Phenotypic and molecular characterization of extended spectrum- and metallo- beta lactamase producing Pseudomonas aeruginosa clinical isolates from Egypt.

BACKGROUND: Antimicrobial resistance among Pseudomonas aeruginosa (P. aeruginosa), a leading cause of nosocomial infections worldwide, is escalating. This study investigated the prevalence of extended-spectrum ß-lactamases (ESBLs) and metallo-ß-lactamases (MBLs) among 104 P. aeruginosa clinical isolates from Alexandria Main University Hospital, Alexandria, Egypt. METHODS: Antimicrobial susceptibility testing was performed using agar dilution technique, or broth microdilution method in case of colistin. ESBL and MBL prevalence was assessed phenotypically and genotypically using polymerase chain reaction (PCR). The role of plasmids in mediating resistance to extended-spectrum ß-lactams was studied via transformation technique using plasmids isolated from ceftazidime-resistant isolates. RESULTS: Antimicrobial susceptibility testing revealed alarming resistance rates to carbapenems, cephalosporins, and fluoroquinolones. Using PCR as the gold standard, phenotypic methods underestimated ESBL production while overestimating MBL production. Eighty-five isolates (81.7%) possessed only ESBL encoding genes, among which 69 isolates harbored a single ESBL gene [blaOXA-10 (n = 67) and blaPER (n = 2)]. Four ESBL-genotype combinations were detected: blaPER + blaOXA-10 (n = 8), blaVEB-1 + blaOXA-10 (n = 6), blaPSE + blaOXA-10 (n = 1), and blaPER + blaVEB-1 + blaOXA-10 (n = 1). Three isolates (2.9%) possessed only the MBL encoding gene blaVIM. Three ESBL + MBL- genotype combinations: blaOXA-10 + blaAIM, blaOXA-10 + blaVIM, and blaPER + blaOXA-10 + blaAIM were detected in 2, 1 and 1 isolate(s), respectively. Five plasmid preparations harboring blaVEB-1 and blaOXA-10 were successfully transformed into chemically competent Escherichia coli DH5α with transformation efficiencies ranging between 6.8 × 10 3 and 3.7 × 10 4 CFU/µg DNA plasmid. Selected tested transformants were ceftazidime-resistant and harbored plasmids carrying blaOXA-10. CONCLUSIONS: The study highlights the importance of the expeditious characterization of ESBLs and MBLs using genotypic methods among P. aeruginosa clinical isolates to hinder the development and dissemination of multidrug resistant strains.

Prevalence of different virulence factors and their association with antimicrobial resistance among Pseudomonas aeruginosa clinical isolates from Egypt.

BACKGROUND: Emergence of multi-drug resistant Pseudomonas aeruginosa, coupled with the pathogen's versatile virulence factors, lead to high morbidity and mortality rates. The current study investigated the potential association between the antibiotic resistance and the production of virulence factors among P. aeruginosa clinical isolates collected from Alexandria Main University Hospital in Egypt. We also evaluated the potential of the phenotypic detection of virulence factors to reflect virulence as detected by virulence genes presence. The role of alginate in the formation of biofilms and the effect of ambroxol, a mucolytic agent, on the inhibition of biofilm formation were investigated. RESULTS: A multi-drug resistant phenotype was detected among 79.8% of the isolates. The most predominant virulence factor was biofilm formation (89.4%), while DNase was least detected (10.6%). Pigment production was significantly associated with ceftazidime susceptibility, phospholipase C production was significantly linked to sensitivity to cefepime, and DNase production was significantly associated with intermediate resistance to meropenem. Among the tested virulence genes, lasB and algD showed the highest prevalence rates (93.3% and 91.3%, respectively), while toxA and plcN were the least detected ones (46.2% and 53.8%, respectively). Significant association of toxA with ceftazidime susceptibility, exoS with ceftazidime and aztreonam susceptibility, and plcH with piperacillin-tazobactam susceptibility was observed. There was a significant correlation between alkaline protease production and the detection of algD, lasB, exoS, plcH and plcN; pigment production and the presence of algD, lasB, toxA and exoS; and gelatinase production and the existence of lasB, exoS and plcH. Ambroxol showed a high anti-biofilm activity (5% to 92%). Quantitative reverse transcriptase polymerase chain reaction showed that alginate was not an essential matrix component in P. aeruginosa biofilms. CONCLUSIONS: High virulence coupled with the isolates' multi-drug resistance to commonly used antimicrobials would increase morbidity and mortality rates among P. aeruginosa infections. Ambroxol that displayed anti-biofilm action could be suggested as an alternative treatment option, yet in vivo studies are required to confirm these findings. We recommend active surveillance of antimicrobial resistance and virulence determinant prevalence for better understanding of coregulatory mechanisms.

Preparation and evaluation of periodontal films based on polyelectrolyte complex formation.

Local intra-pocket drug delivery devices can provide an effective concentration of the antimicrobial agent at the site of action with avoidance of undesirable side effects. This study explored the application of chitosan-alginate and chitosan-pectin polyelectrolyte complex (PEC) films as drug release regulators for tetracycline HCl (Tc) to treat periodontal pockets. Periodontal films with 1:1 Tc:PEC ratio were prepared using 1:1 chitosan (Ch) to sodium alginate (A) or 1:3 Ch to pectin (P). The scanning electron microscope showed acceptable film appearance and differential scanning calorimetry analysis confirmed complex formation. The in vitro release studies for both films showed a burst drug release, followed by prolonged release for 70 h. A prolonged antibacterial activity of both films against Staphylococcus aureus ATCC 6538 was observed over a period of 21 days. Aging studies indicated that the five months storage period in freezer did not significantly influence the drug release profile or the antibacterial activity of both films. Clinical evaluation showed a significant reduction in pocket depth (p < 0.0001) to their normal values (≤3 mm). PEC films could be exploited as a prolonged drug release devices for treatment of periodontal pockets.

Comparative study to investigate the effect of meloxicam or minocycline HCl in situ gel system on local treatment of periodontal pockets.

In situ gelling formulations allow easy application to the target area. Gelation is induced by physiological stimuli at the site of application where the formula attains semisolid properties and exerts sustained drug release. In situ gelling formulations containing either 3% meloxicam (Mx) or 2% minocycline HCl (MH) were prepared for local application into the periodontal pockets. Gel formulations were based on the thermosensitive Pluronic(®) (Pl) and the pH-sensitive Carbopol(®) (C) polymers. C gels were prepared in combination with HPMC (H) to decrease its acidity. The total percent drug released from Pl formulae was 21.72% after 1 week for Mx and 85% after 3 days for MH. Their release kinetics data indicated anomalous non-Fickian behavior that could be controlled by both diffusion and chain relaxation. Addition of MH to C/H gels (1:2.5) resulted in liquefaction, followed by drug precipitation. Regarding C/H gel containing Mx, it showed a prolonged release rate up to 7 days with an initial burst effect; the kinetics data revealed Fickian-diffusion mechanism. The in vitro antibacterial activity studies for MH gel in Pl revealed that the drug released exceeded the minimum inhibitory concentration (MIC) of MH against Staphylococcus aureus ATCC 6538; placebo gel showed no effect on the microorganism. Clinical evaluation of Pl gels containing either Mx or MH showed significant improvement in chronic periodontitis patients, manifested by decrease in pocket depth and gingival index and increase in bone density.

Role of plasmid carrying bla NDM in mediating antibiotic resistance among Acinetobacter baumannii clinical isolates from Egypt.

We investigated antibiotic resistance levels among bla NDM -positive (n = 9) and -negative (n = 65) A. baumannii clinical isolates collected in 2010 and 2015 from Alexandria Main University Hospital, Egypt using disc diffusion and minimum inhibitory concentration (MIC) determination. Plasmids from bla NDM -positive isolates were transformed into a carbapenem-susceptible A. baumannii (CS-AB) isolate to assess the role of plasmid transfer in mediating carbapenem resistance. Imipenem, meropenem, and ertapenem MIC90 values against bla NDM -positive isolates were 128, > 256, and 256 µg/mL, respectively. Plasmid isolation and polymerase chain reaction revealed that bla NDM was plasmid mediated. The plasmids were electroporated into the cells of a CS-AB isolate at an efficiency of 1.3 × 10-8 to 2.6 × 10-7, transforming them to bla NDM -positive carbapenem-resistant cells with an imipenem MIC increase of 256-fold. In addition to carbapenem resistance, the bla NDM -positive isolates also exhibited higher levels of cephalosporins, tetracycline, aminoglycosides, fluoroquinolones, and colistin resistance than the bla NDM -negative isolates. Acquisition of bla NDM -carrying plasmids dramatically increased imipenem resistance among A. baumannii isolates. Intriguingly, bla NDM -positive isolates also showed a high degree of resistance to antibiotics of different classes. The potential co-existence of different resistance determinants on A. baumannii plasmids and their possible transfer owing to the natural competence of the pathogen are especially alarming. More effective infection control and antibiotic stewardship programs are needed to curb the spread and treat such infections in both hospital and community settings.

Phenotypic and genotypic characterization of carbapenem-resistant Acinetobacter baumannii isolates from Egypt.

Background: Antibiotic use is largely under-regulated in Egypt leading to the emergence of resistant isolates. Carbapenems are last resort agents to treat Acinetobacter baumannii infections resistant to other classes of antibiotics. However, carbapenem-resistant isolates are emerging at an alarming rate. This study aimed at phenotypically and molecularly characterizing seventy four carbapenem-unsusceptible A. baumannii isolates from Egypt to detect the different enzymes responsible for carbapenem resistance. Methods: Carbapenemase production was assessed by a number of phenotypic methods: modified Hodge test (MHT), carbapenem inactivation method (CIM), combined disc test (CDT), CarbAcineto NP test and boronic acid disc test. Polymerase chain reaction (PCR) was used to screen the isolates for the presence of some genes responsible for resistance to carbapenems, as well as some insertion sequences. Results: PCR amplification of class D carbapenemases revealed the prevalence of blaOXA-51 and blaOXA-23 in 100% of the isolates and of blaOXA-58 in only one isolate (1.4%). blaVIM and blaNDM-1 belonging to class B metallo-ß-lactamases were present in 100 and 12.1% of the isolates, respectively. The prevalence of ISAba1, ISAba2 and ISAba3 was 100, 2.7 and 4.1%, respectively. None of the tested isolates carried blaOXA-40 , blaIMP , blaSIM , blaSPM , blaGIM or the class A blaKPC . Taking PCR as the gold standard method for the detection of different carbapenemases, the sensitivities of the MHT, CIM, CDT, CarbAcineto NP test and boronic acid disc/imipenem or meropenem test for this particular collection of isolates were 78.4, 68.9, 79.7, 95.9, and 56.8% or 70.3%, respectively. Conclusions: The widespread detection of carbapenem-resistant A. baumannii (CR-AB) has become a real threat to the efficacy of treatment regimens. Among the studied cohort of CR-AB clinical isolates, blaOXA-51 , blaOXA-23 and blaVIM were the most prevalent, followed by blaNDM-1 and blaOXA-58 . The genotypic detection of carbapenemases among CR-AB clinical isolates using PCR was most conclusive, followed closely by the phenotypic testing using CarbAcineto NP test.

Microbiological assessment of the ultraviolet screening effect of sunscreen preparations.

The purpose of this study was the development of a microbiological method for the assessment of the ultraviolet (UV) screening effect of sunscreen preparations and determination of their sun protection factor. The method is based on the lethal effect of UV radiation on Escherichia coli (E. coli ) and the protective ability of sunscreens. The time of UV exposure required for the reduction of the E. coli viable count by 90% (decimal reduction time, DRT) was used as the photoprotection assessment parameter. The method was tested by assessing the effect of selected experimental variables on the DRT. The suitability of the method as a quality control tool for sunscreen preparations was then checked by assessing the influence of selected formulation variables on the photoprotective effect of a series of o/w emulsion formulations with different compositions. The method proved valid for detecting changes in the photoprotective effect of a market sunscreen product as a result of modifying experimental conditions. It also proved valid for ranking market sunscreen products according to their UV screening effect. Equally important, the method could successfully detect changes in the photoprotective effect of sunscreen test formulations as a function of the concentration and type of the sunscreen agents.

Dissemination of multiple carbapenem-resistant clones of Acinetobacter baumannii in the Eastern District of Saudi Arabia.

It has previously been shown that carbapenem-resistant Acinetobacter baumannii are frequently detected in Saudi Arabia. The present study aimed to identify the epidemiology and distribution of antibiotic resistance determinants in these bacteria. A total of 83 A. baumannii isolates were typed by pulsed-field gel electrophoresis (PFGE), and screened by PCR for carbapenemase genes and insertion sequences. Antibiotic sensitivity to imipenem, meropenem, tigecycline, and colistin were determined. Eight different PFGE groups were identified, and were spread across multiple hospitals. Many of the PFGE groups contained isolates belonging to World-wide clone 2. Carbapenem resistance or intermediate resistance was detected in 69% of isolates. The bla VIM gene was detected in 94% of isolates, while bla OXA-23-like genes were detected in 58%. The data demonstrate the co-existence and wide distribution of a number of clones of carbapenem-resistant A. baumannii carrying multiple carbapenem-resistance determinants within hospitals in the Eastern Region of Saudi Arabia.

Tetrazolo[1,5-a]quinoline as a potential promising new scaffold for the synthesis of novel anti-inflammatory and antibacterial agents.

Three series of tetrazolo[1,5-a]quinoline derivatives have been synthesized. The first series was synthesized starting by the condensation of tetrazolo[1,5-a]quinoline-4-carboxaldehyde 2 with substituted thiosemicarbazides, followed by cyclization of the resulting thiosemicarbazones 3 with malonic acid in the presence of acetyl chloride to give pyrimidyl derivatives 4a-c. The second series was prepared by the condensation of the latter compounds 4a-c with the selected aromatic aldehydes to afford the arylidene derivatives 5a-f. The third series 7a-c was synthesized by condensation of tetrazolo[1,5-a]quinoline-4-carboxaldehyde 2 with the appropriate acetophenone, followed by cyclocondensation of the formed alpha,beta-unsaturated ketones with thiourea. The newly synthesized compounds were evaluated for their anti-inflammatory and antimicrobial activities. Four compounds were proved to be as active as indomethacin in animal models of inflammation.

Clonal diversity of Acinetobacter baumannii from diabetic patients in Saudi Arabian hospitals.

Carbapenem-resistant Acinetobacter baumannii (CR-AB) represents a major health-care problem, causing high rates of morbidity and mortality. This study investigated the clonality of CR-AB isolated from diabetic patients from different regions in Saudi Arabia, as well as the relatedness of the ß-lactamase genes. A total of 64 non-repetitive CR-AB clinical isolates were collected from 16 different regions in Saudi Arabia from intensive care patients. Isolates were identified phenotypically by the Vitek 2 compact system and genotypically by amplification of the blaOXA-51-like gene. The target sequences were amplified by PCR and the clonal diversity of the isolates was explored by PFGE. Resistance studies revealed that the prevalence of imipenem and meropenem resistance was 92% and 96%, respectively, while the vast majority of the isolates were susceptible to tigecycline and colistin. In addition, blaVIM and blaOXA-23 were the most prevalent genes in the isolates under investigation, while ISAba1 was the most dominant insertion sequence. PFGE results showed 13 clusters; clone H was dominant, comprising 20 isolates from four hospitals, followed by clones C and F, comprising 11 isolates each from three and six hospitals, respectively. Moreover, the current study signified the clonal diversity of CR-AB in Saudi Arabia and showed the ability of some clones to infect patients in many different cities.

A framework for a competency based medical curriculum in saudi arabia.

BACKGROUND: We recently adopted a competency based curriculum based on the CanMEDs model. This shift required the cross-mapping of all key CanMEDs competencies with the competencies for higher education in Saudi Arabia as per the Saudi National Commission for Academic Accreditation & Assessment (NCAAA) guidelines. OBJECTIVES: To formulate competencies for our curriculum and to create a framework aligned with NCAAA, CanMEDs and Saudi Meds. METHODS: After finalization of program outcomes, the program goals were cross-mapped with CanMEDs and Saudi Meds competencies and then the CanMEDs competencies were reverse mapped with our outcomes. Finally benchmarking of outcomes with the programs of the Universities of Manitoba and Toronto was done. RESULTS: We were able to cross-map and match major outcomes of our program with both the CanMEDs and the Saudi Meds frameworks, ensuring that the outcomes are in line with NCAAA, CanMEDs and Saud Meds. Also, our program objectives were bench marked with two of the Canadian medical schools. CONCLUSION: We propose that our framework can be a model for other universities in Saudi Arabia to consider when shifting to a competency based curriculum.

Identification of the Anabaena sp. strain PCC7120 cyanophycin synthetase as suitable enzyme for production of cyanophycin in gram-negative bacteria like Pseudomonas putida and Ralstonia eutropha.

The cyanophycin synthetase gene cphA1 encoding the major cyanophycin synthetase (CphA) of Anabaena sp. strain PCC7120 was expressed in Escherichia coli conferring so far the highest specific CphA activity to E. coli (6.7 nmol arginine per min and mg protein). CphA1 and cphA genes of Synechocystis sp. strains PCC6803 and PCC6308 and Synechococcus strain MA19 were also expressed in wild types and polyhydroxyalkanoate-negative (PHA) mutants of Pseudomonas putida and Ralstonia eutropha. Recombinant strains of these bacteria expressing cphA1 accumulated generally more cyanophycin (23.0 and 20.0% of cellular dry matter, CDM, respectively) than recombinants expressing any other cphA (6.8, 9.0, or 15.8% of CDM for P. putida strains and 7.3, 12.6, or 14.1% of CDM for R. eutropha). Furthermore, PHA-negative mutants of P. putida (9.7, 10.0, 17.5, or 24.0% of CDM) and R. eutropha (8.9, 13.8, 16.0, or 22.0% of CDM) accumulated generally more cyanophycin than the corresponding PHA-positive parent strains (6.8, 9.0, 15.8, and 23.0% of CDM for P. putida strains and 7.3, 12.6, 14.1, or 20.0% of CDM for R. eutropha strains). Recombinant strains of Gram-positive bacteria (Bacillus megaterium, Corynebacterium glutamicum) were not suitable for cyanophycin production due to accumulation of less cyanophycin and retarded release of cyanophycin. PHA-negative mutants of P. putida and R. eutropha expressing cphA1 of Anabaena sp. strain PCC7120 are therefore preferred candidates for industrial production of cyanophycin.