The rising incidence of extensively drug-resistant (XDR) Klebsiella pneumoniae, including carbapenem- and colistin-resistant strains, leads to the limitation of available effective antibiotics. Miang, known as chewing tea, is produced from Camellia sinensis var. assamica or Assam tea leaves fermentation. Previous studies revealed that the extract of Miang contains various phenolic and flavonoid compounds with numerous biological activities including antibacterial activity. However, the antibacterial activity of Miang against XDR bacteria especially colistin-resistant strains had not been investigated. In this study, the compositions of phenolic and flavonoid compounds in fresh, steamed, and fermented Assam tea leaves were examined by HPLC, and their antibacterial activities were evaluated by the determination of the MIC and MBC. Pyrogallol was detected only in the extract from Miang and showed the highest activities with an MIC of 0.25 mg/mL and an MBC of 0.25–0.5 mg/mL against methicillin-susceptible Staphylococcus aureus, methicillin-resistant S. aureus, Escherichia coli ATCC 25922, colistin-resistant E. coli, and colistin-resistant K. pneumoniae. The effects on morphology and proteomic changes in K. pneumoniae NH54 treated with Miang extract were characterized by SEM and label-free quantitative shotgun proteomics analysis. The results revealed that Miang extract caused the decrease in bacterial cell wall integrity and cell lysis. The up- and downregulated expression with approximately a 2 to >5-fold change in proteins involved in peptidoglycan synthesis and outer membrane, carbohydrate, and amino acid metabolism were identified. These findings suggested that Miang containing pyrogallol and other secondary metabolites from fermentation has potential as an alternative candidate with an antibacterial agent or natural active pharmaceutical ingredient against XDR bacteria including colistin-resistant bacteria.

The aim of this study was to identify and characterize multidrug resistance genes and the genetic contexts of integrons found in extensively drug resistant (XDR) Pseudomonas aeruginosa PA99 clinical isolate from Thailand. The sequencing of P. aeruginosa PA99 genomic DNA was done by using Pacific Biosciences RS II sequencing platform. The generated reads were de novo assembled by Canu version 1.4 and the annotation was performed using Prokka v1.12b. The complete genome sequence was subjected for identification of sequence type, serotype, integrons, and antimicrobial resistance genes by MLST 2.0, PAst 1.0, INTEGRALL, Resfinder 4.1, and CARD 3.2.5, respectively. Pseudomonas aeruginosa PA99 genome consisted of a 6,946,480-bp chromosomal DNA with 65.9% GC and belonged to ST964 and serotype O4. Twenty-one antimicrobial resistance genes conferring XDR phenotype were identified. Of special note were carbapenem resistance genes (bla_IMP-1, bla_PAO, bla_OXA-21, and bla_OXA-396) and colistin resistance gene basR with L71R mutation. Integron analysis revealed that P. aeruginosa PA99 harbored five class 1 integrons: two copies of In994 (bla_IMP-1), an In1575 (aadB), and two novel integrons, In2083 (bla_OXA-21 - aac(6')-Ib3 - aac(6')-Ib-cr - ere(A)1∆2 - dfrA1r) and In2084 (bla_IMP-1 - aac(6')-Ib3 - aac(6')-Ib-cr). To the best of our knowledge, this is the first report of two novel class I integrons designated by INTEGRALL as In2083 and In2084 found in XDR-P. aeruginosa PA99 clinical isolate from Thailand. The characterization of genetic contexts of In2083 and In2084 provide the evidence of the assorting of resistance genes to evolve as novel integrons.

This study aimed to characterize the mobile genetic context of an ∼ 82-kb chromosomal region showing co-localization of heavy-metal and antibiotic resistance island in an XDR-Enterobacter cloacae NH77. Genomic DNA was sequenced using the Pacific Biosciences RS II platform. Long reads were de novo assembled with Canu v1.4 and annotated using Prokka v1.12b. The complete genome was analyzed to identify sequence type, antimicrobial resistance genes, integrons, insertion sequences and plasmid type using MLST 2.0, ResFinder 4.7.2, CARD 4.0.1, INTEGRALL and PlasmidFinder 2.1. Comparative analyses were performed using BLAST against Enterobacteriaceae genomes available in the NCBI GenBank database. E. cloacae NH77 belonged to ST995 and was classified as XDR. The genome consisted of a 5,040,532-bp chromosome and a 41,179-bp IncN2 plasmid carrying bla_NDM-1. A 49,286-bp chromosomal resistance gene island (RGI) contained two class 1 integrons (In191 and In705) associated with 15 antimicrobial resistance genes. BLAST analysis showed the high similarity of the ∼49-kb RGI to sequences found in the chromosomes of E. hormaechei and E. asburiae and an E. coli plasmid. The ∼49-kb RGI was located immediately downstream of a Tn7-sil-pco cluster, forming an ∼82-kb composite mobile resistance island. All sequences contained pcoSE locating at the end of the ∼49-kb RGI. This study describes an XDR-E. cloacae ST995 clinical isolate containing an ∼82-kb chromosomal resistance island comprising Tn7-sil-pco and a ∼49-kb RGI. The mobile genetic context suggests that pcoSE may function as a recombination target site for the ∼49-kb RGI.