Differential dose-response effect of cyclosporine A in regulating apoptosis and autophagy markers in MCF-7 cells.

Cyclosporine A (CsA) is an immunosuppressant primarily used at a higher dosage in transplant medicine and autoimmune diseases with a higher success rate. At lower doses, CsA exhibits immunomodulatory properties. CsA has also been reported to inhibit breast cancer cell growth by downregulating the expression of pyruvate kinase. However, differential dose-response effects of CsA in cell growth, colonization, apoptosis, and autophagy remain largely unidentified in breast cancer cells. Herein, we showed the cell growth-inhibiting effects of CsA by preventing cell colonization and enhancing DNA damage and apoptotic index at a relatively lower concentration of 2 µM in MCF-7 breast cancer cells. However, at a higher concentration of 20 µM, CsA leads to differential expression of autophagy-related genes ATG1, ATG8, and ATG9 and apoptosis-associated markers, such as Bcl-2, Bcl-XL, Bad, and Bax, indicating a dose-response effect on differential cell death mechanisms in MCF-7 cells. This was confirmed in the protein-protein interaction network of COX-2 (PTGS2), a prime target of CsA, which had close interactions with Bcl-2, p53, EGFR, and STAT3. Furthermore, we investigated the combined effect of CsA with SHP2/PI3K-AKT inhibitors showing significant MCF-7 cell growth reduction, suggesting its potential to use as an adjuvant during breast cancer therapy.

ß-Lactam Resistance in ESKAPE Pathogens Mediated Through Modifications in Penicillin-Binding Proteins: An Overview.

Bacteria acquire ß-lactam resistance through a multitude of mechanisms among which production of ß-lactamases (enzymes that hydrolyze ß-lactams) is the most common, especially in Gram-negatives. Structural changes in the high-molecular-weight, essential penicillin-binding proteins (PBPs) are widespread in Gram-positives and increasingly reported in Gram-negatives. PBP-mediated resistance is largely achieved by accumulation of mutation(s) resulting in reduced binding affinities of ß-lactams. Herein, we discuss PBP-mediated resistance among ESKAPE pathogens that cause diverse hospital- and community-acquired infections globally.

Hybrid Plasmids Encoding Antimicrobial Resistance and Virulence Traits Among Hypervirulent Klebsiella pneumoniae ST2096 in India.

Background: Hypervirulent variants of Klebsiella pneumoniae (HvKp) were typically associated with a broadly antimicrobial susceptible clone of sequence type (ST) 23 at the time of its emergence. Concerningly, HvKp is now also emerging within multidrug-resistant (MDR) clones, including ST11, ST15, and ST147. MDR-HvKp either carry both the virulence and resistance plasmids or carry a large hybrid plasmid coding for both virulence and resistance determinants. Here, we aimed to genetically characterize a collection of MDR-HvKp ST2096 isolates haboring hybrid plasmids carrying both antimicrobial resistance (AMR) and virulence genes. Methods: Nine K. pneumoniae ST2096 isolated over 1 year from the blood sample of hospitalized patients in southern India that were MDR and suspected to be HvKp were selected. All nine isolates were subjected to short-read whole-genome sequencing; a subset (n = 4) was additionally subjected to long-read sequencing to obtain complete genomes for characterization. Mucoviscosity assay was also performed for phenotypic assessment. Results: Among the nine isolates, seven were carbapenem-resistant, two of which carried blaNDM-5 on an IncFII plasmid and five carried blaOXA-232 on a ColKP3 plasmid. The organisms were confirmed as HvKp, with characteristic virulence genes (rmpA2, iutA, and iucABCD) carried on a large (~320 kbp) IncFIB-IncHI1B co-integrate. This hybrid plasmid also carried the aadA2, armA, blaOXA-1, msrE, mphE, sul1, and dfrA14 AMR genes in addition to the heavy-metal resistance genes. The hybrid plasmid showed about 60% similarity to the IncHI1B virulence plasmid of K. pneumoniae SGH10 and ~70% sequence identity with the first identified IncHI1B pNDM-MAR plasmid. Notably, the hybrid plasmid carried its type IV-A3 CRISPR-Cas system which harbored spacer regions against traL of IncF plasmids, thereby preventing their acquisition. Conclusion: The convergence of virulence and AMR is clinically concerning in K. pneumoniae. Our data highlight the role of hybrid plasmids carrying both AMR and virulence genes in K. pneumoniae ST2096, suggesting that MDR-HvKp is not confined to selected clones; we highlight the continued emergence of such genotypes across the species. The convergence is occurring globally amidst several clones and is of great concern to public health.

Emergence of Multidrug Resistant Hypervirulent ST23 Klebsiella pneumoniae: Multidrug Resistant Plasmid Acquisition Drives Evolution.

Background: In recent years, the emergence of multidrug resistant hypervirulent K. pneumoniae (MDR hvKp) isolates poses severe therapeutic challenge to global public health. The present study used the complete genome sequence of two MDR hvKp isolates belonging to ST23 to characterize the phylogenetic background and plasmid diversity. Methods: Two hvKp isolates from patients with bacteremia were sequenced using Ion Torrent PGM and Oxford Nanopore MinION platforms and assembled by hybrid genome assembly approach. Comparative genomics approaches were used to investigate the population structure, evolution, virulence, and antimicrobial resistance of MDR hvKp strains. Results: The study isolates exhibited typical features of hvKp phenotypes associated with ST23. The convergence of multidrug resistance and hypervirulence were attributed by the presence of multiple plasmids including a 216 kb virulence plasmid and MDR plasmids belonging to IncA/C2, IncFIB, IncX3, and ColKP3 groups. The insertion of catA1 gene into virulence plasmid was observed along with genetic factors such as aerobactin, salmochelin, and rmpA2 that confer hvKp's hypervirulent phenotype. The core genome single nucleotide polymorphism (SNP) phylogenetic analyses of the isolates showed the evolution of ST23 hvKp was predominantly driven by ICEKp acquisitions. Conclusion: To the best of our knowledge, this is the first report of MDR hvKp isolates of ST23 with insertion of catA1 gene into the virulence plasmid which presents the possibility of hotspot integration sites on the plasmids to aid acquisition of AMR genes. ST23 is no longer confined to susceptible strains of hvKp. Our findings emphasize the need for more studies on recombinant events, plasmid transmission dynamics and evolutionary process involving hvKp.

First hybrid complete genome of Aeromonas veronii reveals chromosome-mediated novel structural variant mcr-3.30 from a human clinical sample.

Recent findings demonstrate the origin of the plasmid-mediated colistin resistance gene mcr-3 from aeromonads. The present study aimed to screen for plasmid-mediated colistin resistance among 30 clinical multidrug-resistant (MDR) Aeromonas spp. PCR was used to screen for the presence of mcr-1, mcr-2, mcr-3 and mcr-4, which revealed mcr-3 in a colistin-susceptible isolate (FC951). All other isolates were negative for mcr. Sequencing of FC951 revealed that the mcr-3 (mcr-3.30) identified was different from previously reported variants and had 95.62 and 95.28 % nucleotide similarity with mcr-3.3 and mcr-3.10. Hybrid assembly using IonTorrent and MinION reads revealed structural genetic information for mcr-3.30 with an insertion of ISAs18 within the gene. Due to this, mcr-3.30 was non-expressive, which makes FC951 susceptible to colistin. Further, in silico sequence and protein structural analysis confirmed the new variant. To the best of our knowledge, this is the first report on a novel mcr-3 variant from India. The significant role of mcr-like genes in different Aeromonas species remains unknown and requires additional investigation to obtains insights into the mechanism of colistin resistance.

Antimicrobial resistance, virulence & plasmid profiles among clinical isolates of Shigella serogroups.

Background & objectives: Bacillary dysentery caused by Shigella spp. remains an important cause of the crisis in low-income countries. It has been observed that Shigella species have become increasingly resistant to most widely used antimicrobials. In this study, the antimicrobial resistance, virulence and plasmid profile of clinical isolates of Shigella species were determined. Methods: Sixty clinical Shigella isolates were subjected to whole-genome sequencing using Ion Torrent platform and the genome sequences were analyzed for the presence of acquired resistance genes, virulence genes and plasmids using web-based software tools. Results: Genome analysis revealed more resistance genes in Shigella flexneri than in other serogroups. Among ß-lactamases, blaOXA-1was predominantly seen followed by the blaTEM-1B and blaEC genes. For quinolone resistance, the qnr S gene was widely seen. Novel mutations in gyr B, par C and par E genes were observed. Cephalosporins resistance gene, blaCTX-M-15 was identified and plasmid-mediated AmpC ß-lactamases genes were found among the isolates. Further, a co-trimoxazole resistance gene was identified in most of the isolates studied. Virulence genes such as ipaD, ipaH, virF, senB, iha, capU, lpfA, sigA, pic, sepA, celb and gad were identified. Plasmid analysis revealed that the IncFII was the most commonly seen plasmid type in the isolates. Interpretation & conclusions: The presence of quinolone and cephalosporin resistance genes in Shigella serogroups has serious implications for the further spread of this resistance to other enteric pathogens or commensal organisms. This suggests the need for continuous surveillance to understand the epidemiology of the resistance.

Virulence gene profiles of Shigella species isolated from stool specimens in India: its association with clinical manifestation and antimicrobial resistance.

Shigella is the major cause of bacillary dysentery worldwide, especially in developing countries. There are several virulence factors essential for the organism to be virulent which are generally present in the virulence plasmid and on chromosomal pathogenicity islands. The present study was undertaken to determine the virulence gene profile of Shigella spp isolated from a clinical specimen and to study their significant association with common clinical symptoms and antimicrobial resistance. Sixty Shigella whole genome sequences, including 22 S. flexneri, 14 S. sonnei, 17 S. boydii and 7 S. dysenteriae were analyzed for the presence of virulence genes. The gene found predominantly in this study were ipaH (90%) followed by sigA (83%), and lpfA (78%) respectively. The virulence genes were significantly higher in S. flexneri, particularly in serotype 2 compared to S. sonnei. Interestingly, a significant association was observed between sigA gene and fever whereas sepA and sigA were found to be associated with diarrhea. Among the studied Shigella isolates, the presence of virulence genes was found higher in isolates resistant to more than three antibiotic classes. The present work revealed the varying incidence of virulence determinants among different Shigella serogroups and shows their contribution to disease severity.

Draft Genome Sequence of Colistin-Resistant Acinetobacter baumannii Strain VB22595 Isolated from a Central Line-Associated Bloodstream Infection.

Acinetobacter baumannii is an important emerging pathogen that causes health care-associated infections. In this study, we determined the genome of a multidrug-resistant clinical strain, VB22595, isolated from a hospital in Southern India. The draft genome indicates that strain VB22595 encodes a genome of ~3.92 Mb in size and does not contain plasmid derived MCR-1 for colistin resistance.