Abdulkader F. Tawfik

Prevalence and Genetic Characteristics of TEM, SHV, and CTX-M in Clinical Klebsiella pneumoniae Isolates from Saudi Arabia

The prevalence and genetic basis of extended-spectrum beta-lactamases (ESBLs) in Klebsiella pneumoniae remains unclear in Saudi Arabia. Therefore, this study was devoted to determine the prevalence and characterize ESBL-producing K. pneumoniae in Al-Qassim area, Saudi Arabia. A total of 430 isolates of K. pneumoniae isolated from clinical samples were collected over 6 months from January to June 2008. These isolates were screened for the presence of ESBLs by double-disk synergy test and re-evaluated by E-test ESBL method. Minimum inhibitory concentrations of 15 antibiotics against ESBL-positive strains were determined by E-test strips. The β-lactamases involved were characterized by polymerase chain reaction assays and DNA sequencing. Conjugation experiments were done and ISEcp1 elements were tested among CTX-M positive isolates. The prevalence of ESBL was 25.6% (110/430) and all ESBL-positive isolates were sensitive to imipenem and tigecycline; however, the resistance rate to gentamicin, amikacin, and ciprofloxacin was 87.3%, 10%, and 9.1%, respectively. Of these, 89.1% produced SHV, 70.9% produced TEM, and 36.4% were CTX-M-producing strains. The prevalence of ESBL SHV SHV-12 and SHV-5 was of 60% and 18.2%, respectively, and various non-ESBL SHV, including SHV-1 (5.5%), -11 (3.6%), and -85 (1.8%), was detected. However, the prevalence of CTX-M-15 and CTX-M-14 was 34.5% and 1.8%, respectively. ISEcp1 element was detected in 60% of bla(CTX-M-15) genes. All bla(CTX-M) genes were transferable; however, most of bla(SHV-12) and bla(SHV-5) were not transferable. TEM-type ESBLs were not detected in any of the isolates. This is the first description of CTX-M-14, SHV-5, SHV-11, and SHV-85 in Saudi Arabia. We have documented the dominance of K. pneumoniae SHV-12 and highlighted the emergence of CTX-M-15 in Saudi Arabia.

Effects of artemisinin, dihydroartemisinin and arteether on immune responses of normal mice

Artemisinin (Qinghaosu) is a potent antimalarial sesquiterpene lactone isolated from the Chinese herb Artemisia annua. Arteether, a potent semisynthetic analogue of dihydroartemisinin is being developed by the World Health Organization as the artemisinin derivative of choice for the treatment of malaria. All three agents in doses of 400 and 600 mg/kg body weight were found to exhibit marked suppression of humoral responses, as measured by the hemolytic plaque assay, with arteether being the most potent. These agents did not alter the delayed-type hypersensitivity response to sheep erythrocytes at the same dose levels. In addition, all three agents were found not to possess any anti-inflammatory activity when tested on carrageenan-induced oedema. These results indicated that these agents have a selective immunosuppressive activity. They did not exhibit immunostimulating activity in contrast to what has been reported for sodium artesunate.

Distribution of Ambler class A, B and D β-lactamases among Pseudomonas aeruginosa isolates

OBJECTIVES We determined the prevalence rate of classes A, B and D β-lactamases among extended-spectrum cephalosporin (ESC)-non-susceptible Pseudomonas aeruginosa clinical isolates from burned patients. METHODS Disc susceptibility testing was performed on 156 P. aeruginosa isolates collected during 2010 at Prince Salman Hospital in Riyadh, Saudi Arabia. Phenotypic screening of ESBLs and MBLs in the isolates resistant to ceftazidime (MIC>8 mg/L) was carried out. Genes encoding ESBLs and MBL were sought by PCR in ESBL- and MBL-producing isolates. RESULTS The resistance rate to ceftazidime was 22.43%. The resistance rates for ESC-non-susceptible P. aeruginosa isolates to piperacillin, piperacillin/tazobactam, cefepime, aztreonam, imipenem, amikacin, gentamicin and ciprofloxacin were 100%, 71.14%, 88.57%, 48.57%, 70.0%, 82.5%, 87.5%, and 90.0% respectively. No resistance was detected to polymyxine B. The prevalence of ESBL and MBL in ESC-non-susceptible P. aeruginosa was 69.44% and 42.85%, respectively. The prevalence of structural genes for VEB-1, OXA-10 and GES ESBLs in P. aeruginosa was 68%, 56% and 20%, respectively. VIM gene was detected in 15 (100%) of MBL-producing isolates. OXA-10 like gene was concomitant with VEB, GES and/or VIM. Eight isolates harbored OXA-10 with VEB (imipenem MIC 6-8 mg/L), while five isolates harbored OXA-10 with VIM (imipenem MIC ≥ 32 mg/L) and one isolate contained OXA-10, VEB and GES (imipenem MIC 8 mg/L). PER was not detected in this study. CONCLUSION VEB-1 and OXA-10 are the predominant ESBL genes and bla(VIM) is the dominate MBL gene in ESC-non-sensitive P. aeruginosa isolates in Saudi Arabia. VEB, OXA-10 and GES ESBLs have not been reported previously in Saudi Arabia and GES has not been reported previously in Middle East and North Africa.

Determination of lomefloxacin in biological fluids by high-performance liquid chromatography and a microbiological method

A high‐performance liquid chromatographic method (HPLC) was developed for the determination of lomefloxacin in plasma and urine and was compared to a microbiological assay. Lomefloxacin and norfloxacin (internal standard) were extracted from plasma and urine samples using chloroform. Measurements were carried out with a fluorescence detector using an excitation wavelength of 280 nm and an emission wavelength of 430 nm with a mercury lamp. Quantification was achieved by the measurement of the peak‐height ratio and the analytical recovery of the drug from plasma and urine was found to be (mean±SD) 99·3 ± 3·74% and 95·7%±3·82%, respectively. In the microbiological assay, E. coli ATCC 1346 was the test organism using an agar diffusion technique. The coefficients of variation for within‐day analysis for both the HPLC method and microbiological assay from plasma samples were less than 7%. The minimum detectable concentration for both the HPLC and the microbiological method was 50 ng/ml and 100 ng/ml, respectively. Both methods were used to determine the lomefloxacin level in plasma following intravenous administration to mice. Excellent agreement was obtained between the results of the two methods. The HPLC method offers significant advantages in accuracy, precision, speed of analysis and turnover‐time.

Extended-spectrum and metallo-beta-lactamases among ceftazidime-resistant Pseudomonas aeruginosa in Riyadh, Saudi Arabia.

Abstract We investigated the extended-spectrum (ESBLs) and metallo-beta-lactamases (MBLs) among Pseudomonas aeruginosa isolates in Saudi Arabia. Disc susceptibility testing was performed on 200 P. aeruginosa isolates collected during 2010 at the Armed Forces Hospital in Riyadh, with MIC testing and phenotypic screening for ESBLs and MBLs carried out on those found to be ceftazidime resistant. Genes for ESBLs and MBLs were sought by PCR. Thirty-nine (19·5%) P. aeruginosa isolates were ceftazidime resistant, mostly with considerable resistance to other antibiotics except colistin. Twenty-three of these 39 (59%) appeared ESBL positive and 16 (41%) had MBLs. blaVEB, and blaGES genes were found in 20 (86·95%), and 5 (21·74%) of 23 ESBL-positive isolates, respectively whilst blaVIM was detected in all 16 MBL-producers. blaOXA-10-like often accompanied blaVEB, blaVIM or blaGES. Several isolates had similar antibiogram and β-lactamase profiles, and may represent outbreaks; nevertheless, the collection was not dominated by any single clone. This dominance of acquired ceftazidime-inactivating beta-lactamases, often in combination is in contrast to the situation in Europe and the USA, where most ceftazidime resistance in P. aeruginosa is attributable to AmpC and efflux.

Antimicrobial resistance pattern and prevalence of metallo-β-lactamases in Pseudomonas aeruginosa from Saudi Arabia

To determine the prevalence of metallo-β-lactamases (MBLs) in Pseudomonas aeruginosa and to investigate whether MBL genes have spread in MBL-producing isolates. A total of 350 clinical isolates of P. aeruginosa were screened for production of MBL. Antibiotic susceptibility testing was determined by E-test strips. Six MBL genes and class 1 integron were tested by polymerase chain reaction (PCR). Positive MBL genes were subjected to sequencing. Matting out assay was carried out. The resistance rates of 350 P. aeruginosa isolates to piperacillin, piperacillin/tazobactam, cefotaxime, ceftazidime, cefepime, aztreonam, imipenem, and ciprofloxacin were 54, 50.3, 48.3, 45.14, 40.3, 30.57, 38.6 and 36.85%, respectively. The results of MBL screening revealed that 20.57% (72/350) of P. aeruginosa produced MBL. Sixty three (46.46%) of 135 imipenem-resistant isolates were not found to produce MBL. MBL-producing isolates were 100% resistant to β-lactams except aztreonam, which showed resistance rate of 63.88%. Only 20.3 and 5.42% of the MBL-producing isolates were resistant to amikacin and polymyxin B, respectively. PCR and deoxyribonuleic acid (DNA) sequencing investigated revealed that all MBL isolates harbour blaVIM-2 gene. High prevalence of imipenem resistant and MBL-producing P. aeruginosa isolates was reported. Imipenem resistance is in increasing and MBL is responsible for 20.57% of the resistance. The blaVIM-2 is the dominant MBL gene in MBL-producing isolates in Saudi Arabia.

Inhibition of Motility and Adherence of Proteus mirabilis to Uroepithelial Cells by Subinhibitory Concentrations of Amikacin

The effect of subinhibitory concentrations of amikacin on Proteus mirabilis motility and adherence to human uroepithelial and to HeLa cells was compared with that of gentamicin. In addition, the effect of both antibiotics on cell surface hydrophobicity was also examined. Exposure of bacterial cells in the logarithmic phase to one fourth of amikacin or gentamicin at one fourth of their respective minimal inhibitory concentrations causes the inhibition of swarming and motility of Proteus strains. Amikacin significantly reduced adhesion of Proteus strains to human uroepithelial cells and gentamicin exerts the same effect to a lesser extent. Such inhibitory concentrations of amikacin or gentamicin had no significant effect on the attachment ability of these strains to HeLa cells compared to the nontreated cells. Treatment of the bacterial cells with amikacin or gentamicin changed significantly the cell surface hydrophobicity towards the hydrophilic state compared to nontreated cells, and it was found to be strain dependent. Since motility and attachment ability are considered as pathogenic traits, these data indicate the impact of amikacin on the virulence factors especially in urinary tract infections with Proteus strains.

Effect of Beta-Lactamase Expression on Susceptibility of Local Isolates of Enterobacter cloacae, Serratia marcescens and Pseudomonas aeruginosasBeta-Lactam Antibiotics

During surveillance studies, a total of 66 strains of gram-negative bacilli (28 Enterobacter cloacae, 20 Serratia marcescens and 18 Pseudomonas aeruginosa) with a reduced susceptibility to third-generation cephalosporins, aztreonam and amikacin were isolated from documented infections. All isolates were highly susceptible to imipenem and sparfloxacin. beta-Lactamase activity was demonstrated in all isolates of E. cloacae and S. marcescens, and in 77% of P. aeruginosa isolates. Inducible beta-lactamase activity was detected in 80, 65 and 40% of E. cloacae, S. marcescens and P. aeruginosa isolates, respectively, when cefoxitin was used as an inducer. More inducible beta-lactamase producers were observed when imipenem was used as an inducer. Isolates of E. cloacae, and to a lesser extent S. marcescens, expressed a wide spectrum of beta-lactamase activities. There was a good correlation between baseline beta-lactamase activity and the respective MIC of ceftazidime, cefotaxime and ceftriaxone, and to a lesser extent aztreonam in E. cloacae and S. marcescens isolates. Only one isolate of E. cloacae demonstrated an extended beta-lactamase activity. The data suggest that the resistance of E. cloacae and S. marcescens isolates to beta-lactam antibiotics is largely dependent upon hyperproduction of beta-lactamase.

Influence of new quinolones on normal immune capabilities.

Ciprofloxacin, pefloxacin, norfloxacin, and ofloxacin were investigated for immunomodulatory activity on humoral and cell-mediated immune responses. Ciprofloxacin and pefloxacin altered the humoral immune responses of mice to sheep red blood cells. This effect was not exhibited by norfloxacin or ofloxacin. All four quinolones did not alter cell-mediated responses. When these antimicrobial agents were tested for their interaction with human polymorphonuclear phagocytic activity, all agents suppressed this activity. In addition, all except norfloxacin showed anti-inflammatory activity.

Antimicrobial activity of artemisinin and its derivatives against anaerobic bacteria.

Artemisinin and nine of its semisynthetic derivatives were tested for antibacterial activity against anaerobic, facultative anaerobic, microaerophilic and aerobic bacteria. Only anaerobic bacteria and gonococci showed sensitivity to artemisinin derivatives. Artemisinin and its deoxy derivatives had no activity at 100 micrograms/ml concentration. The newly synthesized methyl diperoxy derivative had the highest activity against all tested anaerobes with a MIC of 9.4 micrograms/ml.