Abdelgawad Hashem

Emergence of carbapenem-resistant Acinetobacter baumannii harboring the OXA-23 carbapenemase in intensive care units of Egyptian hospitals

BACKGROUND Healthcare-associated infections are a worldwide threat to hospitalized patients, especially those in intensive care units. The prevalence of these infections in Egypt, and their antimicrobial resistance patterns and mechanisms, were investigated in this study. METHODS A total of 547 cases of healthcare-associated infections were investigated. Causative agents were identified and antimicrobial susceptibility determined. Carbapenem-resistant Acinetobacter baumannii isolates were further investigated for their resistance mechanism via the modified Hodge test, inhibitor-potentiated disk diffusion test, synergy with carbonyl cyanide chlorophenylhydrazone, and PCR. Moreover, clonal linkage was examined via enterobacterial repetitive intergenic consensus (ERIC)-PCR. RESULTS Klebsiella spp was the most prevalent species in the isolates examined (217; 40%). Although A. baumannii represented only 10% of the total isolates, it showed the highest percentage of carbapenem resistance (74%). PCR showed that 100% of the resistant isolates carried both blaOXA-51 and blaOXA-23 genes, 85% carried the class 1 integrase genes, and only 2.5% carried metallo-beta-lactamase (blaVIM). ERIC-PCR indicated that isolates from different hospitals were genetically linked. CONCLUSIONS These findings represent the first report of the alarming spread of OXA-23 carbapenemase in A. baumannii in Egyptian intensive care units. The spread of such strains has serious health consequences and requires the application of strict infection control measures.

Biochemical studies on immobilized fungal β-glucosidase

β-Glucosidase from Aspergillus niger was immobilized on sponge by covalent binding through a spacer group (glutaraldehyde). Sponge-immobilized enzyme had the highest immobilization yield (95.67%) and retained 63.66% of the original activity exhibited by the free enzyme. The optimum pH of the immobilized enzyme remains almost the same as for the free enzyme (pH 4.0). The optimum temperature for β-glucosidase activity was increased by 10 oC after immobilization. The activation energy (Ea) of the immobilized β-glucosidase was lower than the free enzyme (3.34 and 4.55 kcal/mol), respectively. Immobilized β-glucosidase exhibited great thermal stability and retained all the initial activity after incubation at 55 oC for 2 h; however, the free enzyme retained 89.25% under the same condition. The calculated half-life (t½) value of heat inactivation of immobilized enzyme at 60, 65 and 70 oC was 213.62, 72.95 and 56.80 min, respectively, whereas at these temperatures the free enzyme was less stable (half-life of 200.0, 55.31 and 49.5 min, respectively). The deactivation rate constant at 65 oC for the immobilized β-glucosidase is 9.5x10-3/ min, which was lower than that of the free form (12.53x10-3/ min). The immobilization process improved the pH stability of the enzyme (immobilized and free enzyme retained 69.35 and 39.86%, respectively, of their initial activity after 45 min at pH 7.5). The effect of some chemical substances on the activity of the immobilized and free β-glucosidase has been investigated. In the presence of sodium dodecyl sulfate (SDS) and p-chloromercuri benzoate (p-CMB) the immobilized enzyme retained 36.13 and 45.34%, respectively, of the initial activity, which is higher than that of free enzyme (13.71 and 1.61%, respectively). The Michaelis constant (Km) value of the free enzyme was 40.0 mM, while the apparent Km value for the immobilized enzyme was 46.51 mM. The maximum reaction rate (vmax) of immobilized β-glucosidase was smaller than that of the free enzyme by 7.69%. Sponge-immobilized β-glucosidase was repeatedly used to hydrolyze cellobiose (5 and 8 cycles with retained activity of 67.32 and 51.04%, respectively).

‘Supermutators’ found amongst highly levofloxacin-resistant E. coli isolates: a rapid protocol for the detection of mutation sites

Fluoroquinolone resistance is gradually acquired through several mechanisms. In particular, chromosomal mutations in the genes encoding topoisomerases II and IV and increased expression of the multidrug efflux pump AcrAB-TolC are the most common mechanisms. In this study, multiplex polymerase chain reaction (PCR) protocols were designed for high-throughput sequencing of the quinolone resistance determining regions of topoisomerases genes (gyrA, parC and parE) and/or the expression regulation systems of multidrug efflux pump AcrAB (acrRAB, marRAB and soxSR). These protocols were applied to sequence samples from five subpopulations of 103 clinical Escherichia coli isolates. These subpopulations were classified according to their levofloxacin susceptibility pattern as follows: highly resistant (HR), resistant (R), intermediate (I), reduced susceptibility (RS) and susceptible (S). All HR isolates had mutations in the six genes surveyed, with two ‘supermutator’ isolates harboring 13 mutations in these six genes. Strong associations were observed between mutations in acrR and HR isolates, parE and R/HR isolates and parC and I/R/HR isolates, whereas surprisingly, gyrA mutations were common in RS/I/R/HR isolates. Further investigation revealed that strong associations were limited to the triple mutations gyrA-S83L/D87N/R237H and HR isolates and the double mutations S83L/D87N and I/R/HR isolates, whereas the single mutation S83L was common in RS/I/R/HR isolates. Interestingly, two novel mutations (gyrA-R237H and acrR-V29G) were located and found to strongly associate with HR isolates. To the best of our knowledge, the gyrA-R237H and acrR-V29G mutations have never been reported and require further investigation to determine their exact role in resistance or ‘fitness’ as defined by their ability to compensate for the organismal cost of gaining resistance.

Isolation and Characterization of Relevant Algal and Bacterial Strains from Egyptian Environment for Potential Use in Photosynthetically Aerated Wastewater Treatment

A number of environmental samples were collected from different locations in Egypt. Briefly, 9 bacterial strains and 4 algal strains were isolated and characterized. All isolated bacterial strains showed a remarkable ability to tolerate and/or biodegrade phenol (aromatic pollutant) and pyridine (hetero-aromatic pollutant). Phenol showed higher toxicity than pyridine to both bacterial and algal isolates. The bacterial isolates were identified as members of Pseudomonas, Chryseomonas, Sphingomonas and Burkholderiae species. The highest biodegradation rate and capacity were reported to bacterial isolate M4, identified as Pseudomonas MT1. This strain was able to degrade up to 1700 and 3000 mg l-1 of phenol and pyridine respectively. Pseudomonas MT1 showed the highest phenol biodegradation rate of 29 mg h-1 and lag phase approximately of 8 h and was optimally grown on 1000 -1250 mg phenol l-1. All algal isolates were morphologically identified as members of the Chlorella genus. None of the isolated algal strains showed biodegradation ability of any of the tested organic pollutants. However, isolates A1, A2 and A4 showed remarkable tolerance to both phenol and/or pyridine. The highest tolerance capacity was reported to algal isolate A4, identified as Chlorella vulgaris MM1 with a toxicity cut off of 500 and 1000 mg l-1 of phenol and pyridine respectively. Both Pseudomonas MT1 and Chlorella vulgaris MM1 had no inhibitory effect on each other. Therefore, they represented potential candidates for the construction of algal bacterial microcosm used for the photosynthetically aerated biological degradation of effluents loaded with various organic pollutants.

Chemical modification of Aspergillus niger β-glucosidase and its catalytic properties.

Aspergillus niger β-glucosidase was modified by covalent coupling to periodate activated polysaccharides (glycosylation). The conjugated enzyme to activated starch showed the highest specific activity (128.5 U/mg protein). Compared to the native enzyme, the conjugated form exhibited: a higher optimal reaction temperature, a lower Ea (activation energy), a higher K m (Michaelis constant) and Vmax (maximal reaction rate), and improved thermal stability. The calculated t 1/2 (half-life) values of heat in-activation at 60 °C and 70 °C were 245.7 and 54.5 min respectively, whereas at these temperatures the native enzyme was less stable (t 1/2 of 200.0 and 49.5 min respectively). The conjugated enzyme retained 32.3 and 29.7%, respectively from its initial activity in presence of 5 mM Sodium Dodecyl Sulphate (SDS) and p -Chloro Mercuri Benzoate ( p -CMB), while the native enzyme showed a remarkable loss of activity (retained activity 1.61 and 13.7%, respectively). The present work has established the potential of glycosylation to enhance the catalytic properties of β-glucosidase enzyme, making this enzyme potentially feasible for biotechnological applications.

Approaches to improve the solubility and availability of progesterone biotransformation by Mucor racemosus

Abstract Substrate solubility in steroid biotransformation is critical for improving the biotransformation of hydrophobic substrates. The present investigation describes the effect of some organic solvents, polyoxyethylene sorbitan monooleate (Tween 80; surfactant) and β-cyclodextrin (β-CD), on the biotransformation of progesterone (PR) by Mucor racemosus. PR was transformed to three derivatives namely; 11α-hydroxyprogesterone (11α-HP) as main product (I), 4-pregnen-18-al-11β, 21-diol-3, 20-dione (Aldosterone) (II) and 20-hydroxy-pregnan-18-oic acid (III) as minor products. Among the tested organic solvents, methanol gave the highest total bioconversion efficiency (T.B.E) of 75.0 ± 2.49%. Tween 80 improved PR biotransformation up to 81.56 ± 2.29%, while with an optimum molar ratio of PR to β-CD of 1:1, the TBE was increased to 87.15 ± 2.17% of the added substrate in presence of β-CD. The interaction between PR and β-CD in the inclusion complex was assessed using differential scanning calorimetry (DSC) and infrared (IR) spectra.

Bioinformatics determination of ETEC signature genes as potential targets for molecular diagnosis and reverse vaccinology

Background Genomes of the model bacterium, Escherichia coli, exhibit high plasticity caused by gene gain/loss via pathoadaptive mutations, genetic rearrangement, and horizontal gene transfer [1,2]. This genetic variability is also translated into a remarkable phenotypic and pathotypic diversity: while some E. coli strains normally inhabit the mammalian colon, other pathotypes cause a wide range of intestinal and extraintestinal diseases that include mild intestinal disturbance but also severe urinary tract infections and outbreaks of shigellosis-like dysentery or cholera-like watery diarrhea [1]. In this study, we focus on enterotoxigenic E. coli (ETEC), one of the worlds deadliest infectious agents, which also represents a serious public health in Egypts rural areas. Our aim is to integrate multiple bioinformatics tools to determine horizontally transferred, pathotype-specific signature genes as targets for specific, high-throughput molecular diagnostic tools and reverse vaccinology screens.