Molecular Characteristics of Colistin Resistance in Acinetobacter baumannii and the Activity of Antimicrobial Combination Therapy in a Tertiary Care Medical Center in Lebanon.

(1) Background: Infections with pan-drug-resistant (PDR) bacteria, such as A. baumannii, are becoming increasingly common, especially in healthcare facilities. In this study, we selected 15 colistin-resistant clinical A. baumannii isolates from a hospital in Beirut, Lebanon, to test combination therapies and determine their sequence types (STs) and the mechanism of colistin resistance using whole-genome sequencing (WGS). (2) Methods: Antimicrobial susceptibility testing via broth microdilution against 12 antimicrobials from different classes and growth rate assays were performed. A checkerboard assay was conducted on PDR isolates using six different antimicrobials, each in combination with colistin. Genomic DNA was extracted from all isolates and subjected to WGS. (3) Results: All isolates were resistant to all tested antimicrobials with the one exception that was susceptible to gentamicin. Combining colistin with either meropenem, ceftolozane-tazobactam, or teicoplanin showed synergistic activity. Sequencing data revealed that 67% of the isolates belonged to Pasteur ST2 and 33% to ST187. Furthermore, these isolates harbored a number of resistance genes, including blaOXA-23. Mutations in the pmrC gene were behind colistin resistance. (4) Conclusions: With the rise in antimicrobial resistance and the absence of novel antimicrobial production, alternative treatments must be found. The combination therapy results from this study suggest treatment options for PDR ST2 A. baumannii-infected patients.

Molecular characterization and differential effects of levofloxacin and ciprofloxacin on the potential for developing quinolone resistance among clinical Pseudomonas aeruginosa isolates.

Background: Fluoroquinolones are some of the most used antimicrobial agents for the treatment of Pseudomonas aeruginosa. This study aimed at exploring the differential activity of ciprofloxacin and levofloxacin on the selection of resistance among P. aeruginosa isolates at our medical center. Methods: 233 P. aeruginosa clinical isolates were included in this study. Antimicrobial susceptibility testing (AST) was done using disk diffusion and broth microdilution assays. Random Amplification of Polymorphic DNA (RAPD) was done to determine the genetic relatedness between the isolates. Induction of resistance against ciprofloxacin and levofloxacin was done on 19 isolates. Fitness cost assay was done on the 38 induced mutants and their parental isolates. Finally, whole genome sequencing was done on 16 induced mutants and their 8 parental isolates. Results: AST results showed that aztreonam had the highest non-susceptibility. RAPD results identified 18 clusters. The 19 P. aeruginosa isolates that were induced against ciprofloxacin and levofloxacin yielded MICs ranging between 16 and 256 µg/mL. Levofloxacin required fewer passages in 10 isolates and the same number of passages in 9 isolates as compared to ciprofloxacin to reach their breakpoints. Fitness cost results showed that 12 and 10 induced mutants against ciprofloxacin and levofloxacin, respectively, had higher fitness cost when compared to their parental isolates. Whole genome sequencing results showed that resistance to ciprofloxacin and levofloxacin in sequenced mutants were mainly associated with alterations in gyrA, gyrB and parC genes. Conclusion: Understanding resistance patterns and risk factors associated with infections is crucial to decrease the emerging threat of antimicrobial resistance.

Transcriptional profiling and genetic analysis of a cystic fibrosis airway-relevant model shows asymmetric responses to growth in a polymicrobial community.

Bacterial infections in the lungs of persons with cystic fibrosis are typically composed of multispecies biofilm-like communities, which modulate clinically relevant phenotypes that cannot be explained in the context of a single species culture. Most analyses to date provide a picture of the transcriptional responses of individual pathogens; however, there is relatively little data describing the transcriptional landscape of clinically relevant multispecies communities. Harnessing a previously described cystic fibrosis-relevant, polymicrobial community model consisting of Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus sanguinis, and Prevotella melaninogenica, we performed an RNA-Seq analysis on the biofilm population to elucidate the transcriptional profiles of the community grown in artificial sputum medium (ASM) as compared to growth in monoculture, without mucin, and in fresh medium supplemented with tobramycin. We provide evidence that, although the transcriptional profile of P. aeruginosa is community agnostic, the transcriptomes of S. aureus and S. sanguinis are community aware. Furthermore, P. aeruginosa and P. melaninogenica are transcriptionally sensitive to the presence of mucin in ASM, whereas S. aureus and S. sanguinis largely do not alter their transcriptional profiles in the presence of mucin when grown in a community. Only P. aeruginosa shows a robust response to tobramycin. Genetic studies of mutants altered in community-specific growth provide complementary data regarding how these microbes adapt to a community context. IMPORTANCE Polymicrobial infections constitute the majority of infections in the cystic fibrosis (CF) airway, but their study has largely been neglected in a laboratory setting. Our lab previously reported a polymicrobial community that can help explain clinical outcomes in the lungs of persons with CF. Here, we obtained transcriptional profiles of the community versus monocultures to provide transcriptional information about how this model community responds to CF-related growth conditions and perturbations. Genetic studies provide complementary functional outputs to assess how the microbes adapt to life in a community.

The impact of vaccination on the burden of invasive pneumococcal disease from a nationwide surveillance program in Lebanon: an unexpected increase in mortality driven by non-vaccine serotypes.

BACKGROUND: The impact of pneumococcal conjugate vaccines (PCVs) on the burden of invasive pneumococcal disease (IPD) and serotype distribution was examined across age groups from data collected by the Lebanese Inter-Hospital Pneumococcal Surveillance Program. METHODS: Between 2005 and 2020, 593 invasive Streptococcus pneumoniae isolates were collected from 79 hospitals throughout Lebanon. Serotypes and antimicrobial resistance (AMR) profiles were identified, and trends compared over 3 eras: PCV7, post-PCV7/ pre-PCV13, and PCV13 eras. RESULTS: The prevalence of PCV7 serotypes decreased significantly from 43.6% in the PCV7 era to 17.8% during the PCV13 era (p<0.001). PCV13-only serotypes remained stable in the PCV13 compared to the post-PCV7 eras, especially serotypes 1 and 3, whereas non-vaccine types (NVT) increased throughout the study period, especially 24 and 16F. The mortality rate increased substantially from 12.5% (PCV7 era) to 24.8% (PCV13 era). A significant decrease in AMR was observed across the three study eras. CONCLUSION: PCVs substantially impacted IPD and AMR in vaccinated and unvaccinated populations despite an increase in mortality driven by NVT. Broadening the recommendation of vaccination to include older age-groups, using higher valency vaccines, and implementing stringent antimicrobial stewardship are likely to further impact the burden of IPD.

A national study through a 'Farm-to-fork' Approach to determine Salmonella dissemination along with the Lebanese poultry production chain.

This cross-sectional study was conducted to determine the prevalence of Salmonella at different stages of the broiler production chain and layer flocks in addition to their antibiotic resistance profile and molecular patterns. Over a period of 3 years, different sample matrices were collected from Lebanese farms, slaughterhouses and retail markets. Out of 672 Salmonella serotyped, 514 were analysed for antimicrobial resistance and 214 for clonality using Pulsed-field gel electrophoresis (PFGE). The results highlighted an important prevalence of Salmonella, 30% in farms, 35.8% in slaughterhouses and 22.4% at retail level. A large diversity of serotypes was identified with predominance among Salmonella Infantis (32.9%), Salmonella Enteritidis (28.4%) and Salmonella Kentucky (21.4%). High resistance to nalidixic acid was revealed in all the isolates. The most prominent resistance was exhibited in S. Kentucky and S. Infantis. The latter was resistant to tetracycline (99%), streptomycin (88.2%) and remarkable multi-drug resistance (MDR) (89.7%). All S. Kentucky isolates were resistant to ciprofloxacin, MDR (62.4%) and 6% were resistant to extended-spectrum cephalosporin (ESCs). One persistent clone of S. Enteritidis was found common between poultry and humans. Similar genomic profiles were detected between farms, slaughterhouses and retail suggesting the dissemination of identical clones throughout the food chain possibly due to weak barriers preventing such transmission.

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

BACKGROUND: Carbapenem-resistant Gram-negative bacteria are a major clinical concern as they cause virtually untreatable infections since carbapenems are among the last-resort antimicrobial agents. ß-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/ß-lactamase inhibitor (ßLI) combinations. In this study, we assessed the in vitro and in vivo phenotypic and molecular efficacies of three ßLIs when combined with different carbapenems against carbapenem-resistant Gram-negative clinical isolates. The chosen ßLIs were (1) Avibactam, against OXA-type carbapenemases, (2) calcium-EDTA, against NDM-1, and (3) Relebactam, against KPC-2. METHODS: Six Acinetobacter baumannii clinical isolates were screened for bla OXA-23-like, bla OXA-24/40, bla OXA-51-like, bla OXA-58, and bla OXA-143-like, and eight Enterobacteriaceae clinical isolates were screened for bla OXA-48, bla NDM-1, and bla KPC-2. The minimal inhibitory concentrations of Imipenem (IPM), Ertapenem (ETP), and Meropenem (MEM) with corresponding ßLIs for each isolate were determined. The efficacy of the most suitable in vitro treatment option against each of bla OXA-48, bla NDM-1, and bla KPC-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/ßLI combinations used under both in vitro and in vivo settings. RESULTS: Combining MEM, IPM, and ETP with the corresponding ßLIs 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 bla OXA-48 and bla KPC-2, respectively. RT-qPCR demonstrated the consistent overexpression of bla OXA-48 upon treatment, without hindering Avibactam's activity, while bla NDM-1 and bla KPC-2 experienced variable expression levels upon treatment under in vitro and in vivo settings despite their effective phenotypic results. CONCLUSION: New carbapenem/ßLI 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.

PulseNet Lebanon: An Overview of Its Activities, Outbreak Investigations, and Challenges.

Background: Foodborne diseases are still a major health issue in Lebanon, although some steps have been taken forward in food safety. To this purpose, PulseNet Lebanon, a foodborne diseases tracking network, was established in 2009, through the collaboration between the Ministry of Public Health (MoPH) and the American University of Beirut (AUB). Materials and Methods: Three papers published regarding the PulseNet project were summarized. Initially, clinical and food samples, collected within the surveillance network scope, were identified by using the respective API for Salmonella and Listeria spp. Salmonella spp. were further serotyped by using the Kauffman and White method. Campylobacter spp. were determined by the 16 S rRNA sequencing method. Antimicrobial susceptibility to a number of antibiotics was determined by using the disk diffusion method for Samonella and Campylobacter spp. Genomic diversity was determined by using pulsed field gel electrophoresis (PFGE) and random amplified polymorphic DNA (RAPD). Results: Results indicated that 290 clinical and 49 food isolates were identified as Salmonella. Serotyping revealed the prevalence of ten and seven serotypes in the clinical and food samples, respectively. Fifty-one isolates from chicken ceca and carcass were identified to be Campylobacter spp. Fifty-nine samples were identified to be Listeria monocytogenes. Antimicrobial susceptibility testing revealed a wide range of resistance among the different samples. PFGE showed a variation in pulsotypes among the Salmonella serotypes. PFGE also linked certain outbreaks to their food sources. This method also demonstrated 13 subtypes with 100% similarity among the L. monocytogenes isolates. Finally, the Camplyobcater spp. were grouped into nine clusters with a minimum similarity of 43.5% using RAPD. Conclusion: This summary of results shows the importance of implementing a "farm-to-fork" approach in the surveillance of foodborne disease outbreaks in Lebanon, allowing the detection of pathogens causing foodborne disease outbreaks in a timely fashion.

NAT1 genotypic and phenotypic contribution to urinary bladder cancer risk: a systematic review and meta-analysis.

N-acetyltransferase 1 (NAT1), a polymorphic Phase II enzyme, plays an essential role in metabolizing heterocyclic and aromatic amines, which are implicated in urinary bladder cancer (BCa). This systematic review investigates a possible association between the different NAT1 genetic polymorphisms and BCa risk. Medline, PubMed, EMBASE, Scopus, Web of Science, OpenGrey, and BASE databases were searched to identify eligible studies. The random-effect model was used to calculate pooled effects estimates. Statistical heterogeneity was tested with Chi-square and I2. Twenty case-control studies, including 5606 cases and 6620 controls, met the inclusion criteria. Pooled odds ratios (OR) analyses showed a statistically significant difference in NAT1*10 versus non-NAT1*10 acetylators in the total sample (OR: 0.87; 95% CI: 0.79-0.96) but was borderline among Caucasians (OR: 0.88 with 95% CI: 0.77-1.01). No statistically significant differences in BCa risk were found for: NAT1*10 versus NAT1*4 wild type (OR: 0.97; 95% CI: 0.78-1.19), NAT1 'Fast' versus 'Normal' acetylators (OR: 1.03; 95% CI: 0.84-1.27), and NAT1 'Slow' versus 'Fast' (OR: 2.32; 95% CI: 0.93-5.84) or 'Slow' versus 'Normal' acetylators (OR: 1.84; 95% CI: 0.92-3.68). When stratifying by smoking status, no statistically significant differences in BCa risk were found for NAT1*10 versus non-NAT1*10 acetylators among the different subgroups. Our study suggests a modest protective role for NAT1*10 and a possible risk contributory role for slow acetylation genotypes in BCa risk. Further research is recommended to confirm these associations.

Increase of blaOXA-23-like in Acinetobacter baumannii at a tertiary care center in Lebanon between 2007 and 2013.

INTRODUCTION: The multi-drug resistant nature of Acinetobacter baumannii isolates have rendered many broad-spectrum antimicrobial agents ineffective against them. The purpose of this retrospective study is to define and compare the molecular characteristics of A. baumannii isolates from patients at a tertiary care center in Lebanon from two outbreaks, the first in 2007-2008, as part of a case-controlled study involving A. baumannii cases admitted to the ICU, and the second in 2013. METHODOLOGY: A total of 148 A. baumannii clinical isolates were collected from various clinical specimens during 2007-2008 and 2013. All A. baumannii isolates were screened for blaOXA-23-like and blaOXA-51-like genes of carbapenem resistance. Additionally, in an effort to assess the degree of the isolates' genomic relatedness, random amplification of polymorphic DNA (RAPD) was performed. RESULTS: There was an increase in the prevalence of blaOXA-23-like and blaOXA-51-like genes between the two time periods; however, only 22% isolate genomic relatedness was calculated between 2007-2008 and 2013. Taking 80% as a margin of compatibility, 31 distinct clusters containing 2 to 11 strains were observed when both time periods were analyzed. CONCLUSION: The presence of numerous clusters accompanied by a predominant increase in the prevalence of blaOXA-23-like between 2007 and 2013 suggests a horizontal transmission of the gene within various strains of the species, contributing to the persistent increase in carbapenem resistance over the years. Therefore, infection control measures are required with compliance among all healthcare workers.

Combating antimicrobial resistance using antimicrobial combination therapy and ß-lactamase inhibitors.

INTRODUCTION: The range of antimicrobial agents used to treat bacterial infections is becoming limited with the constant increase in antimicrobial resistance (AMR). Several genetic factors underlie AMR, including ß-lactamase-encoding genes such as blaCTXM-15 that confers resistance to third-generation cephalosporins, and blaOXA-48, blaNDM-1, and blaKPC-2 that confer resistance to carbapenems. Remaining treatment approaches for such resistant infections include antimicrobial combination therapy and the use of ß-lactamase inhibitors. This study assesses the molecular effects of such treatment approaches on antimicrobial resistant Enterobacteriaceae clinical isolates in vitro and in vivo. METHODOLOGY: Nine clinical Enterobacteriaceae isolates were included in the study. One harboring blaCTXM-15, one harboring blaOXA-48, one harboring blaKPC-2, two harboring blaNDM-1 and blaCTXM-15, and four harboring blaOXA-48 and blaCTXM-15. Minimal inhibitory concentrations were determined for carbapenems with ß-lactamase inhibitors: avibactam, Ca-EDTA, and relebactam. Synergism between antibiotic combinations was determined by double disc diffusion when using colistin with several antibiotics. In vitro and in vivo gene expression levels were done on these combinations with and without inhibitors. RESULTS: The use of meropenem, imipenem, and ertapenem with the selected ß-lactamase inhibitors restored isolate susceptibility in 100%, 87.5%, and 25% of the cases, respectively. Antimicrobial synergism was mostly detected between colistin and meropenem, fosfomycin, or tigecycline. Survival studies revealed the survival of most mice receiving antimicrobial combination therapy with inhibitors as compared to the controls. Overall gene expression levels of resistance genes were variable depending on treatment. CONCLUSIONS: The threat of antibiotic resistant bacterial infections remains viable; however, different approaches to therapy are available.

Increased blaOXA-23-like prevalence in Acinetobacter baumannii at a tertiary care center in Lebanon (2007-2013).

INTRODUCTION: Acinetobacter baumannii has become one of the most feared organisms in hospital-acquired infections during the past decades. Their multi-drug resistant profiles have rendered many broad-spectrum antibiotics ineffective. The purpose of this retrospective study is to describe and compare molecular characteristics of A. baumannii isolated from patients at a tertiary care center in Lebanon from two outbreaks, the first in 2007-2008 as part of a case-controlled study involving Acinetobacter baumannii cases admitted to the ICU and the second in 2013. METHODOLOGY: A total of 148 A. baumannii clinical isolates were collected from various clinical specimens during 2007-2008 and 2013. All A. baumannii isolates were subjected to PCR amplification of blaOXA-23-like and blaOXA-51-like genes of carbapenem resistance. Random amplification of polymorphic DNA (RAPD) was also performed to assess their genomic relatedness. RESULTS: There was an increase in the prevalence of blaOXA-23-like and blaOXA-51-like between the two time periods; however, only with 22% genomic relatedness between 2007-2008 and 2013 isolates. Taking 80% as margin of compatibility, 31 distinct clusters containing 2 to 11 strains were observed in both time periods. CONCLUSION: The presence of numerous clusters accompanied by a predominant increase in the prevalence of blaOXA-23-like gene between 2007 and 2013 suggests a horizontal transmission of the gene within various strains of the species, constituting a primary factor in the continued increase of carbapenem resistance over the years. As such, infection control measures ought to be taken with the highest priority and compliance among all involved healthcare workers is of utmost importance.