Spread of CTX-M-15 Extended Spectrum β-lactamases Encoding Genes Among Enterobacteriaceae in the Middle Eastern Region

The CTX-M type enzymes have become the most prevalent extended spectrumβ-lactamases (ESBLs) worldwide. Among the CTX-M type enzymes, CTX-M-15 is themost widespread and has been reported from all continents. It has been recoveredfrom different Enterobacteriaceae and has been isolated from both community andhospital acquired infections. This review primarily highlights the prevalence of CTXM-15 in addition to other ESBLs in the Middle East. Detection of any type of ESBL isof importance in therapeutic treatment

Frequency of conjugative transfer of plasmid-encoded ISEcp1 - blaCTX-M-15 and aac(6')-lb-cr genes in Enterobacteriaceae at a tertiary care center in Lebanon - role of transferases

<p>Abstract</p> <p>Background</p> <p>The frequency of transfer of genes encoding resistance to antimicrobial agents was determined by conjugation in ESBL-producing and/or fluoroquinolone or aminoglycoside resistant Enterobacteriaceae clinical isolates at a tertiary care center in Lebanon. In addition, the role of <it>tra </it>genes encoding transferases in mediating conjugation was assessed.</p> <p>Methods</p> <p>Conjugation experiments were done on 53 ESBL-producing and/or fluoroquinolone resistant <it>E. coli </it>and <it>K. pneumoniae </it>and ESBL-producing <it>S. sonnei </it>isolates. Antimicrobial susceptibility testing on parent and transconjugant isolates, and PCR amplifications on plasmid extracts of the resistance-encoding genes: <it>bla</it>_CTX-M-15with the <it>ISEcp1 </it>insertion sequence, the <it>aac(6')-lb-cr </it>and <it>qnr</it>S genes, as well as <it>tra </it>encoding transferases genes were done. Random amplified polymorphic DNA (RAPD) analysis was performed to demonstrate whether conjugative isolates are clonal and whether they are linked epidemiologically to a particular source.</p> <p>Results</p> <p>Antimicrobial susceptibility testing on transconjugants revealed that 26 out of 53 (49%) ESBL-producing <it>Enterobacteriaceae </it>were able to transfer antimicrobial resistance to the recipients. Transfer of high-level resistance to the transconjugants encoded by the <it>bla</it>_CTX-M-15gene downstream the <it>ISEcp1 </it>insertion sequence against 3rd generation cephalosporins, and of low-level resistance against ciprofloxacin, and variable levels of resistance against aminoglycosides encoded by <it>aac(6')-lb-cr </it>gene, were observed in transconjugants. <it>tra </it>encoding transferase genes were detected exclusively in conjugative isolates.</p> <p>Conclusion</p> <p>In conclusion, the frequency of transfer of antimicrobial resistance in non clonal <it>Enterobacteriaceae </it>at the tertiary care center by conjugation was 49%. Conjugation occurred in isolates expressing the <it>tra </it>encoding transferase genes. Multiple conjugative strains harboring the plasmid encoded antimicrobial resistant genes were circulating in the medical center. Molecular epidemiology analysis showed that conjugative isolates are neither clonal nor linked to a particular site and transfer of antimicrobial resistance is by horizontal transfer of plasmids.</p

Detection of a highly prevalent and potentially virulent strain of Pseudomonas aeruginosa from nosocomial infections in a medical center

BACKGROUND: We correlated genotypes, virulence factors and antimicrobial susceptibility patterns of nosocomially identified Pseudomonas aeruginosa isolates from clinical specimens to those of environmental isolates encountered in the same units of a medical center. Antibiotic susceptibility testing, RAPD analysis and detection of enzymatic activities of extracellular virulence factors, were done on these isolates. RESULTS: Data showed that most of the clinical and environmental isolates were susceptible to tested antimicrobial agents. RAPD analysis determined the presence of 31 genotypes, with genotype 1 detected in 42% of the clinical isolates and 43% of the environmental isolates. Enzymatic activity testing showed that genotype 1 produced all virulence factors tested for. CONCLUSION: In conclusion, our data demonstrated the predominant prevalence of a potentially virulent P. aeruginosa genotype, circulating in a number of units of the medical center and emphasize the need to reinforce infection control measures

Evaluating the Efficacies of Carbapenem/β-Lactamase Inhibitors Against Carbapenem-Resistant Gram-Negative Bacteria in vitro and in vivo

BackgroundCarbapenem-resistant Gram-negative bacteria are a major clinical concern as they cause virtually untreatable infections since carbapenems are among the last-resort antimicrobial agents. β-Lactamases implicated in carbapenem resistance include KPC, NDM, and OXA-type carbapenemases. Antimicrobial combination therapy is the current treatment approach against carbapenem resistance in order to limit the excessive use of colistin; however, its advantages over monotherapy remain debatable. An alternative treatment strategy would be the use of carbapenem/β-lactamase inhibitor (βLI) combinations. In this study, we assessed the in vitro and in vivo phenotypic and molecular efficacies of three βLIs when combined with different carbapenems against carbapenem-resistant Gram-negative clinical isolates. The chosen βLIs were (1) Avibactam, against OXA-type carbapenemases, (2) calcium-EDTA, against NDM-1, and (3) Relebactam, against KPC-2.MethodsSix Acinetobacter baumannii clinical isolates were screened for blaOXA-23-like, blaOXA-24/40, blaOXA-51-like, blaOXA-58, and blaOXA-143-like, and eight Enterobacteriaceae clinical isolates were screened for blaOXA-48, blaNDM-1, and blaKPC-2. The minimal inhibitory concentrations of Imipenem (IPM), Ertapenem (ETP), and Meropenem (MEM) with corresponding βLIs for each isolate were determined. The efficacy of the most suitable in vitro treatment option against each of blaOXA-48, blaNDM-1, and blaKPC-2 was assessed via survival studies in a BALB/c murine infection model. Finally, RT-qPCR was performed to assess the molecular response of the genes of resistance to the carbapenem/βLI combinations used under both in vitro and in vivo settings.ResultsCombining MEM, IPM, and ETP with the corresponding βLIs restored the isolates’ susceptibilities to those antimicrobial agents in 66.7%, 57.1%, and 30.8% of the samples, respectively. Survival studies in mice revealed 100% survival rates when MEM was combined with either Avibactam or Relebactam against blaOXA-48 and blaKPC-2, respectively. RT-qPCR demonstrated the consistent overexpression of blaOXA-48 upon treatment, without hindering Avibactam’s activity, while blaNDM-1 and blaKPC-2 experienced variable expression levels upon treatment under in vitro and in vivo settings despite their effective phenotypic results.ConclusionNew carbapenem/βLI combinations may be viable alternatives to antimicrobial combination therapy as they displayed high efficacy in vitro and in vivo. Meropenem/Avibactam and Meropenem/Relebactam should be tested on larger sample sizes with different carbapenemases before progressing further in its preclinical development

Correlation between Group B Streptococcal Genotypes, Their Antimicrobial Resistance Profiles, and Virulence Genes among Pregnant Women in Lebanon

The antimicrobial susceptibility profiles of 76 Streptococcus agalactiae (Group B Streptococci [GBS]) isolates from vaginal specimens of pregnant women near term were correlated to their genotypes generated by Random Amplified Polymorphic DNA analysis and their virulence factors encoding genes cylE, lmb, scpB, rib, and bca by PCR. Based on the distribution of the susceptibility patterns, six profiles were generated. RAPD analysis detected 7 clusters of genotypes. The cylE gene was present in 99% of the isolates, the lmb in 96%, scpB in 94.7%, rib in 33%, and bca in 56.5% of isolates. The isolates demonstrated a significant correlation between antimicrobial resistance and genotype clusters denoting the distribution of particular clones with different antimicrobial resistance profiles, entailing the practice of caution in therapeutic options. All virulence factors encoding genes were detected in all seven genotypic clusters with rib and bca not coexisting in the same genome

Genotypes and serotype distribution of macrolide resistant invasive and non- invasive Streptococcus pneumoniae isolates from Lebanon

<p>Abstract</p> <p>Background</p> <p>This study determined macrolide resistance genotypes in clinical isolates of <it>Streptococcus pneumoniae </it>from multiple medical centers in Lebanon and assessed the serotype distribution in relation to these mechanism(s) of resistance and the source of isolate recovery.</p> <p>Methods</p> <p>Forty four macrolide resistant and 21 macrolide susceptible <it>S. pneumoniae </it>clinical isolates were tested for antimicrobial susceptibility according to CLSI guidelines (2008) and underwent molecular characterization. Serotyping of these isolates was performed by Multiplex PCR-based serotype deduction using CDC protocols. PCR amplification of macrolide resistant <it>erm </it>(encoding methylase) and <it>mef </it>(encoding macrolide efflux pump protein) genes was carried out.</p> <p>Results</p> <p>Among 44 isolates resistant to erythromycin, 35 were resistant to penicillin and 18 to ceftriaxone. Examination of 44 macrolide resistant isolates by PCR showed that 16 isolates harbored the <it>erm</it>(B) gene, 8 isolates harbored the <it>mef </it>gene, and 14 isolates harbored both the <it>erm</it>(B) and <it>mef </it>genes. There was no amplification by PCR of the <it>erm</it>(B) or <it>mef </it>genes in 6 isolates. Seven different capsular serotypes 2, 9V/9A,12F, 14,19A, 19F, and 23, were detected by multiplex PCR serotype deduction in 35 of 44 macrolide resistant isolates, with 19F being the most prevalent serotype. With the exception of serotype 2, all serotypes were invasive. Isolates belonging to the invasive serotypes 14 and 19F harbored both <it>erm</it>(B) and <it>mef </it>genes. Nine of the 44 macrolide resistant isolates were non-serotypable by our protocols.</p> <p>Conclusion</p> <p>Macrolide resistance in <it>S. pneumoniae </it>in Lebanon is mainly through target site modification but is also mediated through efflux pumps, with serotype 19F having dual resistance and being the most prevalent and invasive.</p

Molecular characterization, toxin detection and resistance testing of human clinical Clostridium difficile isolates from Lebanon

Clostridium (Clostridioides) difficile is the main cause for nosocomial diarrhoea in industrialised nations. Epidemiologic data on the pathogen’s occurrence in other world regions are still scarce. In this context we characterized with phenotypic and molecular genetic methods C. difficile isolates stemming from hospitalised patients with diarrhoea in Lebanon. From 129 stool samples of symptomatic patients at a tertiary care University hospital in Lebanon, a total of 107 C. difficile strains were cultivated and underwent ribotyping, toxin gene detection and antibiotic resistance testing. Ribotype 014 (RT014, 16.8%) predominated, followed by RT002 (9.3%), RT106 (8.4%) and RT070 (6.5%). Binary toxin gene-positive isolates (RT023, RT078 and RT126) were rarely detected and RT027 was absent. Interestingly, within one isolate only the toxin A gene (tcdA) was detected. Multiple-locus variable-number tandem repeat analysis (MLVA) revealed strong strain diversity in most RTs. The isolates were sensitive to metronidazole and vancomycin, and only a small proportion of strains displayed resistance against moxifloxacin, rifampicin, and clarithromycin (5.6%, 1.9%, and 2.8%), respectively. The data indicate that the genetic strain composition of Lebanese strains differs markedly from the situation seen in Europe and North America. Especially the epidemic RTs seen in the latter regions were almost absent in Lebanon. Interestingly, most strains showed almost no resistance to commonly used antibiotics that are suspected to play a major role in the development of C. difficile infection, despite frequent use of these antibiotics in Lebanon. Thus, the role of antimicrobial resistance as a major driving force for infection development remains uncertain in this area

Emerging ceftazidime-avibactam resistance against carbapenem resistant Escherichia coli and Klebsiella pneumoniae in Lebanon

Introduction: Ceftazidime-avibactam (CZA) has been introduced as a novel drug to essentially combat the rising trends of carbapenem resistant Enterobacteriaceae. In the absence of in vitro data about the activity of this drug against carbapenem resistant (CR) Escherichia coli and Klebsiella pneumoniae in Lebanon, this study was warranted.&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; &nbsp; Methodology: A total of 150 isolates, identified using the MALDI-TOF,&nbsp; encompassing 50 CR E. coli, 60 CR K. pneumoniae, and 10 isolates each of extended-spectrum Beta-lactamases (ESBLs), and non-CR multidrug-resistant (MDR) of each species were analyzed.&nbsp; The minimum inhibitory concentration (MIC) for CZA was determined by the E-test (Liofilchem, Roseto degli Abruzzi, Italy). In addition, the disk diffusion (DD) test was used to determine the activity of CZA and of the antimicrobials routinely used to test for such pathogens. &nbsp; Results:&nbsp; The CZA activity against the 50 CR E. coli showed an MIC50 ≥ 256 μg/mL, an MIC90 ≥ 256 μg/mL, and an MIC range of 0.023 to ≥ 256 μg/mL, reflecting a susceptibility of 40 %. As For the 60 CR K. pneumoniae isolates, the MIC50 was ≥ 256 μg/mL, MIC90 ≥ 256 μg/mL, and the MIC range was 0.094 to ≥ 256 μg/mL, reflecting a susceptibility of 35%. However, uniform CZA susceptibility (100%) was detected against ESBL and MDR isolates of both species, being comparable or higher to the routinely used antimicrobials. &nbsp; &nbsp; Conclusion: Although CZA was recently introduced into this country, it was surprising to note this humble susceptibility of CZA against CR E. coli and CR K. pneumoniae. To explain such findings, it is worth pursuing investigations related to antimicrobial utilization in clinical practice and antimicrobial stewardship. Moreover, genotypic determination is needed to be revealed to help explain the observed phenotypic resistance &nbsp; &nbsp