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.

ANTIBACTERIAL EFFECT OF NANNOCHLOROPSIS OCULATA AS ROOT CANAL STERILIZATION MATERIAL ON STREPTOCOCCUS MUTANS BIOFILM

ABSTRACTBackground: Streptococcus mutans in the most frequent microbiota that causes pulp necrosis because of caries. The microorganism that is colonized and embedded in the biofilm matrix is resistant to antimicrobials compared to planktonic cells. Root canal sterilization materials must have good biocompatibility with tissues. Nannochloropsis oculata is an algae that contains various compounds such as terpenoids, alkaloids, and flavonoids that have potential as antibacterial and antioxidant and can be used as alternative to root canal sterilization. Method: This research was true experimental laboratory research with post-test only control group design. The antibacterial potential of Nannochloropsis oculata was tested using the biofilm method, divided into 5 groups. The control group was: K- (aquadest), K+ (calcium hydroxide), and the treatment group was given Nannochloropsis oculata: P1 (0.625%), P2 (1.25%), and P3 (2.5 %). Congo Red method test was to determine the formation of biofilm that shows black strains on agar. While biofilm test with Microtiter Plate Assay to measure the value of biofilm that were inhibited in Optical Density (OD) value in the ELISA Reader. The lower the value, the more biofilm inhibited, with OD value, inhibition percentage could counted Result: The result of all treatment groups were increasing in percentage inhibition value shows inhibition in biofilm growth (p <0.05). Conclusion: Nannochloropsis oculata had an antibacterial effect on the biofilm of Streptococcus mutans

GC-MS Analysis and Antimicrobial Assessment of Syzygium cumini (L.) Skeels Seed Ethanol Extract

People use plants to treat infections, and this has led to search of antimicrobials from medicinal plants. In this work, we evaluated the ethanol extract of Syzygium cumini seeds for their antibacterial and antifungal activities. Extraction was performed by maceration method using ethanol. The antimicrobial efficacy of the extract was assessed by agar well diffusion method against ten bacterial species, Bacillus cereus, Bacillus subtilis, Enterococcus faecalis, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Salmonella typhi, Serratia marcescens, Staphylococcus aureus and Streptococcus mutans, and five fungal species, Aspergillus flavus, Aspergillus fumigatus, Aspergillus niger, Candida albicans and Mucor sp. Minimum inhibitory concentrations (MICs) of the extract were determined by resazurin microtiter plate assay. Phytochemicals in the extract was identified by gas chromatography mass spectrometry (GC-MS) information. In agar well diffusion method, Gram-negative bacteria such as P. aeruginosa and S. marcescens, Gram-positive bacteria such as B. subtilis and E. faecalis and fungi A. fumigatus were more susceptible showing larger zones of inhibition. In resazurin method, low MICs were recorded for bacteria, B. cereus (<7.8 µg) and P. aeruginosa (15.6 µg) and fungi, A. fumigatus (31.2 µg). Fifteen compounds were identified by GC-MS profiling of the extract. The antimicrobial activity of the extract can be rightly related to the secondary metabolites in the ethanol extract of Syzygium cumini seeds.

Detection of CpG Methylation in G-Quadruplex Forming Sequences Using G-Quadruplex Ligands

Genomic DNA methylation is involved in many diseases and is expected to be a specific biomarker for even the pre-symptomatic diagnosis of many diseases. Thus, a rapid and inexpensive detection method is required for disease diagnosis. We have previously reported that cytosine methylation in G-quadruplex (G4)-forming oligonucleotides develops different G4 topologies. In this study, we developed a method for detecting CpG methylation in G4-forming oligonucleotides based on the structural differences between methylated and unmethylated G4 DNAs. The differences in G4 topologies due to CpG methylation can be discriminated by G4 ligands. We performed a binding assay between methylated or unmethylated G4 DNAs and G4 ligands. The binding abilities of fluorescent G4 ligands to BCL-2, HRAS1, HRAS2, VEGF G4-forming sequences were examined by fluorescence-based microtiter plate assay. The differences in fluorescence intensities between methylated and unmethylated G4 DNAs were statistically significant. In addition to fluorescence detection, the binding of G4 ligand to DNA was detected by chemiluminescence. A significant difference was also detected in chemiluminescence intensity between methylated and unmethylated DNA. This is the first study on the detection of CpG methylation in G4 structures, focusing on structural changes using G4 ligands.

Evaluating Five Escherichia coli Derivative Strains as a Platform for Arginine Deiminase Overproduction

Aims: This study attempted to evaluate the five host strains, including BL21 (DE3), Rosetta (DE3), DH5α, XL1-BLUE, and SHuffle, in terms of arginine deiminase (ADI) production and enzyme activity. Background: Escherichia coli is one of the most preferred host microorganisms for the production of recombinant proteins due to its well-characterized genome, availability of various expression vectors, and host strains. Choosing a proper host strain for the overproduction of a desired recombinant protein is very important because of the diversity of genetically modified expression strains. Various E. coli cells have been examined in different patent applications. Method: ADI was chosen as a bacterial enzyme that degrades L-arginine. It is effective in the treatment of some types of human cancers like melanoma and hepatocellular carcinoma (HCC), which are arginine-auxotrophic. Five mentioned E. coli strains were cultivated. The pET-3a was used as the expression vector. The competent E. coli cells were obtained through the CaCl2 method. It was then transformed with the construct of pET3a-ADI using the heat shock strategy. The ADI production levels were examined by 10% SDS-PAGE analysis. The ability of host strains for the expression of the requested recombinant protein was compared. The enzymatic activity of the obtained recombinant ADI from each studied strain was assessed by a colorimetric 96-well microtiter plate assay. Result: All the five strains exhibited a significant band at 46 kDa. BL21 (DE3) produced the highest amount of ADI protein, followed by Rosetta (DE3). The following activity assay showed that ADI from BL21 (DE3) and Rosetta (DE3) had the most activity. Conclusion: There are some genetic and metabolic differences among the various E. coli strains, leading to differences in the amount of recombinant protein production. The results of this study can be used for the efficacy evaluation of the five studied strains for the production of similar pharmaceutical enzymes. The strains also could be analyzed in terms of proteomics.

Mucosa-Associated Escherichia coli in Colorectal Cancer Patients and Control Subjects: Variations in the Prevalence and Attributing Features

Accumulating evidence indicates that specific strains of mucosa-associated Escherichia coli (E. coli) can influence the development of colorectal carcinoma. This study aimed to investigate the prevalence and characterization of mucosa-associated E. coli obtained from the colorectal cancer (CRC) patients and control group. At two referral university-affiliated hospitals in northwest Iran, 100 patients, 50 with CRC and 50 without, were studied over the course of a year. Fresh biopsy specimens were used to identify mucosa-associated E. coli isolates after dithiothreitol mucolysis. To classify the E. coli strains, ten colonies per sample were typed using enterobacterial repetitive intergenic consensus-based PCR (ERIC-PCR). The strains were classified into phylogroups using the quadruplex PCR method. The PCR method was used to examine for the presence of cyclomodulin, bfp, stx1, stx2, and eae-encoding genes. The strains were tested for biofilm formation using the microtiter plate assay. CRC patients had more mucosa-associated E. coli than the control group ( p < 0.05 ). Enteropathogenic Escherichia coli (EPEC) was also found in 23% of CRC strains and 7.1% of control strains ( p < 0.05 ). Phylogroup A was predominant in control group specimens, while E. coli isolates from CRC patients belonged most frequently to phylogroups D and B2. Furthermore, the frequency of cyclomodulin-encoding genes in the CRC patients was significantly higher than the control group. Around 36.9% of E. coli strains from CRC samples were able to form biofilms, compared to 16.6% E. coli strains from the control group ( p < 0.05 ). Noticeably, cyclomodulin-positive strains were more likely to form biofilm in comparison to cyclomodulin-negative strains ( p < 0.05 ). In conclusion, mucosa-associated E. coli especially cyclomodulin-positive isolates from B2 and D phylogroups possessing biofilm-producing capacity colonize the gut mucosa of CRC patients.

1318. Clinical and Molecular Characteristics of Hypermucoviscous Klebsiella pneumoniae Causing Pneumonia in Korea

Background Invasive Klebsiella pneumoniae (K. pneumoniae) was emerged in Asia, well-known for community-onset liver abscess. Healthcare-associated pneumonia caused by hypervirulent K. pneumoniae has been reported in recent studies. The purpose of this study was to evaluate the clinical and molecular characteristics of hypervirulent K. pneumoniae compared with classic K. pneumoniae in respiratory infection. Methods The study was performed on 163 K. pneumoniae isolates of respiratory infections collected from Keimyung University of Dongsan Medical Center from November 2013 to November 2015; group A, as classic K. pneumoniae and group B, as hypervirulent K. pneumoniae. Hypermucoviscous phenotype was confirmed with string test. Capsular serotypes, rmpA, magA, allS, mrkD, entB, kfu, and iutA were identified using specific primers by polymerase chain reaction. The biofilm mass was determined using the microtiter plate assay measured by optical density (OD, 570nm). Results A total 163 patients were analyzed, 100 (61.3%) of group A and 68 (38.7%) of group B. Community-acquired pneumonia was observed in 49.2% of group B and 18.0% of group A (p=0.001). Underlying diseases except chronic lung disease were more associated with group A. Mean age (72.6±11.7 vs. 68.8±12.5 years, p=0.051) and antimicrobial resistant rates were higher in group A. Mechanical ventilators (21.0% vs. 36.5%, p=0.030) was more associated with group B. Concordances of initial antibiotics (57.5% vs. 92.1%, p=0.001) were more observed in group B. Biofilm formation and infection related 30-day mortality showed no differences between the two groups. Conclusion Contrary to our expectations, hypervirulent K. pneumoniae was more associated with community-acquired pneumonia in this study. Compared to classic K. pneumoniae, hypervirulent K. pneumoniae showed more association with severe pneumonia and less association with underlying diseases. In respiratory infection, biofilm formation was not different according to hypermucoviscousity. Disclosures All Authors: No reported disclosures

Synergistic Antibiofilm Effect of Thymol and Piperine in Combination with Aminoglycosides Antibiotics against Four Salmonella enterica Serovars

Biofilms related to human infection have high levels of pathogenicity due to their resistance to antimicrobial agents. The discovery of antibiofilm agents is necessary. One approach to overcome this problem is the use of antibiotics agents’ combination. This study aimed to determine the efficacy of the combination of natural products thymol and piperine with three aminoglycosides antibiotics, amikacin, kanamycin, and streptomycin against biofilm-forming Salmonella enterica. The microtiter plate assay method was used to evaluate the biofilm-producing capacity of the isolates. Minimum inhibitory concentration (MIC) and minimum bactericidal concentration were determined by the broth microdilution method. The inhibition of biofilm formation and biofilm eradication was determined using the microtiter broth method. The checkerboard method was used to determine the combined effects of natural products with aminoglycosides antibiotics. All the tested isolates showed various levels of biofilm formation. Overall, combinations provided 43.3% of synergy in preventing the biofilm formation and 40% of synergy in eradicating preformed biofilms, and in both cases, no antagonism was observed. The combination of thymol with kanamycin showed a synergistic effect with 16- to 32-fold decrease of the minimum biofilm eradication concentration (MBEC) of kanamycin. The interaction of piperine with amikacin and streptomycin also revealed a synergistic effect with 16-fold reduction of the minimum biofilm inhibitory concentration (MBIC). The combination of thymol with the three antibiotics showed a strong synergistic effect in both inhibiting the biofilm formation and eradicating the preformed biofilm. This study demonstrates that thymol and piperine potentiate the antibiofilm activity of amikacin, kanamycin, and streptomycin. These combinations are a promising approach therapeutic to overcome the problem of Salmonella enterica biofilm-associated infections. In addition, these combinations could help reduce the concentration of individual components, thereby minimizing the nephrotoxicity of aminoglycosides antibiotics.

BSA Adsorption and Antibiofilm Properties of (N-Cyclohexylacrylamide-co-Acrylamide/AMPSNa) Hydrogels

The hydrogels poly (N-cyclohexylacrylamide-co-acrylamide/2-acrylamido-2-methyl-1-propanesodiumsulfonate) i.e.poly(NCA-co-AM/AMPSNa) (HG41,HG42,HG43&HG44) were synthesised via free-radical copolymerization of NCA and (AM) in a fixed proportion (50:50), but varying the ionic monomer-AMPSNa (0.1g,0.3g,0.5 d 0.7g) in a medium of mixture of water and methanol at 60°C in an oil bath, Potassium persulfate (KPS) was used as an initiator, while N,N'-methylene-bisacrylamide (MBA) was used as a cross-linker. FT-IR spectral data, SEM, XRD, and TGA techniques were used to characterise the synthesised hydrogel (HG43). On changing pH(3.0, 5.0, 7.0 & 9.0), the amount of Bovine Serum Albumin (BSA) adsorption efficiency by these hydrogels was evaluated. At pH 5.0, which is near to BSA's isoelectric point (4.7)., the maximum adsorption was found. BSA adsorption increased as the amount of AMPSNa increased. SEM and XRD were used to examine the BSA-adsorbed hydrogel (HG43). The antibiofilm abilities of the hydrogel (HG43) by Microtiter plate Assay(MTP), Fluorescence microscopy and SEMl against Staphylococcus aureus and Pseudomonas aureginosa displayed outstanding efficacy.

Biocide's susceptibility and frequency of biocide resistance genes and the effect of exposure to sub-inhibitory concentrations of sodium hypochlorite on antibiotic susceptibility of Stenotrophomonas maltophilia

Background: The overused of biocides in healthcare-facilities poses risk for emergence and spread of antibiotic resistance among nosocomial pathogens. Hospital-acquired infections due to S. maltophilia particularly in the immunocompromised patients have been increased. The objective of this study was to evaluate the susceptibility of S. maltophilia clinical isolates to commonly used biocides in hospitals, as well the frequency of biocides resistance gene among them. This study also intended to assess the effect of exposure of S. maltophilia isolates to sub-inhibitory concentrations of sodium hypochlorite upon the antimicrobial susceptibility patterns. Methods: This study included 97 S. maltophilia isolates. Biofilm formation was determined by microtiter plate assay. The susceptibility tests of five biocides were studied against all S. maltophilia isolates by microbroth dilution method. Susceptibility of isolates to antibiotics by disk diffusion method were compared before and after exposure to sub-inhibitory concentrations of sodium hypochlorite. Presence of qacE, qacEΔ1, SugE genes was screened by PCR. Results: Based on minimum inhibitory and bactericidal concentrations of biocides sodium hypochlorite 5% and ethyl alcohol 70% were the strongest and weakest against S. maltophilia isolates, respectively. The frequency of sugE gene resistance genes was found to be high (90.7%) in our clinical S. maltophilia isolates. None of the isolates carried qacE and qacEΔ1 gene. Exposure to sub-inhibitory concentration of sodium hypochlorite showed significantly change the susceptibility of isolates towards ceftazidime (P = .019), ticarcillin/clavulanate (P = .009). and chloramphenicol (P = .028). Conclusions: This study demonstrated that exposure to sub-inhibitory concentration of sodium hypochlorite leads to reduced antibiotic susceptibility and development of multidrug-resistant S. maltophilia strains.