Molecular Characterization of Carbapenem/Colistin-Resistant Acinetobacter baumannii Clinical Isolates from Egypt by Whole-Genome Sequencing.

PURPOSE: The rise of carbapenem-resistant A. baumannii (CRAB) is considered a public health problem limiting the treatment options. Our current work studied the emergence and mechanisms of colistin-resistance among CRAB isolates in Egypt. MATERIALS AND METHODS: Seventeen clinically recovered A. baumannii were identified and screened for their antimicrobial susceptibilities using VITEK-2 system. Colistin susceptibility was evaluated using broth microdilution, and characterization of carbapenem/colistin resistance determinants was performed using whole-genome sequencing (Illumina MiSeq). RESULTS: About 52.9% (9/17) were colistin-resistant. PCR results revealed that all isolates carried bla OXA-51-like genes, bla OXA-23-like was detected in 82.3% (14/17) and bla NDM in 23.5% (4/17). Two isolates harboured bla GES-35 and bla OXA-23. Furthermore, genome analysis of seven isolates revealed six belonged to international clone 2 (IC2) while the remaining isolate was a singleton (ST158), representing a clone circulating in Mediterranean/Middle Eastern countries. CONCLUSION: The emergence and high incidence of colistin-resistance among CRAB clinical isolates in Egypt are alarming because it further limits therapy options and requires prudent antimicrobial stewardship and stringent infection control measures. Whole-genome sequence analyses suggest that the resistance to colistin was associated with multiple mutations in the pmrCAB genes. The high incidence of the high-risk lineage IC2 harbouring bla OXA-23-like as well as bla NDM is also of concern.

Reliability of phenotypic methods for detection of colistin resistance among carbapenem-resistant Acinetobacter baumannii clinical isolates from Egypt.

INTRODUCTION: Acinetobacter baumannii is a challenging pathogen responsible for serious nosocomial infections. Colistin resistance in carbapenem-resistant A. baumannii strains is a critical health problem as it limits the available therapeutic options. The current work aimed to study the reliability of several phenotypic methods for the detection of colistin resistance among carbapenem-resistant A. baumannii isolates in Egypt. METHODS: A total of 22 carbapenem-resistant A. baumannii isolates were recovered. Colistin minimum inhibitory concentrations (MICs) were determined using broth microdilution (BMD) and compared to agar dilution (AD), automated system (VITEK-2) and gradient test (E-test) and were analyzed by statistical methods. RESULTS: Phenotypic testing showed that nine of 22 isolates (40.9%) were colistin-resistant by BMD and seven of them were also resistant by AD, with the categorical agreement (CA) of 72.7% and essential agreement (EA) of 90.9%. Colistin MIC results ranged from 1-8 µg/mL and 1-32 µg/mL by both AD and BMD respectively. Detection of colistin resistance by gradient test and automated system showed high very major error (VME) rates (40.9%) compared to BMD with a lack of CA between them. AD gave moderate agreement with BMD by 90.9% EA, 72.7% CA and only 9.1% VME. CONCLUSIONS: In delineating colistin breakpoints BMD followed by AD method are defined as the only reliable phenotypic methods for colistin resistance evaluation. More rapid and reliable tests, other than BMD and AD, are required for the convenient detection of colistin resistance in the routine clinical microbiology laboratory daily workflow.

High-risk clones and novel sequence type ST4497 of Klebsiella pneumoniae clinical isolates producing different alleles of NDM-type and other carbapenemases from a single tertiary-care centre in Egypt.

Enterobacteria producing NDM carbapenemases represent a severe diagnostic and therapeutic challenge in healthcare settings. Infections caused by NDM-positive strains are usually associated with high mortality rates and very limited treatment options. A total number of 33 carbapenem-resistant Klebsiella pneumoniae (CRKP) isolates were included in this study, comprising 30 recovered from clinical diagnostic samples and 3 cultured from screening rectal swabs taken at patient admission. Bacterial identification was performed by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF/MS) and antibiotic susceptibility testing was performed by reference broth microdilution and a commercial automated method. Isolates were investigated for carbapenemase production using the ß-CARBA test, the modified carbapenem inactivation method (mCIM) and, for the 30 clinical isolates, by MALDI-TOF/MS, using the MBT STARⓇ-Carba IVD Kit. Carbapenem resistance genes were characterised by PCR and sequencing. Seven different blaNDM gene variants were identified in 94% of the isolates, whilst three variants of blaOXA-48-like were detected in 27% of the isolates. Most CRKP corresponded to high-risk clones (ST147, ST11 and ST15). Novel ST4497 is reported for the first time in this study as well as the first emergence of K. pneumoniae ST231 producing OXA-232 in Egypt. These results indicate an ongoing evolution of the blaNDM genes in our area and confirm the need for a maintained surveillance system in order to monitor the spread of these mobile blaNDM genes.

CTX-M-15-producing Escherichia coli clinical isolates in Cairo (Egypt), including isolates of clonal complex ST10 and clones ST131, ST73, and ST405 in both community and hospital settings.

In Egypt, little is known about the genetic background of Escherichia coli isolates harboring extended-spectrum ß-lactamase (ESBL). Five hundred twenty Enterobacteriaceae were prospectively collected (May 2007-August 2008) at the Theodor Bilharz Research Institute (Cairo). Among the collected Enterobacteriaceae, 56% (n=291) were E. coli and 32% (n=165) Klebsiella pneumoniae. A total of 16% (n=3) of all isolates were ESBL, 19% (n=55) of the E. coli and 14% (n=23) of the K. pneumoniae. The proportion of E. coli ESBL producers did not differ significantly between in and outpatients (20% vs. 17%) but was significantly different for non-E. coli ESBL producers (18.5% vs. 1.2%: p=0.0001). The majority of E. coli ESBL producers (75%) was isolated from urine. All the ESBL-producing Enterobacteriaceae available for molecular study (n=74) produced CTX-M-15. Among the CTX-M-15-producing E. coli isolates; 40% belonged to phylogenetic group A, 32% to D, and 26% to B2. ERIC-2 PCR profiles were obtained for all these E. coli isolates and multilocus sequence typing for those belonging to group B2. Genotyping analyses showed strain diversity; however, some clusters had profiles indistinguishable from that of previously published clones. Multilocus sequence typing showed that 75% of E. coli group B2 belonged to clone ST131. This indicates that a new country in Africa is adversely affected by clones of E. coli-producing CTX-M-15.

Herpes simplex virus type-2 in Egyptian patients with bladder cancer or cystitis.

The present study was designed to investigate the prevalence of herpes simplex virus type-2 (HSV-2) in Egyptian patients with bladder cancer or cystitis and to evaluate the performance of different diagnostic HSV-2 assays. The study included 50 patients: 27 with bladder cancer (group I), 23 with cystitis (group II) and 20 subjects as controls (group III). HSV-2 DNA was detected using polymerase chain reaction (PCR) on bladder tissue and buffy coat cells (BCC). Electron microscopic studies (EMS) on BCC and ELISAs for IgM, IgG and specific glycoprotein G-2 (gG-2) IgG were performed. HSV-2 DNA was detected by PCR on bladder tissue biopsies in 29.6% and 21.7% of group I and II respectively and it was also detected by PCR on BCC in 22.2% and 21.7% of group I and II respectively. EMS revealed HSV like particles in 16.6% of cases. IgG, specific gG-2 IgG and IgM were detected in 30%, 16% and 6% of cases respectively. The different assays were evaluated in relation to PCR on bladder tissue biopsies. The gG-2-based ELISA and EMS on BCC were found to be highly specific (97.3% and 100% respectively), with similar low sensitivity of approximately 54%. PCR on BCC was the most sensitive assay. The association of HSV-2 with bladder cancer is suggested especially in schistosomal patients.

SEN virus infection in Egyptian patients with chronic hepatitis C and patients undergoing hemodialysis.

The SEN virus has been tentatively linked to transfusion-associated non-A to E hepatitis. The aim of the present study was to 1) determine the prevalence of SEN virus among Egyptian patients with hepatitis C virus (HCV)-related chronic liver disease and patients undergoing hemodialysis and 2) demonstrate the clinical effect of SEN virus infection on coexistent hepatitis C in terms of severity and probability of developing hepatocellular carcinoma. Polymerase chain reaction was used to detect SEN virus-D and SEN virus-H DNA in serum samples of 74 patients with HCV-related chronic liver disease, 45 uremic patients undergoing maintenance hemodialysis, and 28 healthy controls. SEN virus-D/H DNA was detected in 13.5% of patients with chronic liver disease, 11.1% of patients undergoing hemodialysis, and 7.1% of healthy controls, with no significant differences between patients and the control group. Clinical and biochemical measures did not significantly differ between SEN virus-infected and noninfected patients in the chronic liver disease group or the hemodialysis group. The rate of SEN virus infection was significantly higher in patients with chronic liver disease and hepatocellular carcinoma (33.3%) than in those with chronic liver disease only (8.5%) (P < .05). In conclusion, SEN virus does not seem to be a common infection in Egyptian patients. It has no apparent influence on the severity of coexistent HCV-related chronic liver disease but could be a risk factor for hepatocellular carcinoma in such patients. Further studies are needed to define the etiopathogenic role of SEN virus infection in the development of hepatocellular carcinoma.