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.

Antimicrobial effects of chlorine dioxide in a hospital setting.

Chlorine dioxide is a powerful disinfectant with strong antibacterial properties. We conducted a study at different sites of the Lebanese American University Medical Center-Rizk Hospital to determine the efficacy of the ECOM air mask in decreasing the particle load. Air cultures were obtained from three different locations, namely the patients' elevator, visitors' elevator and mobile clinic and the number of colonies grown on each type of agar was determined. We also measured particle counts at the three sites both at baseline and after placement of the ECOM air mask. After 7 days of ECOM air mask use, the numbers of colonies grown on all types of media was decreased by 20-100% versus the baseline values. The counts of particles of different diameters (0.3, 0.5 and 5 µm) were decreased at all three sampled sites. This study highlighted the efficacy of the ECOM air mask. The utility of the gaseous form of ClO2 as an antiseptic in the hospital setting appears promising.

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.

Targeted Central Nervous System Irradiation with Proton Microbeam Induces Mitochondrial Changes in Caenorhabditis elegans.

Fifty percent of all patients with cancer worldwide require radiotherapy. In the case of brain tumors, despite the improvement in the precision of radiation delivery with proton therapy, studies have shown structural and functional changes in the brains of treated patients with protons. The molecular pathways involved in generating these effects are not completely understood. In this context, we analyzed the impact of proton exposure in the central nervous system area of Caenorhabditis elegans with a focus on mitochondrial function, which is potentially implicated in the occurrence of radiation-induced damage. To achieve this objective, the nematode C. elegans were micro-irradiated with 220 Gy of protons (4 MeV) in the nerve ring (head region) using the proton microbeam, MIRCOM. Our results show that protons induce mitochondrial dysfunction, characterized by an immediate dose-dependent loss of the mitochondrial membrane potential (ΔΨm) associated with oxidative stress 24 h after irradiation, which is itself characterized by the induction of the antioxidant proteins in the targeted region, observed using SOD-1::GFP and SOD-3::GFP strains. Moreover, we demonstrated a two-fold increase in the mtDNA copy number in the targeted region 24 h after irradiation. In addition, using the GFP::LGG-1 strain, an induction of autophagy in the irradiated region was observed 6 h following the irradiation, which is associated with the up-regulation of the gene expression of pink-1 (PTEN-induced kinase) and pdr-1 (C. elegans parkin homolog). Furthermore, our data showed that micro-irradiation of the nerve ring region did not impact the whole-body oxygen consumption 24 h following the irradiation. These results indicate a global mitochondrial dysfunction in the irradiated region following proton exposure. This provides a better understanding of the molecular pathways involved in radiation-induced side effects and may help in finding new therapies.

The under investigated facet of the COVID-19 pandemic: Molecular analysis of secondary bacterial infections at a COVID dedicated intensive care unit within a tertiary care center in Lebanon.

Background: The coronavirus disease 2019 (COVID-19) caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has spread worldwide. Secondary bacterial infections are associated with unfavorable outcomes in respiratory viral infections. This study aimed at determining the prevalence of secondary bacterial infections in COVID-19 patients admitted at a tertiary medical center in Lebanon. Methodology: From May till November, 2020, a total of 26 Gram-negative isolates were recovered from 16 patients during the course of their COVID-19 infection with Escherichia coli being the most prevalent. The isolates were assessed for their antimicrobial susceptibility by broth microdilution against 19 antimicrobial agents from different classes. Whole genome sequencing of 13 isolates allowed the mining of antimicrobial resistance (AMR) determinants as well as mobile genetic elements and sequence types (ST). Finally, broth microdilution with three different efflux pump inhibitors [theobromine, conessine and PheArg-ß-naphthylamide (PAßN)] was done. Results: Antimicrobial susceptibility testing showed that out of the 26 Gram-negative isolates, 1 (4%) was extensively drug resistant and 14 (54%) were multi-drug resistant (MDR). Whole genome sequencing results revealed a plethora of AMR determinants among the 13 sequenced isolates. Moreover, the 9 Enterobacterales and 4 Pseudomonas aeruginosa sequenced isolates belonged to 9 and 2 different ST, respectively. Using a variety of efflux pump inhibitors we demonstrated that only PAßN had a significant effect when combined with levofloxacin, and the latter regained its activity against two P. aeruginosa isolates. Conclusion: The identification of carbapenem and colistin resistant Gram-negative bacilli causing secondary bacterial infections in critical patients diagnosed with COVID-19 should be of high concern. Additionally, it is crucial to monitor and track AMR, post-COVID pandemic, in order to better understand the effect of this disease on AMR exacerbation.

Mitochondrial stress in advanced fibrosis and cirrhosis associated with chronic hepatitis B, chronic hepatitis C, or nonalcoholic steatohepatitis.

BACKGROUND AND AIMS: Hepatitis B virus (HBV) infection causes oxidative stress (OS) and alters mitochondria in experimental models. Our goal was to investigate whether HBV might alter liver mitochondria also in humans, and the resulting mitochondrial stress might account for the progression of fibrosis in chronic hepatitis B (CHB). APPROACH AND RESULTS: The study included 146 treatment-naïve CHB mono-infected patients. Patients with CHB and advanced fibrosis (AF) or cirrhosis (F3-F4) were compared to patients with no/mild-moderate fibrosis (F0-F2). Patients with CHB were further compared to patients with chronic hepatitis C (CHC; n = 33), nonalcoholic steatohepatatis (NASH; n = 12), and healthy controls ( n = 24). We detected oxidative damage to mitochondrial DNA (mtDNA), including mtDNA strand beaks, and identified multiple mtDNA deletions in patients with F3-F4 as compared to patients with F0-F2. Alterations in mitochondrial function, mitochondrial unfolded protein response, biogenesis, mitophagy, and liver inflammation were observed in patients with AF or cirrhosis associated with CHB, CHC, and NASH. In vitro , significant increases of the mitochondrial formation of superoxide and peroxynitrite as well as mtDNA damage, nitration of the mitochondrial respiratory chain complexes, and impairment of complex I occurred in HepG2 cells replicating HBV or transiently expressing hepatitits B virus X protein. mtDNA damage and complex I impairment were prevented with the superoxide-scavenging Mito-Tempo or with inducible nitric oxide synthase (iNOS)-specific inhibitor 1400 W. CONCLUSIONS: Our results emphasized the importance of mitochondrial OS, mtDNA damage, and associated alterations in mitochondrial function and dynamics in AF or cirrhosis in CHB and NASH. Mitochondria might be a target in drug development to stop fibrosis progression.

Facial microbial flora in bearded versus nonbearded men in the operating room setting: A single-center cross-sectional STROBE-compliant observational study.

Beards are controversial in the operating room setting because of the possible retention and shedding of pathogens. Surgical site infection poses a significant burden on healthcare systems. All male healthcare workers who entered the operating room were approached to participate in the study. Four facial swab samples were anonymously collected and a hygiene practice questionnaire was administered. Sample A was taken from the upper and lower lips, sample B from cheeks, and samples C and D were collected by 20 and 40 cm shedding below the face. Colony-forming units (CFUs) and minimum inhibitory concentrations (MICs) of meropenem resistance were determined for samples A and B. Random samples from A, B, C, and D, in addition to meropenem-resistant isolates were cultured with chlorohexidine. Sixty-one bearded and 19 nonbearded healthcare workers participated in the study. 98% were positive for bacterial growth with CFU ranging between 30 × 104 and 200 × 106 CFU/mL. Bacterial growth was significantly higher in bearded participants (P < .05). Eighteen (27.1%) isolates were resistant to meropenem; of these which 14 (77.8%) were from bearded participants, this was not statistically significant. Chlorohexidine was effective in inhibiting the growth of all strains including the meropenem-resistant isolates. Bearded men in the operating room had a significantly higher facial bacterial load. Larger-scale resistance studies are needed to address facial bacterial resistance among healthcare workers in the operating room. This study aimed to estimate the facial microbial load and identify strains and antimicrobial resistance profiles in bearded versus nonbearded male healthcare workers in the operating room of a tertiary hospital in the Middle East.

Acquired resistome and plasmid sequencing of mcr-1 carrying MDR Enterobacteriaceae from poultry and their relationship to STs associated with humans.

OBJECTIVES: To investigate the acquired resistome in 18 colistin-resistant Escherichia coli isolated from different poultry farms in Lebanon, analyse Inc plasmids associated with mcr and assess potential transmission to humans. METHODS: A total of 18 E. coli were recovered from poultry faeces collected from different poultry farms in Lebanon. Broth microdilution (BMD) assay was performed to determine the antimicrobial resistance profiles. WGS was used to identify the genetic determinants behind the resistance in these isolates. RESULTS: BMD results showed that all of the 18 isolates were colistin resistant. Furthermore, resistance to trimethoprim/sulfamethoxazole was the most recorded among the isolates and only one isolate was resistant to cefepime. Sequencing results showed that the isolates were distributed into seven different STs and that the most abundant was ST1140. The number of antimicrobial resistant determinants ranged from 4 to 21 among the 18 isolates, with tet(A) and floR being the most frequent. Moreover, a total of 15 different plasmid replicon types were identified. The mcr-1 gene was shown to be predominantly located on IncX4 plasmids. Additionally, two isolates harboured the IncI2-type self-conjugative plasmid. CONCLUSIONS: The findings show that mcr and other important resistance determinants occur in MDR E. coli isolated poultry farms in Lebanon. The occurrence of mcr on mobile plasmids and the zoonotic potential and clinical relevance of some strains highlight a risk of transmission to humans.

Diversity of Sequence Types and Impact of Fitness Cost among Carbapenem-Resistant Acinetobacter baumannii Isolates from Tripoli, Libya.

We investigated the molecular epidemiology of 21 carbapenem-resistant Acinetobacter baumannii isolates from Libya and assessed their relative fitness. Core genome multilocus sequence typing (MLST) revealed five interhospital transmission clusters. Three clusters were associated with the international clones (IC) IC1, IC2, and IC7. Carbapenem-resistance was associated with blaOXA-23, blaGES-11, or blaNDM-1. Compared to that of A. baumannii DSM 30008, the doubling time was similar over 10 h, but after 16 h, half the isolates grew to higher densities, suggesting a fitness advantage.

An unequivocal superbug: PDR Klebsiella pneumoniae with an arsenal of resistance and virulence factor genes.

INTRODUCTION: Infections caused by extensively-drug resistant (XDR) and pan-drug resistant (PDR) Klebsiella pneumoniae represent an emerging threat due to the high associated mortality. This study aimed to characterize two carbapenem resistant K. pneumoniae strains from the same patient, the first being PDR (referred to as IMP 1078b) and the second being XDR (referred to IMP 1078s) isolated from the same patient. METHODOLOGY: Antimicrobial susceptibility testing was done for the 2 K. pneumoniae isolates, followed by carbapenem/ß-lactamase inhibitor combination assay, and fitness cost against cefepime and meropenem. Then, whole-genome sequence analysis was performed to decipher the molecular mechanisms behind the high level of resistance recorded in both isolates. Finally, qRT-PCR was done for ß-lactam resistant genes. RESULTS: This is the first report about a K. pneumoniae isolate harboring 47 antimicrobial resistance genes and having type IV pilli (Yersinia) and the fimbrial adherence determinant Stb (Salmonella) as virulence factors. Further analysis on both isolates are discussed within the article. CONCLUSION: The co-existence of a high number of antimicrobial resistant (AMR) genes and virulence factor genes may lead to a life threatening invasive and untreatable infection.

Prevalence and molecular epidemiology of carbapenem-resistant Gram-negative bacilli and their resistance determinants in the Eastern Mediterranean Region over the last decade.

Carbapenem resistance in Enterobacteriaceae, Acinetobacter baumannii and Pseudomonas aeruginosa is increasing worldwide, which has led the World Health Organization (WHO) to list these bacteria in the critical priority pathogens group. Infections by such pathogens pose a serious threat to hospitalised patients and are associated with clinical and economic consequences. What worsens the case is the weak pipeline of available antimicrobial agents to treat such infections and the absence of new drugs. The aim of this review was to shed light on all studies tackling carbapenem resistance in Enterobacteriaceae, A. baumannii and P. aeruginosa in the Eastern Mediterranean region, with indication for each country, description of studies timeline, prevalence of carbapenem resistance, and carbapenem resistance-encoding genes detected in these countries.

Dissemination of Multidrug-Resistant and mcr-1 Gram-Negative Bacilli in Broilers, Farm Workers, and the Surrounding Environment in Lebanon.

Poultry are currently regarded as reservoirs from which multidrug resistance can be readily transferred to the surrounding ecosystem. The aim of this study was to explore the prevalence of extended spectrum beta-lactamases (ESBLs) and ampC and mcr-1 Gram-negative bacilli in chicken, farmers, and environment in Lebanon. In May 2017, we revisited the farm where the first mcr-1 was detected in 2015 in Lebanon. Overall 200 chicken fecal swabs, 6 farmers' fecal samples, and 41 environmental samples were collected. Real time (RT)-PCR was performed for beta-lactamases and mcr genes' screening using newly designed primers/probes. Multilocus sequence typing (MLST) was also performed. ESBL/ampCs were found in 118 samples from chicken, 4 from workers, and all environmental ones. mcr-1 was detected in all chicken and farmers' samples from which 314 and 7 strains were isolated, respectively. Three mcr-1 Escherichia coli strains were isolated from litter and feed. Compared to 2015, the prevalence of ESBL/ampC producers, TEM and CTX-M genes increased significantly in 2017. Main spectra profiles dendrogram of isolated E. coli strains in 2015/2017 and MLST revealed the presence of different clones and sequence types. The evolution of resistance appears to be multi-clonal and related to the diffusion of plasmids carrying ESBL and mcr-1 genes. More work is needed to quantify the magnitude of this emerging problem in Lebanon.

Antimicrobial Activity of Polyphenols and Alkaloids in Middle Eastern Plants.

Antibiotic-resistant microorganisms have been an ever-growing concern over the past years. This has led researchers to direct their attention onto plants to be able to discover new possible antimicrobial compounds. The Middle East encompasses a wide spectrum of plant diversity with over 20,000 different species in habitats ranging from deserts to snow-capped mountains. Several plant secondary metabolites and their derivatives have been identified as possible antimicrobial agents. Among the secondary metabolites studied, alkaloids and polyphenols have shown strong antimicrobial activity. Polyphenols are one of the most numerous and diverse group of secondary metabolites; their antioxidant properties provide the basis for antimicrobial effects. Alkaloids provided the underlying structure for the development of several antibiotics with a diverse range of action. The ability of some plant secondary metabolites to act as resistance-modifying agents is a promising field in mitigating the spread of bacterial resistance.

Recurrent Asymptomatic Sigmoid Diverticular Perforation in a Patient with Pemphigus Vulgaris on Immunosuppressive Therapy: A Case Report.

BACKGROUND Perforation of the colon is associated with high mortality and requires early diagnosis. However, the diagnosis of perforation from atypical causes can be a diagnostic challenge. This report is of a rare case of recurrent sigmoid colonic perforation in a patient with diverticular disease who did not present with an acute abdomen but who had pemphigus vulgaris treated with immunosuppressive therapy. CASE REPORT A 57-year-old man with pemphigus vulgaris was treated with steroids, non-steroidal anti-inflammatory drugs (NSAIDS), and azathioprine. He had episodes of abdominal bloating but denied any other symptoms. He was diagnosed with spontaneous sigmoid diverticular perforation without presenting with an acute abdomen. CONCLUSIONS Diverticular perforation can be asymptomatic in patients on immunosuppressive therapy. Therefore, there should be a high index of suspicion for bowel perforation in patients with abdominal symptoms who are treated for skin diseases, such as pemphigus vulgaris, and are on steroids and other immunosuppressive treatments.

Epidemiology of multi drug resistant organisms in pigs in Lebanon.

INTRODUCTION: Livestock are nowadays considered potent reservoirs of multi drug resistance. Enteric resistant organisms in animals can be transmitted to humans and be causative agents of infections with therapeutic challenges. The aim of this study is to determine the prevalence of multi drug resistant organisms in Lebanese swine farms. METHODOLOGY: In May 2017, 94 fecal samples were collected from pigs in the south of Lebanon. Three media supplemented with cefotaxime, ertapenem, colistin were used for the screening of ESBL, carbapenemase producers and colistin resistance respectively. MALDI-TOF was used for bacterial identification. Double disk synergy test, ampC disk test and carpa np test were used for the detection of ESBL, ampC and carbapenemase producers respectively. RT-PCR was performed for the screening of beta lactamase and mcr colistin resistance genes. RESULTS: 77/94 fecal samples, showed growth on the medium supplemented with cefotaxime. In total 111 strains were isolated: 94% were identified as E.coli, 6% other organisms such as E. fergusonii and K. pneumoniae. Phenotypic tests showed that 72% of isolated strains were ESBL producers while 28% were ampC beta lactamase producers. RT-PCR analysis revealed that blaCTX-M was present in 45% of isolated strains, blaTEM in 26% and blaSHV in 10%. In parallel, 22 colistin resistant E.coli strains and 1 K.pneumoniae carrying mcr-1 were isolated. CONCLUSIONS: This study showed the importance of swine farms as reservoirs of resistance in Lebanon. The emergence of colistin resistance in pigs is worrying. A re-evaluation of antibiotic consumption in pigs is therefore warranted.

Multi Drug resistant Organisms in Chicken farms and their Surrounding Environment.

INTRODUCTION: Chicken farms are nowadays regarded as reservoirs of multi-drug resistance. Studies have shown that resistant organisms can be readily transferred from animals to their surrounding ecosystem. The aim of this study is to determine if any link exists between the prevalence of multi-drug resistance in chicken farms and their surrounding environment. METHODOLOGY: In May-2017, 200 fecal swabs were collected from a chicken farm in Lebanon. Fecal samples from six workers and 41 environmental samples surrounding the farm were also taken. Three different selective media were used for the screening of multi-drug resistant and colistin resistant organisms. MALDI-TOF was used for bacterial identification. Double disk synergy test and ampC disk test were used for the screening of ESBL and ampC producers respectively. Furthermore, RT-PCR was performed for the detection of beta lactamase and mcr colistin resistance genes. RESULTS: In chicken, 315 E.coli strains were isolated: 53% were ESBL/ampC co-producers, 27% ampC and 42.5% mcr-1 positive isolates. Furthermore, 29 K.pneumoniae harboring mcr-1 were also isolated. In workers, ESBL producing E.coli were detected in 4/6 workers whereas mcr-1 carrying E.coli were detected in all workers. In the environment, ESBLs and mcr-1 positives were detected in 95% and 7% of the samples respectively. RT-PCR revealed the detection of B-lactamase genes in all samples at different rates. CONCLUSIONS: This study showed a relatively high prevalence of ESBL and mcr-1 positive isolates in chicken and their environment. MLST is in progress to determine if any link exists between multi-drug resistant organisms in these ecosystems investigated.