Correlation of CRISPR/Cas and Antimicrobial Resistance in Klebsiella pneumoniae Clinical Isolates Recovered from Patients in Egypt Compared to Global Strains.

The CRISPR/Cas system has been long known to interfere with the acquisition of foreign genetic elements and was recommended as a tool for fighting antimicrobial resistance. The current study aimed to explore the prevalence of the CRISPR/Cas system in Klebsiella pneumoniae isolates recovered from patients in Egypt in comparison to global strains and correlate the CRISPR/Cas to susceptibility to antimicrobial agents. A total of 181 clinical isolates were PCR-screened for cas and selected antimicrobial resistance genes (ARGs). In parallel, 888 complete genome sequences were retrieved from the NCBI database for in silico analysis. CRISPR/Cas was found in 46 (25.4%) isolates, comprising 18.8% type I-E and 6.6% type I-E*. Multidrug resistance (MDR) and extensive drug resistance (XDR) were found in 73.5% and 25.4% of the isolates, respectively. More than 95% of the CRISPR/Cas-bearing isolates were MDR (65.2%) or XDR (32.6%). No significant difference was found in the susceptibility to the tested antimicrobial agents among the CRISPR/Cas-positive and -negative isolates. The same finding was obtained for the majority of the screened ARGs. Among the published genomes, 23.2% carried CRISPR/Cas, with a higher share of I-E* (12.8%). They were confined to specific sequence types (STs), most commonly ST147, ST23, ST15, and ST14. More plasmids and ARGs were carried by the CRISPR/Cas-negative group than others, but their distribution in the two groups was not significantly different. The prevalence of some ARGs, such as blaKPC, blaTEM, and rmtB, was significantly higher among the genomes of the CRISPR/Cas-negative strains. A weak, nonsignificant positive correlation was found between the number of spacers and the number of resistance plasmids and ARGs. In conclusion, the correlation between CRISPR/Cas and susceptibility to antimicrobial agents or bearing resistance plasmids and ARGs was found to be nonsignificant. Plasmid-targeting spacers might not be naturally captured by CRISPR/Cas. Spacer match analysis is recommended to provide a clearer image of the exact behavior of CRISPR/Cas towards resistance plasmids.

Propranolol restores susceptibility of XDR Gram-negative pathogens to meropenem and Meropenem combination has been evaluated with either tigecycline or amikacin.

BACKGROUND: Infection with extensive-drug-resistant (XDR) carbapenem-resistant (CR) Gram-negative bacteria (GNB) are viewed as a serious threat to human health because of the limited therapeutic options. This imposes the urgent need to find agents that could be used as adjuvants or combined with carbapenems to enhance or restore the susceptibility of XDR CR- GNB. Therefore, this study aimed to examine the effect of propranolol (PR) in combination with Meropenem (MEM) on the susceptibility profile of XDR CR-GNB recovered from severely infected patients as well as to evaluate combining MEM with either tigecycline (TGC) or amikacin (AK). METHODS: A total of 59 non-duplicate CR- GNB were investigated for carbapenemase production by the major phenotypic methods. Molecular identification of five major carbapenemase-coding genes was carried out using polymerase chain reactions (PCR). Antimicrobial susceptibility tests were carried out using standard methods. Phenotypic and genotypic relatedness was carried out using the heatmap and ERIC PCR analysis. PR, 0.5 -1 mg/mL against the resulting non-clonal XDR CR-GNB pathogens were evaluated by calculating the MIC decrease factor (MDF). A combination of MEM with either AK or TGC was performed using the checkerboard assay. RESULTS: A total of 21 (35.6%) and 38 (64.4%) CR-GNB isolates were identified as enterobacterial isolates (including 16 (27.1%) Klebsiella Pneumoniae and 5 (8.5%) Escherichia coli) and non-fermentative bacilli (including, 23 (39%), Acinetobacter baumannii, and 15 (25.4%) Pseudomonas aeruginosa). The heatmap and ERIC PCR analysis resulted in non-clonal 28 XDR CR isolates. PR, at a concentration of 0.5 mg /ml, decreased MICs values of the tested XDR CR isolates (28; 100%) and restored susceptibility of only 4 (14.3%) isolates. However, PR (1 mg/mL) when combined with MEM has completely (28; 100%) restored the susceptibility of the tested XDR CR- GNB to MEM. The MEM + AK and MEM + TGC combination showed mostly additive effects (92.8% and 71.4%, respectively). CONCLUSION: PR at a concentration of 1 mg/mL restored the susceptibility of XDR CR- GNB to MEM which is considered a promising result that should be clinically investigated to reveal its suitability for clinical use in patients suffering from these life-threatening pathogens.

Streptomyces griseus KJ623766: A Natural Producer of Two Anthracycline Cytotoxic Metabolites ß- and γ-Rhodomycinone.

BACKGROUND: This study aimed to produce, purify, structurally elucidate, and explore the biological activities of metabolites produced by Streptomyces (S.) griseus isolate KJ623766, a recovered soil bacterium previously screened in our lab that showed promising cytotoxic activities against various cancer cell lines. METHODS: Production of cytotoxic metabolites from S. griseus isolate KJ623766 was carried out in a 14L laboratory fermenter under specified optimum conditions. Using a 3-(4,5-dimethylthazol-2-yl)-2,5-diphenyl tetrazolium-bromide assay, the cytotoxic activity of the ethyl acetate extract against Caco2 and Hela cancer cell lines was determined. Bioassay-guided fractionation of the ethyl acetate extract using different chromatographic techniques was used for cytotoxic metabolite purification. Chemical structures of the purified metabolites were identified using mass, 1D, and 2D NMR spectroscopic analysis. RESULTS: Bioassay-guided fractionation of the ethyl acetate extract led to the purification of two cytotoxic metabolites, R1 and R2, of reproducible amounts of 5 and 1.5 mg/L, respectively. The structures of R1 and R2 metabolites were identified as ß- and γ-rhodomycinone with CD50 of 6.3, 9.45, 64.8 and 9.11, 9.35, 67.3 µg/mL against Caco2, Hela and Vero cell lines, respectively. Values were comparable to those of the positive control doxorubicin. CONCLUSIONS: This is the first report about the production of ß- and γ-rhodomycinone, two important scaffolds for synthesis of anticancer drugs, from S. griseus.