The spread of multidrug-resistant Pseudomonas aeruginosa isolates constitutes a serious clinical challenge. Bacterial efflux machinery is a crucial mechanism of resistance among P. aeruginosa. Efflux inhibitors such as phenylalanine arginyl β-naphthylamide (PAβN) promote the bacterial susceptibility to antimicrobial agents. The pathogenesis of P. aeruginosa is coordinated via quorum sensing (QS). This study aims to find out the impact of efflux pump inhibitor, PAβN, on QS and virulence attributes in clinical isolates of P. aeruginosa. P. aeruginosa isolates were purified from urine and wound samples, and the antimicrobial susceptibility was carried out by disc diffusion method. The multidrug-resistant and the virulent isolates U16, U21, W19 and W23 were selected. PAβN enhanced their susceptibility to most antimicrobial agents. PAβN reduced QS signalling molecules N-3-oxo-dodecanoyl-l-homoserine lactone and N-butyryl-l-homoserine lactone without affecting bacterial viability. Moreover, PAβN eliminated their virulence factors such as elastase, protease, pyocyanin and bacterial motility. At the transcription level, PAβN significantly (P<0.01) diminished the relative expression of QS cascade (lasI, lasR, rhlI, rhlR, pqsA and pqsR) and QS regulated-type II secretory genes lasB (elastase) and toxA (exotoxin A) compared to the control untreated isolates U16 and U21. In addition, PAβN eliminated the relative expression of pelA (exopolysaccharides) in U16 and U21 isolates. Hence, P. aeruginosa-tested isolates became hypo-virulent upon using PAβN. PAβN significantly blocked the QS circuit and inhibited the virulence factors expressed by clinical isolates of P. aeruginosa. PAβN could be a prime substrate for development of QS inhibitors and prevention of P. aeruginosa pathogenicity.

Background: Pathogenic Escherichia coli is responsible for serious diseases; i.e.: Peritonitis, colitis, and urinary tract infections (UTIs) and even cancer, resulting in human morbidity and mortality. Environmental strains are increasingly spreading through food and dairy products, contributing to the pathogenetic burden of E. coli infections. Objectives: This study was performed to compare phylogeny, virulence factors, pathogenicity islands (PAIs), and pathotypes inbetween clinical and environmental E. coli isolates. Methods: A total of 105 clinical (72) and environmental (33) E. coli isolates were collected. All isolates were subjected to phylogenetic typing using a new quadruplex polymerase chain reaction (PCR). Wide array of virulence genes (VGs) and PAI markers were assessed for both subtypes, as well as, the distribution of different pathotypes among the phylogenetic groups. Results: Seven phylogenetic groups were detected; clinical isolates were more prevalent in phylogenetic groups B2 (22.2%) and D (23.6%), whereas environmental isolates were in groups A (24.2%) and B1 (60.6%). Majority of VGs were higher in clinical E. coli isolates. Environmental isolates showed higher percentage of some other VGs including; stx2 and hlyA. PAI markers were widespread among both categories, showing high extra-intestinal pathogenic E. coli (ExPEC) PAIs combination in environmental isolates. Enteropathogenic E. coli (EPEC) was the most widespread pathotype in clinical isolates versus enterohemorrhagic E. coli (EHEC) in environmental ones. Conclusions: Escherichia coli pathogenicity armoury was not only confined to clinical isolates, but to environmental ones as well. Therefore, environmental E. coli isolates can serve as reservoirs for transmission of E. coli pathogenicity.

The development of microbial resistance requires a novel approach to control microbial infection. This study implies the microbial synthesis of nanometals and assessment of their antivirulent activity against Pseudomonas aeruginosa. Streptomyces isolate S91 was isolated from soil with substantial ability for growth at high salts concentrations. The cell-free supernatant of S91was utilized for the synthesis of Au-NPs and Se-NPs. The 16S rRNA sequence analysis of Streptomyces S91 revealed that S91 had a high similarity (98.82%) to Streptomyces olivaceous. The biosynthesized Au-NPs and Se-NPs were characterized using a UV-Vis spectrophotometer, dynamic light scattering, transmission electron microscopy, energy dispersive X-ray diffraction and Fourier-transform infrared spectroscopy. The quorum sensing inhibitory (QSI) potential of Au-NPs and Se-NPs and the antivirulence activity was examined against P. aeruginosa. The QSI potential was confirmed using RT-PCR. The synthesized Au-NPs and Se-NPs were monodispersed spherical shapes with particle size of 12.2 and 67.98 nm, respectively. Au-NPs and Se-NPs eliminated QS in P. aeruginosa at a concentration range of 2.3–18.5 µg/mL for Au-NPs and 2.3–592 µg/mL for Se-NPs. In addition, Au-NPs and Se-NPs significantly inhibited QS-related virulence factors, such as pyocyanin, protease and, elastase in P. aeruginosa. At the molecular level, Au-NPs and Se-NPs significantly suppressed the relative expression of QS genes and toxins. Hence, the biosynthesized Au-NPS and Se-NPS could be substantial inhibitors of QS and virulence traits of P. aeruginosa.

Over the past decades, Escherichia coli (E. coli) have acquired extensive resistance to antibiotics; especially β- lactams. This study aimed to investigate the frequency of Extended-spectrum β-lactamase (ESBL) and carbapenemase producers among E. coli isolates and their correlation with serotypes, phylogenetic background, and pathogenicity associated islands. A total of 105 E. coli strains were isolated and subjected to antimicrobial susceptibility testing against β-lactam antibiotics. All isolates showed a high resistance profile. Resistant isolates were tested for ESBL and carbapenemase production. Fifty-three and 18 isolates were positive for ESBL and carbapenemase producers, respectively. ESBL and carbapenemase genes were detected by PCR. TEM gene was the most prevalent gene among all isolates followed by SHV and CTX-M15. In carbapenemase-producers, OXA-48 and IMP were the predominant genes. Enteropathogenic E. coli (EPEC) and Enterohemorrhagic E. coli (EHEC) were the major producers of ESBL and carbapenemase, respectively as indicated by serodiagnosis. They were further assessed for the presence of pathogenicity islands (PAIs) and phylogenetic background. The most predominant DEC PAI and ExPEC PAI were HPI and IICFT073. Most clinically ESBL-producers were group D and B2 while environmentally ones were group B1 and A. On contrary, clinically carbapenemase-producers belonged to group C and D. In conclusion, our study confirms the importance of phylogenetic group D, B2, and C origin for antibiotic resistance in E. coli. Ultimately, our findings support the fact that environmental isolates contribute to the local spread of E. coli pathogenicity in Egypt and these isolates maybe serve as reservoirs for transmission of resistance.

Aim Quorum sensing (QS) inhibition is a promising strategy to suppress bacterial virulence and control infection caused by Gram‐negative and Gram‐positive bacteria. This study explores the QS inhibiting activity of the non‐steroidal anti‐inflammatory drugs (NSAIDs) in Acinetobacter baumannii. Methods and Results Ketoprofen, piroxicam and indomethacin revealed QS inhibition via elimination of violacein production of the reporter strain Chromobacterium violaceum ATCC 12472 without affecting bacterial growth. The minimal inhibitory concentration (MIC) of ketoprofen, piroxicam and indomethacin was determined against A. baumannii strains ATCC 17978, ATCC 19606, A1, A11 and A27 by the microbroth dilution method. The MICs of ketoprofen against tested isolates were 0.7–6.25 mg ml−1, piroxicam MICs were 1.25–2.5 mg ml−1, and indomethacin MICs were 3.12–12.5 mg ml−1. Those compounds significantly inhibited QS‐associated virulence factors such as biofilm formation, and surface motility, as well as, significantly increased bacterial tolerance to oxidative stress without affecting bacterial growth. On the molecular level, the three compounds significantly inhibited the transcription of QS regulatory genes abaI/abaR and biofilm‐regulated genes cusD and pgaB. Molecular docking analysis revealed the potent binding affinity of the three compounds with AbaI via hydrogen and/or hydrophobic bonds. Conclusion These results indicate that NSAIDs, ketoprofen, piroxicam and indomethacin, could be potential inhibitors of the QS and could suppress the QS‐related virulence factors of A. baumannii. Significance and Impact Ketoprofen, piroxicam and indomethacin could provide promising implications and strategies for combating the virulence and pathogenesis of A. baumannii.

Background Proteus mirabilis is an opportunistic pathogen, causing a variety of community-acquired and nosocomial illnesses. It poses a potential threat to patients via the production of β-lactamases, which decrease the efficacy of antimicrobial treatment and impair the management of its pathogenicity. Hence, this study was established to determine the prevalence of extended-spectrum β-lactamases (ESBLs), AmpC, and carbapenemases of P. mirabilis isolated from various clinical specimens. Results Proteus mirabilis was identified in 20.7% (58/280) of specimens. ESBL producers were present at a rate of 51.7% (30/58). All AmpC-positive isolates (n = 20) produced ESBLs as well, so 66.7% of ESBL-producing isolates coproduced AmpC enzymes. The modified Hodge test confirmed carbapenemase production in six out of seven imipenem nonsusceptible isolates. Of these, only two (5.7%) isolates were also ESBL-and AmpC-positive. Antibiotic resistance reached the highest level for cotrimoxazole (62.1%, n = 36/58 isolates) and the lowest for imipenem (12.1%, n = 7/58 isolates). The levels of multidrug-resistant (MDR) was 41.4% among the tested isolates. The blaSHV (83.3%), blaAmpC (80%), and blaVIM-1 (50%) were the most detected genes in phenotypically confirmed ESBL-, AmpC-, and carbapenemase-producing isolates, respectively. Besides, more than a half of the tested P. mirabilis strains (53%) coproduced ESBLs and AmpC. Moreover, two isolates coproduced ESBLs and AmpC together with carbapenemases. Furthermore, dendrogram analysis showed great genetic divergence based on the 21 different enterobacterial repetitive intergenic consensus (ERIC) patterns (P1–P21) through the 34 β-lactamase producers. ERIC analysis distinguished clonal similarities between isolates 21 and 22 in P2 and 9 and 10 in P4, which were isolated from the same clinical source and possessed similar patterns of β-lactamase-encoding genes. Conclusion Hence, there is an urgent need to monitor hospitalized patients and improve healthcare in order to reduce the incidence of infection and outbreaks of infection with antibiotic-resistant Proteus.

Background: Fluconazole (FLZ), a potent antifungal medication, is characterized by poor water solubility that reduced its antifungal efficacy. Objective: This study aimed to prepare FLZ-loaded polymeric nanoparticles (NPs) by using different polymers and techniques as a mean of enhancing the antifungal activity of FLZ. Methods: NP1, NP2, and NP3 were prepared by the double emulsion/solvent evaporation method using PLGA, PCL, and PLA, respectively. The ionotropic pre-gelation technique was applied to prepare an alginate/chitosan-based formulation (NP4). Particle size, zeta potential, encapsulation efficiency, and loading capacity were characterized. FT-IR spectra of FLZ, the polymers, and the prepared NPs were estimated. NP4 was selected for further in-vitro release evaluation. The broth dilution method was used to assess the antifungal activity of NP4 using a resistant clinical isolate of Candida albicans. Results: The double emulsion method produced smaller-sized particles (<390 nm) but with much lower encapsulation efficiency (< 12%). Alternatively, the ionic gelation method resulted in nanosized particles with a markedly higher encapsulation efficiency of about 40%. The FT-IR spectroscopy confirmed the loading of the FLZ molecules in the polymeric network of the prepared NPs. The release profile of NP4 showed a burst initial release followed by a controlled pattern up to 24 hours with a higher percent released relative to the free FLZ suspension. NP4 was able to reduce the value of MIC of FLZ by 20 times. Conclusion: The antifungal activity of FLZ against C. albicans was enhanced markedly via its loading in the alginate/chitosan-based polymeric matrix of NP4.

No abstract