial agents are a major challenge for physicians and healthcare workers regarding the eradication of either hospital or community-based infections. These strains with emerging resistance are a serious issue for patients in the intensive care unit (ICU). Antibiotic resistance has increased because of the acquirement of mobile genetic elements such as transposons, plasmids, and integrons and causes the prevalence of multidrug resistance strains (MDR). In addition, an increase in carbapenem resistance, which is used as last line antibiotic treatment to eliminate infections with multidrug-resistant Gram-negative bacteria, is a major concern. Carbapenems resistant A. baumannii (CR-Ab) is a worldwide problem. Because these strains are often resistant to all other commonly used antibiotics. Therefore, pathogenic multi-drug resistance A. baumannii (MDR-Ab) associated infections become hard to eradicate. Plasmid-mediated resistance causes outbreaks of extensive drug-resistant. A. baumannii (XDR-Ab). In addition, recent outbreaks relating to livestock and community settings illustrate the existence of large MDR-Ab strain reservoirs within and outside hospital settings. The purpose of this review, proper monitoring, prevention, and treatment are required to control (XDR-Ab) infections. Attachment, the formation of biofilms and the secretion of toxins, and low activation of inflammatory responses are mechanisms used by pathogenic A. baumannii strain. This review will discuss some aspects associated with antibiotics resistance in A. baumannii as well as cover briefly phage therapy as an alternative therapeutic treatment.

Detection of gyrA and parC Mutations and Prevalence of Plasmid-Mediated Quinolone Resistance Genes in Klebsiella pneumoniae.

BACKGROUND AND AIM: Recently, the extensive use of quinolones led to increased resistance to these antimicrobial agents, with different rates according to the organism and the geographical region. The aim of this study was to detect the resistance rate of Klebsiella pneumoniae Iraqi isolates toward quinolone antimicrobial agents, to determine genetic mutations in gyrA and parC, to screen for efflux-pump activity, and to screen the presence of plasmid-mediated quinolone resistance (PMQR) genes. METHODS: Forty-three K. pneumoniae isolates were confirmed phenotypically and genotypically by Vitek 2 system and species specific primers by PCR using the targeting rpo gene followed by sequencing. Antibiotic susceptibility test was carried out using disc diffusion method. Quinolone resistant isolates were subjected to ciprofloxacin MIC testing, and cartwheel method to screen for efflux pump activity. The presence of the plasmid mediated quinolone resistance genes qepA, qnrB, qnrS, and aac(6)Ib was tested by PCR. Sequencing of gyrA and parC was performed. RESULTS: We observed a high rate of resistance to ceftriaxone, gentamicin ciprofloxacin, and levofloxacin. Low rate of resistance was detected against amikacin and azithromycin. Ciprofloxacin MIC results revealed that 96.1% of the isolates had MICs >256 µg/mL, 83.4% had MICs >512 µg/mL while 34.6% had MIC >1024 µg/mL. Testing of isolates against ciprofloxacin mixed with EtBr at various concentrations resulted in decreased resistant. Sequencing results showed that Ser83Leu was the most common mutation in gyrA that was observed in all quinolone resistant isolates, followed by Asp87Asn. Ser80Ile mutation in parC was observed in 77.7% of the tested isolates. The prevalence of PMQR genes was 92.5% aac (6)-Ib, 51.8% qnrB, 40.7% qepA, and 37% qnrS. CONCLUSION: Quinolone resistance is common in K. pneumoniae isolates in Baghdad. The frequent mutation in gyrA and parC, and the presence of PMQR genes is alarming.

Prevalence of Genes Involved in Colistin Resistance in Acinetobacter baumannii: First Report from Iraq.

Background and Aim: Colistin is increasingly being used as a "last-line" therapy to treat infections caused by multidrug-resistant (MDR) Acinetobacter baumannii isolates, when essentially no other options are available in these days. The aim of this study was to detect genes associated with colistin resistance in A. baumannii. Methods: One hundred twenty-one isolates of A. baumannii were collected from clinical and environmental samples during 2016 to 2018 in Baghdad. Isolates were diagnosed as A. baumannii by using morphological tests, Vitek-2 system, 16SrRNA PCR amplification, and sequencing. Antibiotic susceptibility test was carried out using disk diffusion method. Phenotypic detection of colistin resistance was performed by CHROMagar™ COL-APSE medium and broth microdilution method for the determination of the minimal inhibitory concentration. Molecular detection of genes responsible for colistin resistance in A. baumannii was performed by PCR. Results: Ninety-two (76%) of the 121 A. baumannii isolates were colistin resistant. Twenty-six (21.5%) of the 121 isolates showed positive growth on CHROMagar Acinetobacter base for MDR. PCR detected mcr-1, mcr-2, and mcr-3 genes in 89 (73.5%), 78 (64.5%), and 82 (67.8%) A. baumannii isolates, respectively. Seventy-eight (64.5%) of the 121 isolates harbored the integron intI2 gene and 81 (66.9%) contained intI3 gene. Moreover, 60 (49.6%) of the 121 isolates were positive for the quorum sensing lasI gene. Conclusion: The presence of a large percentage of colistin-resistant A. baumannii strains in Baghdad may be due to the presence of mobile genetic elements, and it is urgent to avoid unnecessary clinical use of colistin.

Broad-Spectrum Bioactivity of Chitosan N-acetylglucosaminohydrolase (Chitosan NAGH) Extracted from Bacillus ligniniphilus.

Background: The genus Bacillus has species with strains that produce Chitosan N-acetylglucosaminohydrolase (NAGH), a hydrolytic enzyme. Objective: A novel bacterium, Bacillus ligniniphilus, was characterized as producing Chitosan NAGH. This study further examine its antibiofilm properties and its possible uses against biofilm-producing bacteria. Methods: Various sea soil samples were evaluated for the presence of Chitosan NAGH. The chosen isolate, Bacillus ligniniphilus 61, was then used to extract and purify Chitosan NAGH using precipitation in ammonium sulfate followed by polyethylene glycol-treated dialysis and gel-permeation chromatography. Biofilm inhibition and antimicrobial activity of Chitosan NAGH was estimated against different bacterial species. Both gene expression profiling of biofilm-related genes and an extracellular polymeric substance (EPS) inhibition assay were performed. Results: The BL61 strain was able to produce much more Chitosan activity than the other strains, as the latter only exhibited antimicrobial activity at low concentration levels; however, they did show as antibiofilm agents at varying proportions. Chitosan NAGH caused a uniform decrease in EPS formation in each isolate. Many biofilm-related genes, e.g., IcaABCD, decreased, but genes related to autoinducer synthetase were not affected by Chitosan NAGH. EPS, which is responsible for polysaccharide formation, was underexpressed at 3-fold down. Conclusions: The current study results allow future researchers to look for better and newer compounds with the antibiofilm property that inhibits the formation of biofilm created by a wide range of bacteria without affecting their growth.

Screening, nutritional optimization and purification for phytase produced by Enterobacter aerogenes and its role in enhancement of hydrocarbons degradation and biofilm inhibition.

In this study, a novel isolate of Enterobacter aerogenes isolated from contaminated soils with hydrocarbons had extracellular phytate-degrading activity. Enterobacter aerogenes isolates were identified by biochemical tests and confirmed by16S rRNA gene products (amplified size 211bp) for genotypic detection. The phytase activity was reached to maximum activity when this isolate was cultivated under the optimal conditions which consisted of using minimal salt medium containing 1%(w/v) rice bran as a sole source for carbon and 2% (w/v) yeast extract at pH 5.5 and temperature of 50°C for 48 h. The phytase had purified to homogeneity by 50% ammonium sulphate precipitation, ion exchange and gel filtration chromatography with 75.7 fold of purification and a yield of 30.35%. The purified phytase is a single peptide with approximate molecular mass of 42 kDa as assessed by SDS-PAGE. The highest degradative ability by Enterobacter aerogenes of black oil, white oil and used engine oil had observed after 72 h of incubation. Rapid degradation of black oil and used engine oil had also observed while slow degradation of white oilat all time of incubation. The purified phytase inhibited biofilm formation ability in a dose-dependent manner for all Gram-negative and Gram-positive biofilm-forming bacteria and a significant difference in cell surface hydrophobicity was observed after exposure of planktonic cells to phytase for hour. The hydrolyzing effect of phytase released by Enterobacter aerogenes for complex salts of phosphorus that are insoluble in the soil led to increase of phosphorus concentrations and enhanced the ability of Enterobacter aerogenes to degrade a specific hydrocarbon in contaminated soil so that the phytase has a promising application in bioremediation of contaminated soils with hydrocarbons.

Acinetobacter baumannii virulence is enhanced by the combined presence of virulence factors genes phospholipase C (plcN) and elastase (lasB).

The ability of multidrug resistance Acinetobacter baumannii to persist in any circumstances regard to the acquisition of many virulence factors genes and antibiotic resistance genes is major concern in the hospitals environments. In this study, thirty A. baumannii isolates were collected from blood infections from hospitalized patients were subjected to screening for virulence factors genes plcN and lasB by conventional PCR. The pathogenicity of representative isolates bearing these gene were tested using galleria mellonella infection assay and adhesion-invasion assay on A549 cell line, and compared with other strain without this gene. Phylogenetic tree revealed that isolates were sorted in two major groups one of them contained two clusters (Group II and III), and another had the other group (Group I). All the 30 A. baumannii isolates were investigated for the presence of virulence factors genes (plc-N and lasB genes) and results showed that, 16 (53.33%) were harboring lasB genes while 7 (23.3%) isolates were harboring plcN gene The presence of any of these gene enhance the killing ability of A. baumannii strain and increased invasiveness in A549 cell line. Increase nosocomial infection with A. baumannii isolates is serious problem especially because of its potency to gain virulence factors genes and its ability to persist in hospital environments. So the shed light in finding which virulence factors these isolates which have is necessary to discover new antimicrobials that targeting the virulence factor of these powerful pathogens.

Chitosanase purified from bacterial isolate Bacillus licheniformis of ruined vegetables displays broad spectrum biofilm inhibition.

A number of bacterial species produces chitosanases which has variety of applications because of its high biodegradability, non-toxicity and antimicrobial assets. In the present study chitosanase is purified from new bacterial species Bacillus licheniformis from spoiled vegetable. This novel strain of Bacillus licheniformis isolated from spoilt cucumber and pepper samples has the ability to produce the chitosanase enzyme when grown on chitosan substrate. Study also examined its antibiofilm properties against diverse bacterial species with biofilm forming ability. The purified chitosanase inhibited the biofilm formation ability for all Gram-negative and Gram-positive biofilm-forming bacteria [biofilm producers] tested in this study in congo red agar and microtiter plate's methods. Highly antibiofilm activity of chitosanase was recorded against Pseudomonas aeruginosa followed by Klebsiella pneumoniae with reduction of biofilm formation upto 22 and 29%, respectively compared with [100] % of control. Biofilm formation has multiple role including ability to enhance resistance and self-protection from external stress. This chitosanase has promising benefit as antibiofilm agent against biofilm forming pathogenic bacteria and has promising application as alternative antibiofilm agents to combat the growing number of multidrug resistant pathogen-associated infections, especially in situation where biofilms are involved.