Genetic Diversity, Antimicrobial Resistance Pattern, and Biofilm Formation in Klebsiella pneumoniae Isolated from Patients with Coronavirus Disease 2019 (COVID-19) and Ventilator-Associated Pneumonia.

INTRODUCTION: Patients with acute respiratory distress syndrome caused by coronavirus disease 2019 (COVID-19) are at risk for superadded infections, especially infections caused by multidrug resistant (MDR) pathogens. Before the COVID-19 pandemic, the prevalence of MDR infections, including infections caused by MDR Klebsiella pneumoniae (K. pneumoniae), was very high in Iran. This study is aimed at assessing the genetic diversity, antimicrobial resistance pattern, and biofilm formation in K. pneumoniae isolates obtained from patients with COVID-19 and ventilator-associated pneumonia (VAP) hospitalized in an intensive care unit (ICU) in Iran. METHODS: In this cross-sectional study, seventy K. pneumoniae isolates were obtained from seventy patients with COVID-19 hospitalized in the ICU of Shahid Beheshti hospital, Kashan, Iran, from May to September, 2020. K. pneumoniae was detected through the ureD gene. Antimicrobial susceptibility testing was done using the Kirby-Bauer disc diffusion method, and biofilm was detected using the microtiter plate assay method. Genetic diversity was also analyzed through polymerase chain reaction based on enterobacterial repetitive intergenic consensus (ERIC-PCR). The BioNumerics software (v. 8.0, Applied Maths, Belgium) was used for analyzing the data and drawing dendrogram and minimum spanning tree. Findings. K. pneumoniae isolates had varying levels of resistance to antibiotics meropenem (80.4%), cefepime-aztreonam-piperacillin/tazobactam (70%), tobramycin (61.4%), ciprofloxacin (57.7%), gentamicin (55.7%), and imipenem (50%). Around 77.14% of isolates were MDR, and 42.8% of them formed biofilm. Genetic diversity analysis revealed 28 genotypes (E1-E28) and 74.28% of isolates were grouped into ten clusters (i.e., clusters A-J). Clusters were further categorized into three major clusters, i.e., clusters E, H, and J. Antimicrobial resistance to meropenem, tobramycin, gentamicin, and ciprofloxacin in cluster J was significantly higher than cluster H, denoting significant relationship between ERIC clusters and antimicrobial resistance. However, there was no significant difference among major clusters E, H, and J respecting biofilm formation. CONCLUSION: K. pneumoniae isolates obtained from patients with COVID-19 have high antimicrobial resistance, and 44.2% of them have genetic similarity and can be clustered in three major clusters. There is a significant difference among clusters respecting antimicrobial resistance.

Algal polysaccharide's potential to combat respiratory infections caused by Klebsiella pneumoniae and Serratia marcescens biofilms.

The growth of respiratory diseases, as witnessed through the SARS and COVID-19 outbreaks, and antimicrobial-resistance together pose a serious threat to humanity. One reason for antimicrobial resistance is formation of bacterial biofilms. In this study the sulphated polysaccharides from green algae Chlamydomonas reinhardtii (Cr-SPs) is tested for its antibacterial and antibiofilm potential against Klebsiella pneumoniae and Serratia marcescens. Agar cup assay clearly indicated the antibacterial potential of Cr-SPs. Minimum inhibitory concentration (MIC50) of Cr-SPs against Klebsiella pneumoniae was found to be 850 µg/ml, and it is 800 µg/ml in Serratia marcescens. Time-kill and colony-forming ability assays suggest the concentration-dependent bactericidal potential of Cr-SPs. Cr-SPs showed 74-100% decrease in biofilm formation in a concentration-dependent manner by modifying the cell surface hydrophobic properties of these bacteria. Cr-SPs have also distorted preformed-biofilms by their ability to interact and destroy the extra polymeric substance and eDNA of the matured biofilm. Scanning electron microscopy analysis showed that Cr-SPs effectively altered the morphology of these bacterial cells and distorted the bacterial biofilms. Furthermore reduced protease, urease and prodigiosin pigment production suggest that Cr-SPs interferes the quorum sensing mechanism in these bacteria. The current study paves way towards developing Cr-SPs as a control strategy for treatment of respiratory tract infections.

Isoflavonoid-Antibiotic Thin Films Fabricated by MAPLE with Improved Resistance to Microbial Colonization.

Staphylococcus aureus (Gram-positive) and Pseudomonas aeruginosa (Gram-negative) bacteria represent major infectious threats in the hospital environment due to their wide distribution, opportunistic behavior, and increasing antibiotic resistance. This study reports on the deposition of polyvinylpyrrolidone/antibiotic/isoflavonoid thin films by the matrix-assisted pulsed laser evaporation (MAPLE) method as anti-adhesion barrier coatings, on biomedical surfaces for improved resistance to microbial colonization. The thin films were characterized by Fourier transform infrared spectroscopy, infrared microscopy, and scanning electron microscopy. In vitro biological assay tests were performed to evaluate the influence of the thin films on the development of biofilms formed by Gram-positive and Gram-negative bacterial strains. In vitro biocompatibility tests were assessed on human endothelial cells examined for up to five days of incubation, via qualitative and quantitative methods. The results of this study revealed that the laser-fabricated coatings are biocompatible and resistant to microbial colonization and biofilm formation, making them successful candidates for biomedical devices and contact surfaces that would otherwise be amenable to contact transmission.

The lipolytic activity of LipJ, a stress-induced enzyme, is regulated by its C-terminal adenylate cyclase domain.

Aim: The confirmation of lipolytic activity and role of Rv1900c in the Mycobacterium physiology Methods:rv1900c/N-terminus domain (rv1900NT) were cloned in pET28a/Escherichia coli, purified by affinity chromatography and characterized. Results: A zone of clearance on tributyrin-agar and activity with pNP-decanoate confirmed the lipolytic activity of Rv1900c. The Rv1900NT demonstrated higher enzyme specific activity, Vmax and kcat, but Rv1900c was more thermostable. The lipolytic activity of Rv1900c decreased in presence of ATP. Mycobacterium smegmatis expressed rv1900c/rv1900NT-altered colony morphology, growth, cell surface properties and survival under stress conditions. The effect was more prominent with Rv1900NT as compared with Rv1900c. Conclusion: The study confirmed the lipolytic activity of Rv1900c and suggested its regulation by the adenylate cyclase domain and role in the intracellular survival of bacteria.

Lay abstract Tuberculosis (TB) remains the top contagious/infectious killer in the world. It is caused by the bacteria Mycobacterium tuberculosis. The bacteria resides/replicates in the immune cell that normally has to eradicate infectious microorganisms. Though the treatment of TB is available, the emergence of drug-resistant bacteria is of major concern. The treatment of drug-resistant TB has been reported to be more difficult due to lengthy and complex treatment regimens. Therefore, there is an urgent need for new and better drugs to treat TB/drug-resistant TB. For this purpose understanding the role of each protein in the physiology of mycobacteria is required. Lipids play a critical role in the intracellular survival of this pathogen in the host. Our study demonstrated that LipJ supported the intracellular survival of bacteria. Therefore, it could be a potential drug target.

Persistent Bacterial Coinfection of a COVID-19 Patient Caused by a Genetically Adapted Pseudomonas aeruginosa Chronic Colonizer.

Pseudomonas aeruginosa is a biofilm-forming opportunistic pathogen which causes chronic infections in immunocompromised patients and leads to high mortality rate. It is identified as a common coinfecting pathogen in COVID-19 patients causing exacerbation of illness. In our hospital, P. aeruginosa is one of the top coinfecting bacteria identified among COVID-19 patients. We collected a strong biofilm-forming P. aeruginosa strain displaying small colony variant morphology from a severe COVID-19 patient. Genomic and transcriptomic sequencing analyses were performed with phenotypic validation to investigate its adaptation in SARS-CoV-2 infected environment. Genomic characterization predicted specific genomic islands highly associated with virulence, transcriptional regulation, and DNA restriction-modification systems. Epigenetic analysis revealed a specific N6-methyl adenine (m6A) methylating pattern including methylation of alginate, flagellar and quorum sensing associated genes. Differential gene expression analysis indicated that this isolate formed excessive biofilm by reducing flagellar formation (7.4 to 1,624.1 folds) and overproducing extracellular matrix components including CdrA (4.4 folds), alginate (5.2 to 29.1 folds) and Pel (4.8-5.5 folds). In summary, we demonstrated that P. aeuginosa clinical isolates with novel epigenetic markers could form excessive biofilm, which might enhance its antibiotic resistance and in vivo colonization in COVID-19 patients.

Clonal dispersion of Acinetobacter baumannii in an intensive care unit designed to patients COVID-19.

INTRODUCTION: SARS-CoV2 pandemic marks the need to pay attention to bacterial pathogens that can complicate the hospital stay of patients in the intensive care unit (ICU). ESKAPE bacteria which includes Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter cloacae are considered the most important, because of their close relationship with the development of ventilator-associated pneumonia (VAP). The aim of this work was to identify and characterize ESKAPE bacteria and to detect their possible clonal spread in medical devices, patients, and medical personnel of the ICU for COVID-19 patients of the Hospital Juarez de Mexico. METHODOLOGY: Genetic identification of ESKAPE bacteria was performed by analyzing the 16S rRNA gene. Resistance assays were performed according to the CLSI guidelines. Assembly of AdeABCRS operon and inhibition assays of pumps efflux in Acinetobacter baumannii isolates were performed. Associated gene involved in biofilm formation (icaA) was performed in isolates belonging to the Staphylococcus genus. Finally, typing by ERIC-PCR and characterization of mobile genetic element SCCmec were done. RESULTS: Heterogeneous distribution of ESKAPE and non-ESKAPE bacteria was detected in various medical devices, patients, and medical personnel. Acinetobacter baumannii and Staphylococcus aureus were the predominant ESKAPE members. The analysis of intergenic regions revealed an important clonal distribution of A. baumannii (AdeABCRS+). Genotyping of SCCmec mobile genetic elements and the icaA gene showed that there is no clonal distribution of S. aureus. CONCLUSIONS: Clonal spread of A. baumannii (AdeABCRS+) highlights the importance of adopting good practices for equipment disinfection, surfaces and management of COVID-19 patients.