Isolation and molecular characterization of Frankia strains resistant to some heavy metals.

Frankia strains isolated from Saudi Arabia, reported for the first time, were identified based on the morphological and molecular tools compared to those isolated from Egypt. All strains displayed typical morphological characterization of Frankia strains represented by branched hyphae, production of vesicles and sporangia. The phylogenetic analysis and relationships among Frankia strains were investigated by comparing 16S rRNA gene sequences. The analysis revealed three genetic groups which formed two clusters. The first cluster was composed of eight Frankia strains subdivided into two genetic groups (one group containing five strains; CgIT3 L2 , CgIS3 N2 , CgIS1 N1, CgIT7N2, and G5; the other group included of three strains: CgIT5L3, CgIS1 N2 , and CcI13). The second cluster was composed of only one genetic group of Frankia strain CgIS3 N1 . The strains in each genetic group exhibited similar genetic distances. All Frankia strains were able to reinfect their host of Casuarina species. For ability of these strains to resist heavy metals, our results proved that all Frankia strains isolated can resist Cu, Co, and Zn at low concentration except Pb which exhibit highly toxic effect at the same concentration used. Frankia strain G5 was proved to be the most resistant strain for heavy metals tested.

Combating Bacterial Biofilm Formation in Urinary Catheter by Green Silver Nanoparticle.

Urinary catheters are commonly associated with urinary tract infections. This study aims to inhibit bacterial colonisation and biofilm of urinary tract catheters. Silicon catheter pieces were varnished with green silver nanoparticles (AgNPs) using Pistacia lentiscus mastic to prevent bacterial colonisation. Pomegranate rind extract was used to synthesize AgNPs. AgNPs were characterized by UV-Vis spectroscopy, X-ray crystallography, and transmission electron microscopy (TEM). Results obtained revealed that the size of most AgNPs ranged between 15-25 nm and they took crystallised metal and oxidised forms. The amounts of released silver ions from 1 cm pieces of catheters coated with AgNPs were estimated for five days and ranged between 10.82 and 4.8 µg. AgNPs coated catheters significantly inhibited the colonisation of catheters by antibiotic-resistant clinical Gram-positive (Staphylococcus epidermidis and Staphylococcus aureus) and Gram-negative (Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis, and Pseudomonas aeruginosa) bacteria. AgNPs-varnish was more active against Gram-negative bacteria than Gram-positive bacteria. The significant inhibitory effect of coated catheters lasted for 72 h for both Gram-positive and Gram-negative bacteria. Varnishing catheters with AgNPs may help to prevent bacterial colonisation and infections.

In vitro Effects of Punica granatum Ellagitannins on Adult Worms of Schistosoma mansoni.

Schistosomiasis ranks second behind malaria in terms of overall morbidity and mortality. We evaluated the lethal effect of Punica granatum ellagitannins, extracted from the fruit rind, placenta and barks of the root and stem, on adult worms of Schistosoma mansoni (S. mansoni). All four ellagitannins were lethal to S. mansoni adult worms. However, while the rind ellagitannins were the most potent, placental ellagitannins were the least. Rind ellagitannins were capable of killing 40% of adult worms at a concentration of 25µg/mL after 5 days. The killing of 100% of the worms was achievable by rind ellagitannins at a concentration of 50µg/mL after 5 days. The LD50S of the rind ellagitannins after 96h and 120h were 41.25 µg/mL and 28.73 respectively. Ellagitannins-treated worms suffered from erosions, wrinkles, swellings and losses, degenerations of the surface tubercles and tegument. In addition, ellagitannins induced deformation and degradation of oral and ventral suckers and degenerations in the muscles of worms. Ellagitannins also caused a separation of coupled worms and reduction of their motility. Data obtained suggest that ellagitannins of pomegranate could be considered as a cheap candidate for the treatment of schistosomiasis.

The First Egyptian Report Showing the Co-Existence of bla NDM-25, bla OXA-23, bla OXA-181, and bla GES-1 Among Carbapenem-Resistant K. pneumoniae Clinical Isolates Genotyped by BOX-PCR.

BACKGROUND AND OBJECTIVE: The emergence of carbapenem-resistant K. pneumoniae (CRKP) continues to escalate and is alarming because of the emergence of pan drug-resistant strains. The objective of this study was to investigate the existence of 12 carbapenemase genes among CRKP clinical isolates. METHODS: Ninety-six Klebsiella spp. clinical isolates were collected. The isolates were identified phenotypically and genotypically. These isolates were screened for susceptibility to 24 different antibiotics. The modified Hodge test (MHT) and the Carba Nordmann/Poirel (NP) test were used to phenotypically screen carbapenem-resistant strains for carbapenemase production. Phenotypic characterization of carbapenemases was performed using the combined disk synergy test (CDST). Additionally, the presence of 12 carbapenemase genes in CRKP isolates was investigated. The DNA sequence of bla NDM and bla GES genes was determined. The BOX-PCR technique was used to determine the clonal relationship between CRKP isolates. RESULTS: All carbapenem-resistant isolates were related to K. pneumoniae. Susceptibility testing showed that 19.79% (19/96) of the collected isolates were carbapenem-resistant. Of the CRKP isolates, 68.42% (13/19) tested positive for the MHT and Carba NP test. CDST showed that 42.11% (8/19), 63.16% (12/19), 47.37% (9/19), and 73.68% (14/19) of the CRKP isolates tested positive for the inhibitory effect of clavulanic acid, sulbactam, phenylboronic acid, and tazobactam, respectively, while 84.21% (16/19) and 68.42% (13/16) tested positive for the inhibitory effect of EDTA and mercaptopropionic acid, respectively. It was found that 10.53% (2/19) of the isolates tested positive for the inhibitory effect of sodium chloride. Molecular investigation of carbapenemases showed that 26.32% (5/19), 73.68% (14/19), 21.05% (4/19), 10.53% (2/19), and 5.26% (1/19) of the isolates tested positive for bla NDM, bla OXA-48, bla OXA-181, bla OXA-51, and bla OXA-23, respectively. None of the isolates tested positive for bla OXA-40 and bla OXA-58. Two allelic variants of bla NDM (bla NDM-1 and bla NDM-25) were detected. BOX-PCR revealed high clonal relatedness between CRKP isolates. CONCLUSION: MHT was more sensitive than Carba NP test for evaluating carbapenemase production and class D carbapenemase genes were the most prevalent of the 12 carbapenemase genes that were evaluated.

Investigation of six plasmid-mediated quinolone resistance genes among clinical isolates of pseudomonas: a genotypic study in Saudi Arabia.

Background: Quinolones are among the most effective antibiotics against Pseudomonas spp. Several chromosomal and/or plasmid-mediated quinolone-resistance mechanisms have been found in Pseudomonas.  Plasmid-mediated quinolone-resistance (PMQR) is mediated by quinolone-resistance (QNR) proteins, modifying enzymes or efflux pumps. Only a few previous studies examined the prevalence of quinolone-resistance in the Kingdom of Saudi Arabia (KSA) and showed it is increasing. Mechanisms of quinolone-resistance among Pseudomonas spp. in the KSA; examined herein; have not been extensively studied. Methods: Ninety-two Pseudomonas isolates were collected and their resistance to seven different types of quinolones was determined by the microbroth dilution method. PMQR mechanisms were examined using a PCR screen to identify six PMQR genes including qnrA, qnrB, qnrD, qnrS, aac(6´)-Ib-cr, and qepA. Clonal relatedness of the quinolone-resistant isolates was determined by ERIC-PCR. Results: Of the isolates, 42.4% (39/92) were resistant to 1-7 of the tested quinolones. Gemifloxacin resistance was the lowest (28.3%) while resistance to the other six quinolones were ≥ 35%. The most common biotype among the 39 quinolone-resistant isolates was resistance to the seven tested quinolones (26/39; 66.7%). qnrD, qnrS, and aac(6´)-Ib-cr were found in 31 (79.5%), 31 (79.5%) and 28 (71.8%) of the 39 isolates, respectively, and all three genes together were found in 22 of the 39 isolates (56.4%). qnrA, qnrB, and qepA were not detected in any of the isolates and two isolates did not harbor any of the six tested genes. The isolates showed 38 different ERIC profiles and only two isolates (Pa16 and Pa17) had an identical profile. Conclusion: This is the first description of PMQR mechanisms among clinical Pseudomonas isolates from the KSA, which appears to be mainly mediated by qnrD, qnrS, and aac(6´)-Ib-cr.

Characterization of phenotypic and genotypic traits of carbapenem-resistant Acinetobacter baumannii clinical isolates recovered from a tertiary care hospital in Taif, Saudi Arabia.

BACKGROUND AND OBJECTIVE: Acinetobacter baumannii (A. baumannii) is a common nosocomial pathogen, which developed multi-drug-resistance to different classes of antibiotics including carbapenems. This study examined ten common carbapenemase genes among 32 carbapenem-resistant A. baumannii clinical isolates recovered from Taif, Saudi Arabia. METHODS: Isolates were phenotypically identified to the genus level by Vitek®2 and API 20NE®. The species level was confirmed by the amplification of bla OXA-51. The susceptibility for 21 different antibiotics was performed by Vitek 2 and modified Kirby-Bauer method. Isolates were genetically screened for 10 carbapenemases. Phylogenetic relatedness between isolates was determined by ERIC-PCR. RESULTS: Genotypically identified A. baumannii represented 100% of the total phenotypically identified Acinetobacter spp. All the carbapenem-resistant isolates were sensitive to polymyxin B and colistin. Among the other antibiotics, ampicillin/sulbactam and tigecycline were the most effective agents. 90.8% of the isolates were resistant to all ten investigated ß-lactams. bla OXA-51, bla IPM, bla NDM and bla OXA-23 were detected in 100%, 87.5%, 62.5% and 59.4% of isolates, respectively. Also, bla VIM and bla OXA-40 were less prevalent and were detected in 9.3% and 3.1% of the isolates, respectively. In addition, bla KPC, bla OXA-48, bla OXA-58, bla OXA-181 were not detected in any isolate. The A. baumannii isolates were categorised into ten genotypes on the basis of the detected carbapenemase genes and ERIC-PCR revealed a remarkable clonal diversity among these isolates. CONCLUSION: Class A and class D carbapenemase genes were the most commonly detected among carbapenem resistant A. baumannii (CRAB) clinical isolates.

Molecular Identification of Aminoglycoside-Modifying Enzymes and Plasmid-Mediated Quinolone Resistance Genes among Klebsiella pneumoniae Clinical Isolates Recovered from Egyptian Patients.

Inappropriate use of antibiotics in clinical settings is thought to have led to the global emergence and spread of multidrug-resistant pathogens. The goal of this study was to investigate the prevalence of genes encoding aminoglycoside resistance and plasmid-mediated quinolone resistance among clinical isolates of Klebsiella pneumoniae. All K. pneumoniae isolates were phenotypically identified using API 20E and then confirmed genotypically through amplification of the specific K. pneumoniae phoE gene. All isolates were genotyped by the enterobacterial repetitive intergenic consensus polymerase chain reaction technique (ERIC-PCR). Antibiotic susceptibility testing was done by a modified Kirby-Bauer method and broth microdilution. All resistant or intermediate-resistant isolates to either gentamicin or amikacin were screened for 7 different genes encoding aminoglycoside-modifying enzymes (AMEs). In addition, all resistant or intermediate-resistant isolates to either ciprofloxacin or levofloxacin were screened for 5 genes encoding the quinolone resistance protein (Qnr), 1 gene encoding quinolone-modifying enzyme, and 3 genes encoding quinolone efflux pumps. Biotyping using API 20E revealed 13 different biotypes. Genotyping demonstrated that all isolates were related to 2 main phylogenetic groups. Susceptibility testing revealed that carbapenems and tigecycline were the most effective agents. Investigation of genes encoding AMEs revealed that acc(6')-Ib was the most prevalent, followed by acc(3')-II, aph(3')-IV, and ant(3'')-I. Examination of genes encoding Qnr proteins demonstrated that qnrB was the most prevalent, followed by qnrS, qnrD, and qnrC. It was found that 61%, 26%, and 12% of quinolone-resistant K. pneumoniae isolates harbored acc(6')-Ib-cr, oqxAB, and qebA, respectively. The current study demonstrated a high prevalence of aminoglycoside and quinolone resistance genes among clinical isolates of K. pneumoniae.

Iodometric and Molecular Detection of ESBL Production Among Clinical Isolates of E. coli Fingerprinted by ERIC-PCR: The First Egyptian Report Declares the Emergence of E. coli O25b-ST131clone Harboring blaGES.

The extensive use of ß-lactam antibiotics has led to emergence and spread of extended-spectrum ß-lactamases (ESBLs). This study was conducted to investigate the prevalence of 7 different ESBL genes (blaTEM, blaSHV, blaCTX-M, blaVEB, blaPER, blaGES, and blaOXA-10) and O25b-ST131 high-risk clone among 61 clinical isolates of Escherichia coli. Also, one broad-spectrum ß-lactamase (blaOXA-1) was investigated. This study was also constructed to evaluate iodometric overlay method in detection of ESBL production. Phenotypic identification of E. coli isolates using API 20E revealed 18 distinct biotypes. DNA fingerprinting using enterobacterial repetitive intergenic consensus polymerase chain reaction (ERIC-PCR) differentiated all isolates into 2 main phylogenetic groups with 60 distinct genetic profiles. Elevated values of minimal inhibitory concentration (MIC)50 and MIC90 for third- and fourth-generation cephalosporins were observed. Phenotypic tests revealed that 85.24% of isolates were ESBL producers. The incidence rates of blaTEM, blaSHV, blaCTX-M, blaGES, blaOXA-1, and blaOXA-10 among E. coli ESBL producer phenotype were 69.23%, 25%, 96.15%, 3.85%, 11.54%, and 48%, respectively. On the other hand, blaVEB and blaPER were not detected. Sequencing of blaTEM and blaSHV revealed that blaTEM-214 and blaSHV-11 were the most prevalent variants. Group characterization of blaCTX-M revealed that blaCTX-M-1 was the most prevalent group of blaCTX-M family. It was found that 30.77% of E. coli ESBL producers belonged to O25b-ST131 clone harboring blaCTX-M-15. This study concluded that iodometric overlay method was 100% sensitive in detection of ESBL production. To our knowledge, this is the first Egyptian study that declares the emergence of E. coli O25b-ST131 harboring blaGES.