The Emergence and Dissemination of Multidrug Resistant Pseudomonas aeruginosa in Lebanon: Current Status and Challenges during the Economic Crisis.

Pseudomonas aeruginosa is a common cause of healthcare-associated infections and chronic airway diseases in non-clinical settings. P. aeruginosa is intrinsically resistant to a variety of antimicrobials and has the ability to acquire resistance to others, causing increasingly recalcitrant infections and elevating public health concerns. We reviewed the literature on multidrug-resistant (MDR) P. aeruginosa isolated from humans (nosocomial and community-associated), animals, and the environment in Lebanon, a country that has been suffering from a surge in antimicrobial resistance (AMR). We identified 24 studies that described the epidemiology and antimicrobial susceptibility profiles of P. aeruginosa. Our analysis showed that the bacterium was predominant in lesions of patients on mechanical ventilation and in burn patients and those with diabetic foot infections and hematological malignancies. We also found that carbapenem resistance in P. aeruginosa isolates in Lebanon involved both enzymatic and non-enzymatic mechanisms but depended predominantly on VIM-2 production (40.7%). Additionally, MDR P. aeruginosa was detected in animals, where a recent study reported the emergence of carbapenemase-producing P. aeruginosa in livestock in Lebanon. Notably, no studies evaluated the contribution of MDR P. aeruginosa in the environment to human infections. Taken together, our findings highlight the need for AMR surveillance programs and a national action plan to combat resistance in Lebanon.

Autophagy: A Novel Mechanism Involved in the Anti-Inflammatory Abilities of Probiotics.

BACKGROUND/AIMS: Deregulation of the complex interaction among host genetics, gut microbiota and environmental factors on one hand and aberrant immune responses on the other hand, are known to be associated with the development of inflammatory bowel disease. Recent studies provided strong evidence that autophagy plays a key role in the etiology of Crohn's disease (CD). Probiotics may exhibit many therapeutic properties, including anti-inflammatory abilities. While successful results have been obtained in ulcerative colitis patients, probiotics remain inefficient in CD for unknown reason. It remains therefore important to better understand their molecular mechanisms of action. METHODS: The activation of autophagy was examined by stimulating bone marrow-derived dendritic cells by the bacteria, followed by confocal microscopy and western blot analysis. The impact of blocking in vitro autophagy was performed in peripheral blood mononuclear cells using 3-methyl adenine or bafilomycin followed by cytokine secretion measurement by ELISA. The role of autophagy in the anti-inflammatory capacities of the bacterial strains was evaluated in vivo using an acute trinitrobenzene sulfonic acid-induced murine model of colitis. The impact of BMDC was evaluated by adoptive transfer, notably using bone marrow cells derived from autophagy-related 16-like 1-deficient mice. RESULTS: We showed that selected lactobacilli and bifidobacteria are able to induce autophagy activation in BMDCs. Blocking in vitro autophagy abolished the capacity of the strains to induce the release of the anti-inflammatory cytokine interleukin-10, while it exacerbated the secretion of the pro-inflammatory cytokine interleukin-1ß. We confirmed in the TNBS-induced mouse model of colitis that autophagy is involved in the protective capacity of these selected strains, and showed that dendritic cells are involved in this process. CONCLUSION: We propose autophagy as a novel mechanism involved in the regulatory capacities of probiotics.

High association of Cryptosporidium spp. infection with colon adenocarcinoma in Lebanese patients.

BACKGROUND: The association between Cryptosporidium and human colon cancer has been reported in different populations. However, this association has not been well studied. In order to add new strong arguments for a probable link between cryptosporidiosis and colon human cancer, the aim of this study was to determine prevalence and to identify species of Cryptosporidium among Lebanese patients. METHODOLOGY AND PRINCIPAL FINDINGS: Overall, 218 digestive biopsies were collected in Tripoli, Lebanon, from three groups of patients: (i) patients with recently diagnosed colon intraepithelial neoplasia/adenocarcinoma before any treatment (n = 72); (ii) patients with recently diagnosed stomach intraepithelial neoplasia/adenocarcinoma before any treatment (n = 21); and (iii) patients without digestive intraepithelial neoplasia/adenocarcinoma but with persistent digestive symptoms (n = 125). DNA extraction was performed from paraffin-embedded tissue. The presence of the parasite in tissues was confirmed by PCR, microscopic observation and immunofluorescence analysis. We identified a high rate (21%) of Cryptosporidium presence in biopsies from Lebanese patients with recently diagnosed colonic neoplasia/adenocarcinoma before any treatment. This prevalence was significantly higher compared to 7% of Cryptosporidium prevalence among patients without colon neoplasia but with persistent gastrointestinal symptoms (OR: 4, CI: 1.65-9.6, P = 0.001). When the comparison was done against normal biopsies, the risk of infection increased 11-fold in the group of patients with colon adenocarcinoma (OR: 11.315, CI: 1.44-89.02, P = 0.003). CONCLUSIONS: This is the first study performed in Lebanon reporting the prevalence of Cryptosporidium among patients with digestive cancer. These results show that Cryptosporidium is strongly associated with human colon cancer being maybe a potential etiological agent of this disease.

Bifidobacteria-derived lipoproteins inhibit infection with coxsackievirus B4 in vitro.

The aim of the present study was to investigate the potential of bifidobacteria in protecting cells from coxsackievirus B4 (CV-B4) infection. Bifidobacterial screening identified two of five strains that protected human epithelial type 2 (HEp-2) cell viability when bifidobacteria were incubated with viral particles prior to inoculation. In contrast, no effect was shown by incubating HEp-2 cells with bifidobacteria prior to CV-B4 inoculation. Cell wall lipoprotein aggregates (LpAs) secreted by the selected strains were assayed for their antiviral activity. The two LpAs exhibited antiviral activity when they were incubated with viral particles prior to inoculation of HEp-2 cells. Recombinant LpA-derived protein exhibited identical antiviral activity. To identify the peptide sequences interacting with the virus particles, LpA proteins were aligned with the peptide sequences of the north canyon rim and puff footprint onto coxsackievirus and adenovirus receptor (CAR). The in silico molecular docking study using CV-B3 as template showed low-energy binding, indicating a stable system for the selected peptides and consequently a likely binding interaction with CV-B. Bifidobacterium longum and Bifidobacterium breve peptides homologous to the viral north rim footprint onto CAR sequence formed hydrogen bonds with several viral residues in the north rim of the canyon, which were already predicted as interacting with CAR. In conclusion, proteins from bifidobacterial LpAs can inhibit infection with CV-B4, likely through binding to the capsid amino acids that interact with CAR.

Identification of Novel Functions for Hepatitis C Virus Envelope Glycoprotein E1 in Virus Entry and Assembly.

Hepatitis C virus (HCV) envelope glycoprotein complex is composed of E1 and E2 subunits. E2 is the receptor-binding protein as well as the major target of neutralizing antibodies, whereas the functions of E1 remain poorly defined. Here, we took advantage of the recently published structure of the N-terminal region of the E1 ectodomain to interrogate the functions of this glycoprotein by mutating residues within this 79-amino-acid region in the context of an infectious clone. The phenotypes of the mutants were characterized to determine the effects of the mutations on virus entry, replication, and assembly. Furthermore, biochemical approaches were also used to characterize the folding and assembly of E1E2 heterodimers. Thirteen out of 19 mutations led to viral attenuation or inactivation. Interestingly, two attenuated mutants, T213A and I262A, were less dependent on claudin-1 for cellular entry in Huh-7 cells. Instead, these viruses relied on claudin-6, indicating a shift in receptor dependence for these two mutants in the target cell line. An unexpected phenotype was also observed for mutant D263A which was no longer infectious but still showed a good level of core protein secretion. Furthermore, genomic RNA was absent from these noninfectious viral particles, indicating that the D263A mutation leads to the assembly and release of viral particles devoid of genomic RNA. Finally, a change in subcellular colocalization between HCV RNA and E1 was observed for the D263A mutant. This unique observation highlights for the first time cross talk between HCV glycoprotein E1 and the genomic RNA during HCV morphogenesis.IMPORTANCE Hepatitis C virus (HCV) infection is a major public health problem worldwide. It encodes two envelope proteins, E1 and E2, which play a major role in the life cycle of this virus. E2 has been extensively characterized, whereas E1 remains poorly understood. Here, we investigated E1 functions by using site-directed mutagenesis in the context of the viral life cycle. Our results identify unique phenotypes. Unexpectedly, two mutants clearly showed a shift in receptor dependence for cell entry, highlighting a role for E1 in modulating HCV particle interaction with a cellular receptor(s). More importantly, another mutant led to the assembly and release of viral particles devoid of genomic RNA. This unique phenotype was further characterized, and we observed a change in subcellular colocalization between HCV RNA and E1. This unique observation highlights for the first time cross talk between a viral envelope protein and genomic RNA during morphogenesis.

Prevalence, antibiotic susceptibility and characterization of antibiotic resistant genes among carbapenem-resistant Gram-negative bacilli and yeast in intestinal flora of cancer patients in North Lebanon.

The emergence and spread of carbapenem-resistant bacteria are a significant clinical and public health concern. The aim of the study is to determine the prevalence of intestinal carriage of carbapenem-resistant bacteria and yeasts in cancer patients under chemotherapy. 41 stool samples collected from cancer patients in Nini hospital in Tripoli, North Lebanon have been analyzed. After isolating yeasts and carbapenem-resistant bacteria, a biochemical identification and antimicrobial susceptibility profile were determined. The mechanism of enzymatic carbapenem-resistance was detected by searching for carbapenemases by both Hodge test and PCR assays. The association of several mechanisms of resistance was also searched. 46.3% (19/41) of patients were colonized by yeast. Candida glabrata (6/19) was the major species. The prevalence of carbapenem-resistant bacteria was 24.4% (10/41) including Escherichia coli (5/10), Enterobacter cloacae (1/10), Enterobacter aerogenes (1/10) Edwardsiella hoshinae (1/10) Pantoea agglomerans (1/10) and Pseudomonas stutzeri (1/10). PCR and sequencing of the amplified fragments revealed that Pseudomonas stutzeri (1/1) carried VIM gene and Enterobacter aerogenes (1/1) and E. coli (1/5) carried OXA-48 gene. The other Enterobacteriaceae were resistant to carbapenems by mechanisms other than a carbapenemase including hyperproduction of cephalosporinase (4/10), extended spectrum beta-lactamases (1/10) and both cephalosporinase and extended spectrum beta-lactamases (2/10). High prevalence of intestinal carriage of carbapenem-resistant bacteria and yeasts were detected in cancer patients under chemotherapy. In order to prevent the development of endogenous infection and the dissemination of antimicrobial resistance, an implementation of antibiotic stewardship programs and infection control measures is required in hospitals particularly in the department of chemotherapy.

Whole-Genome Sequence of a blaOXA-48-Harboring Raoultella ornithinolytica Clinical Isolate from Lebanon.

We analyzed the whole-genome sequence of ablaOXA-48-harboringRaoultella ornithinolyticaclinical isolate from a patient in Lebanon. The size of theRaoultella ornithinolyticaCMUL058 genome was 5,622,862 bp, with a G+C content of 55.7%. We deciphered all the molecular mechanisms of antibiotic resistance, and we compared our genome to other availableR. ornithinolyticagenomes in GenBank. The resistome consisted of 9 antibiotic resistance genes, including a plasmidicblaOXA-48gene whose genetic organization is also described.

First description of gastroenteritis viruses in Lebanese children: a pilot study.

Human enteric viruses are important causes of acute gastroenteritis in infants and children. The role of rotaviruses, adenoviruses, human caliciviruses and astroviruses in the development of severe acute gastroenteritis requiring hospitalization of infants and young children in North Lebanon was investigated. Stool specimens collected between April and May 2010 from 79 Lebanese infants and children hospitalized for severe acute gastroenteritis, were screened for enteric viruses by immunoassays and internally controlled multiplex PCR assay. Out of 79 stool samples, 38 (48%) were positive for rotavirus, and 5 (6%) were positive for norovirus genogroup II. Enteric adenoviruses, sapoviruses and human astroviruses were not detected. Children with severe rotavirus gastroenteritis were younger than those with severe norovirus gastroenteritis. These results highlight the importance of rotavirus and norovirus as causes of severe gastroenteritis in Lebanese children, and the need to incorporate routine screening tests for norovirus infection in clinical settings.