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.

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Klebsiella pneumoniae is considered an important opportunistic multidrug-resistant pathogen. Extended spectrum β-lactamases (ESBLs) and expression of a multitude of virulence factors may work in a harmony resulting in treatment failure. This study was undertaken to compare the virulence characteristics and genetic relatedness between ESBL and non-ESBL producing K. pneumoniae. Methods. Antibiotic sensitivity test of all isolates was determined by disc diffusion assay. Phenotypic and genotypic detection of ESBL were done. Various virulence factors and some virulence factor-associated genes were screened. Random amplified polymorphic DNA (RAPD) was employed to investigate the genetic fingerprints of ESBL from non-ESBL producing K. pneumoniae. Results. 50% of isolates were ESBL producers. A significant association was observed between ESBL production and biofilm (strong and moderate), serum resistance, and iss gene. Moreover, significant association between non-ESBL producers and hypermucoviscosity was identified. Dendogram analysis of RAPD profile classified K. pneumoniae isolates into four clusters (a, b, c, and d). Seventy-six percent of ESBL producers belonged to cluster a. In conclusion, this study suggests a correlation between ESBL production and some virulence factors. Therefore, success of treatment depends mainly on increased clinicians awareness and enhanced testing by laboratories to reduce the spread of these isolates.

Introduction: Emergence of carbapenem resistance in Pseudomonas aeruginosa increases the therapeutic dilemma. In this study, we investigated various mechanisms involved in the resistance of P. aeruginosa clinical isolates to carbapenems. Methodology: P. aeruginosa isolates were isolated from different clinical samples. The antimicrobial susceptibility was evaluated by disc diffusion method. Carbapenemases were detected among carbapenem resistant isolates. Expression level of mexB and oprD was determined by real-time PCR. Molecular relatedness among isolates was detected based on pulsed-field gel electrophoresis (PFGE). Results: Ninety P. aeruginosa isolates were purified from clinical specimens. High levels of resistance to imipenem and meropenem were detected in 16 isolates. PCR analysis of carbapenemases indicated the prevalence of Verona integron-encoded metallo-beta-lactamase (VIM); four isolates produced only VIM enzymes (VIM-1 or VIM-2), while the remaining twelve co-produced both VIM-1 or VIM-2 and NDM enzymes. Additionally, real-time PCR analysis elucidated high expression levels of mexB in seven of the carbapenem resistant isolates and low expression of oprD in seven isolates. The identified carbapenem-resistant isolates were clustered into eleven PFGE profiles where clusters E1 and E2 involved isolates exhibiting multiple carbapenemase genes (blaNDM-1, blaVIM-1 and blaVIM-2). Conclusion: Various mechanisms underlying carbapenem resistance have been detected in our P. aeruginosa cohort of isolates. Emergence of P. aeruginosa as a reservoir of multiple carbapenemases is increasing over time limiting the treatment options to this serious infection. This increases the urgency for infection control practices to reduce the incidence of this infection.

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The increasing incidence of β-lactam resistance due to AmpC β-lactamases in Egypt necessitated this study which aimed to evaluate four different phenotypic methods for detection of AmpC β-lactamases among some clinical isolates of Enterobacteriaceae and compare these results with those obtained using polymerase chain reaction. The distribution of five AmpC β-lactamases genes (AmpC, CIT-M, Fox-1, ACC-1, ACT-1) were determined among the clinical isolates. Among 180 clinical isolates of Enterobacteriaceae, only 57 isolates were AmpC producers by phenotypic methods and 108 were AmpC producers by polymerase chain reaction. Phenotypic methods adapted in this study gave variable results with the most discriminatory results given by direct inoculation of both the enzyme extract and the bacterial culture in the wells. Of these, the best results were given by enzyme inoculation methods where 43 isolates exhibited positive result by this method. The distribution of AmpC β-lactamases gene among the clinical isolates showed that AmpC gene predominated in Escherichia coli. Fox gene was predominantly present in Enterobacter cloacae and E. coli isolates. ACT-1 predominated in E. cloacae. In contrast, enzymes from CIT-M and ACC-1 group were rarely present in Enterobacteriaceae.

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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.