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

Decrease in Shiga toxin expression using a minimal inhibitory concentration of rifampicin followed by bactericidal gentamicin treatment enhances survival of Escherichia coli O157:H7-infected BALB/c mice

<p>Abstract</p> <p>Background</p> <p>Treatment of <it>Escherichia coli </it>O157:H7 infections with antimicrobial agents is controversial due to an association with potentially fatal sequelae. The production of Shiga toxins is believed to be central to the pathogenesis of this organism. Therefore, decreasing the expression of these toxins prior to bacterial eradication may provide a safer course of therapy.</p> <p>Methods</p> <p>The utility of decreasing Shiga toxin gene expression in <it>E. coli </it>O157:H7 with rifampicin prior to bacterial eradication with gentamicin was evaluated <it>in vitro </it>using real-time reverse-transcription polymerase chain reaction. Toxin release from treated bacterial cells was assayed for with reverse passive latex agglutination. The effect of this treatment on the survival of <it>E. coli </it>O157:H7-infected BALB/c mice was also monitored.</p> <p>Results</p> <p>Transcription of Shiga toxin-encoding genes was considerably decreased as an effect of treating <it>E. coli </it>O157:H7 <it>in vitro </it>with the minimum inhibitory concentration (MIC) of rifampicin followed by the minimum bactericidal concentration (MBC) of gentamicin (> 99% decrease) compared to treatment with gentamicin alone (50-75% decrease). The release of Shiga toxins from <it>E. coli </it>O157:H7 incubated with the MIC of rifampicin followed by addition of the MBC of gentamicin was decreased as well. On the other hand, the highest survival rate in BALB/c mice infected with <it>E. coli </it>O157:H7 was observed in those treated with the <it>in vivo </it>MIC equivalent dose of rifampicin followed by the <it>in vivo </it>MBC equivalent dose of gentamicin compared to mice treated with gentamicin or rifampicin alone.</p> <p>Conclusions</p> <p>The use of non-lethal expression-inhibitory doses of antimicrobial agents prior to bactericidal ones in treating <it>E. coli </it>O157:H7 infection is effective and may be potentially useful in human infections with this agent in addition to other Shiga toxin producing <it>E. coli </it>strains.</p

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

HLA Allele Associations and V-Beta T-Lymphocyte Expansions in Patients With Psoriasis, Harboring Toxin-Producing Staphylococcus aureus

HLA alleles have been associated with psoriasis. Toxin-producing strains of Staphylococcus aureus behave as superantigens, and if present in patients, might play a role in the exacerbation of psoriatic lesions by activating certain V-beta (Vβ) T-lymphocyte subsets. Allele frequencies in 22 patients and 22 controls (alleles determined by DNA/SSP typing) were used to calculate a relative risk of 4.7 (P < .05) for HLA-Cw6. S aureus was isolated from the throat of 11 patients. Enterotoxins A and C were detected by agglutination in the culture filtrate of one isolate. The enterotoxin A and/or C genes were detected by PCR in 9 isolates, and transcripts were detected by RT-PCR in 7 of them. None of the isolates from controls harbored enterotoxin genes. Vβ expansions were detected by RT-PCR in all 22 patients. Low or no Vβ expansions were obtained in controls. The association of HLA-Cw6 with psoriasis in Lebanese concurs with that reported for other ethnic groups. Toxin-producing isolates that colonize patients might play a role in the exacerbation of psoriatic lesions

Molecular characterization of staphylococcal cassette chromosome mec and virulence encoding genes in methicillin-resistant staphylococci at a medical center in Lebanon

Background: Methicillin-resistant staphylococci (MRS) are major human pathogens accounting for most hospital-acquired (HA) and community acquired (CA) infections worldwide. The recent increase in MRS in a medical center in Lebanon elicited the determination of SCCmec types, genotypes, and prevalence of Panton-Valentine leucociden (PVL) and toxic shock syndrome toxin-1 (TSST-1) among the MRS isolates.   Methods: Thirty-six MRS isolates collected between October 2010 and September 2011 at a medical center, Lebanon were typed using phenotypic and genotypic methods. Antimicrobial susceptibility was determined using the disk diffusion agar method. SCCmec typing was performed by multiplex PCR and sequence analysis. The prevalence of the genes encoding PVL and TSST-1 virulence factors and their transcription levels, were determined respectively by PCR and semi-quantitative real-time PCR. The genomic relatedness of the isolates was assessed by random amplified polymorphic DNA (RAPD) analysis.Results: Antimicrobial susceptibility revealed three distinct antibiotypes. The predominant SCCmec type found among the MRS isolates was type IVa (51%). Twenty-nine percent harbored SCCmec type III and 14% harbored SCCmec type II. One isolate harbored SCCmec type IVc, and another  harbored SCCmec type I. All methicillin-resistant Staphylococcus aureus (MRSA) isolates were negative for the gene encoding for PVL, and two were positive for the gene encoding for TSST-1. RAPD analysis demonstrated high genomic diversity among the MRS isolates.Conclusion: This study demonstrated the SCCmec types and the clonality of the MRS strains, allowing the differentiation between HA and CA-MRS strains. CA-MRS have  increased  in the hospital environment and rendered highly resistant to erythromycin and clindamycin

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

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

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