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Bloodstream infections (BSIs) caused by carbapenem-resistant Klebsiella pneumoniae (CRKP) are potentially life-threatening and an urgent threat to public health. The present study aims to clarify the characteristics of carbapenemase-encoding and virulent plasmids, and their interactions with the host bacterium. A total of 425 Kp isolates were collected from the blood of BSI patients from nine Chinese hospitals, between 2005 and 2019. Integrated epidemiological and genomic data showed that ST11 and ST307 Kp isolates were associated with nosocomial outbreak and transmission. Comparative analysis of 147 Kp genomes and 39 completely assembled chromosomes revealed extensive interruption of acrR by ISKpn26 in all Kp carbapenemase-2 (KPC-2)-producing ST11 Kp isolates, leading to activation of the AcrAB-Tolc multidrug efflux pump and a subsequent reduction in susceptibility to the last-resort antibiotic tigecycline and six other antibiotics. We described 29 KPC-2 plasmids showing diverse structures, two virulence plasmids in two KPC-2-producing Kp, and two novel multidrug-resistant (MDR)-virulent plasmids. This study revealed a multifactorial impact of KPC-2 plasmid on Kp, which may be associated with nosocomial dissemination of MDR isolates.

Resistance-nodulation-division (RND) efflux pumps are important determinants of multidrug resistance in Gram-negative bacteria. As one of the typical members of the RND superfamily, Escherichia coli OqxAB multidrug efflux pump confers resistance to antimicrobial agents, such as olaquindox and fluoroquinolone. In the present study, site-directed mutagenesis and antimicrobial susceptibility measurement assay were applied to identify the crucial residues within OqxB, the transporter component of the OqxAB efflux pump system. It was found that alanine substitution of proton translocation pathway residues D410, D411, and R976 resulted in a complete loss of the transport function. Further studies revealed that the charge property of these residues is important for proper function of OqxB. Alanine replacement of residues involved in substrate-binding domains, including V141, F180, Y330, and F626, exhibited different responses toward different antimicrobial agents. Conservative replacement of Y330, F626, and F180 with amino acids having similar aromatic ring structure resulted in full or partial recovery of the efflux function. Molecular docking analysis demonstrated that olaquindox may form hydrogen bonds with F626, Y330, and V141, whereas only Y330 and F180 may interact with ciprofloxacin, implicating the different roles played by these residues when transporting different kinds of substrates. Graphical Abstract [Figure: see text].

Background Bloodstream infection (BSI) caused by carbapenem-resistant Enterobacteriaceae are potentially life-threatening related to poorer outcomes. Colistin is considered one of the last-resort treatments against human infections caused by multidrug-resistant (MDR) Gram-negative bacteria. Therefore, emergence of strains from the blood that co-harboring mcr and carbapenem resistance genes were considered as a serious problem. Purpose In this study, two mcr-9-harboring MDR Enterobacter cloacae isolates BSI034 and BSI072 recovered from BSI patients were identified, one of which co-harbored mcr-9 and bla NDM-1. The genetic characteristics of the MDR plasmid needed to be clarified. Methods S1-PFGE and Southern blotting were conducted to determine the location of mcr-9. Whole-genome sequencing was performed to obtain the complete genome and plasmid sequences. The resistome and virulence genes of the strains, accompanied by the genetic characteristics of mcr-9- and bla NDM-1-harboring plasmids, were analyzed. Results Whole-genome sequencing showed that BSI034 harbored mcr-9-carrying IncHI2-type pBSI034-MCR9 and bla NDM-1-carrying IncX3-type pBSI034-NDM1. The 278,517 bp pBSI034-MCR9 carried mcr-9 along with the other 19 resistance genes. mcr-9 was flanked by IS903B (1057 bp) and IS26 (820 bp) in the same orientation. In addition to resistance genes, strain BSI034 also carried a chromosome-located Yersinia high-pathogenicity island, which harbored genes of yersiniabactin biosynthesis operon ybtSXQPAUTE, irp1/2, and fyuA. Conclusion We described the complete genome and mcr-9/bla NDM-1-co-harboring plasmid of E. cloacae from a BSI patient. Notable differences were observed within mosaic modules between pBSI034-MCR9 and other mcr-9-harboring plasmids due to extensive recombination via horizontal gene transfer.

Mycobacterium grossiae sp. nov. of type strain PB739T is a Gram-positive acid–alcohol–fast rod-shaped bacterium, which was recently isolated from a 76-year-old male who suffered from a 1-year history of hemoptysis. This strain was described as novel species in Mycobacterium genus. In this study, its genome was completely sequenced by PacBio technology, analyzed, and compared with other selected complete genome sequences of Mycobacterium to elucidate the distinct pathogenic features of the strain. The genomic analysis revealed that the genome of PB739T consists of one circular DNA chromosome of 5,637,923 bp with a GC content of 70.48% and one plasmid of 43,679 bp with a GC content of 66.24%. The entire genome contains 5434 predicted coding genes, 48 tRNAs, and 6 rRNA genes. Genome and comparative genomics against M. grossiae SCH identified three tandem short-chain dehydrogenase (SDR) genes which only exist in PB739T. These three tandem SDR genes locate in a Genomic island which was identified by Island Viewer. These SDR genes were predicted to be horizontally transferred from a Streptomyces ancestor based on phylogeny. Analysis of the mutant ΔSDR confirmed the relationship between these tandem genes with biofilm and pathogenicity. This report will provide us with an extended understanding of M. grossiae at the genomic level and would be helpful for understanding the evolution of Mycobacterium genus.

Purpose: The discovery of the plasmid-mediated colistin resistance genes, mcr, revealed a mechanism of transmission of colistin resistance, which is a major, global public health concern especially among individuals infected with carbapenem-resistant Gram-negative bacteria. To monitor the spread and epidemiology of mcr genes, a convenient and reliable method to detect mcr genes in clinical isolates is needed, especially in the primary care institutions. This study aimed to establish a restriction endonuclease-based multiplex loop-mediated isothermal amplification (multi-LAMP) assay to detect mcr genes (mcr-1 to mcr-5) harbored by colistin-resistant bacteria. Methods: A triple-LAMP assay for mcr-1, mcr-3, and mcr-4 and a double-LAMP assay for mcr-2 and mcr-5 were established. The sensitivity and specificity of the LAMP reactions were determined via electrophoresis and visual detection. Results: The sensitivity of the LAMP assay was 10-fold greater than that of PCR, with high specificity among the screened primers. Specific mcr genes were distinguished in accordance with band numbers and the fragment length of the digested LAMP amplification products. Furthermore, the LAMP assay was confirmed as a rapid and reliable diagnostic technique upon application for clinical samples, and the results were consistent with those of conventional PCR assay. Conclusion: The multi-LAMP assay is a potentially promising method to detect mcr genes and will, if implemented, help prevent infections by drug-resistant bacteria in primary-care hospitals due to rapid and reliable surveillance. To our knowledge, this is the first study to report the application of LAMP to detect mcr-2 to mcr-5 genes and the first time that multi-LAMP has been applied to detect mcr genes.

The emergence of carbapenem-resistant Klebsiella pneumoniae (CRKP) isolates in Egyptian hospitals has been reported. However, the genetic basis and analysis of the plasmids associated with carbapenem-resistant hypervirulent K. pneumoniae (CR-HvKP) in Egypt have not been presented. Therefore, we attempted to decipher the plasmid sequences that are responsible for transferring the determinants of carbapenem resistance, particularly blaNDM-1 and blaKPC-2. Out of 34 K. pneumoniae isolates collected from two tertiary hospitals in Egypt, 31 were CRKP. Whole-genome sequencing revealed that our isolates were related to 13 different sequence types (STs). The most prevalent ST was ST101, followed by ST383 and ST11. Among the CRKP isolates, one isolate named EBSI036 has been reassessed by Nanopore sequencing. Genetic environment analysis showed that EBSI036 carried 20 antibiotic resistance genes and was identified as a CR-HvKP strain: it harbored four plasmids, namely, pEBSI036-1-NDM-VIR, pEBSI036-2-KPC, pEBSI036-3, and pEBSI036-4. The two carbapenemase genes blaNDM-1 and blaKPC-2 were located on plasmids pEBSI036-1-NDM-VIR and pEBSI036-2-KPC, respectively. The IncFIB:IncHI1B hybrid plasmid pEBSI036-1-NDM-VIR also carried some virulence factors, including the regulator of the mucoid phenotype (rmpA), the regulator of mucoid phenotype 2 (rmpA2), and aerobactin (iucABCD and iutA). Thus, we set out in this study to analyze in depth the genetic basis of the pEBSI036-1-NDM-VIR and pEBSI036-2-KPC plasmids. We report a high-risk clone ST11 KL47 serotype of a CR-HvKP strain isolated from the blood of a 60-year-old hospitalized female patient from the intensive care unit (ICU) in a tertiary care hospital in Egypt, which showed the cohabitation of a novel hybrid plasmid coharboring the blaNDM-1 and virulence genes and a blaKPC-2-carrying plasmid. IMPORTANCE CRKP has been registered in the critical priority tier by the World Health Organization and has become a significant menace to public health. The emergence of CR-HvKP is of great concern in terms of both disease and treatment. In-depth analysis of the carbapenemase-encoding and virulence plasmids may provide insight into ongoing recombination and evolution of virulence and multidrug resistance in K. pneumoniae. Thus, this study serves to alert contagious disease clinicians to the presence of hypervirulence in CRKP isolates in Egyptian hospitals.

The emergence of carbapenem-resistant Klebsiella pneumoniae (CRKP) isolates in Egyptian hospitals has been reported. However, the genetic basis and the analysis of the plasmids associated with CR-hypervirulent-KP (CR-HvKP) in Egypt are not presented. Therefore, we attempt to decipher the plasmids sequences, which are responsible for transferring the determinants of carbapenem-resistance, particularly the blaNDM-1 and blaKPC-2. Out of 34 K. pneumoniae isolates collected from two tertiary hospitals in Egypt, 31 were CRKP. Whole-genome sequencing revealed that our isolates were related to 13 different sequence types (STs). The most prevalent ST was ST101, followed by ST383, and ST11. Among the CRKP isolates, one isolate named EBSI036 has been reassessed using Nanopore sequencing. Genetic environment analysis showed that EBSI036 carried 20 antibiotic resistance genes and was identified as CR-HvKP strain, it harboured four plasmids, namely; pEBSI036-1-NDM-VIR, pEBSI036-2-KPC, pEBSI036-3, and pEBSI036-4. The two carbapenemase genes, blaNDM-1 and blaKPC-2, were located on plasmids pEBSI036-1-NDM-VIR and pEBSI036-2-KPC, respectively. The IncFIB:IncHI1B hybrid plasmid pEBSI036-1-NDM-VIR also carried some virulence factors, including regulator of the mucoid phenotype (rmpA), the regulator of mucoid phenotype 2 (rmpA2), and aerobactin (iucABCD, iutA). Thus, we set out this study to analyse in-depth the genetic basis of pEBSI036-1-NDM-VIR and pEBSI036-2-KPC plasmids. We reported for the first time a high-risk clone ST11 KL47 serotype of CR-HvKP strain isolated from the blood of a 60-year-old hospitalised female patient from the ICU in a tertiary-care hospital in Egypt, which showed the cohabitation of a novel hybrid plasmid coharbouring the blaNDM-1 and virulence genes, besides a blaKPC-2-carrying plasmid.IMPORTANCECRKP had been registered in the critical priority tier by the World Health Organization and became a significant menace to public health. Therefore, we set out this study to analyse in-depth the genetic basis of pEBSI036-1-NDM-VIR and pEBSI036-2-KPC plasmids. Herein, we reported for the first time (to the best of our knowledge) a high-risk clone ST11 KL47 serotype of CR-HvKP strain isolated from the blood of a 60-year-old hospitalised female patient in a tertiary-care hospital from the ICU in Egypt, which showed the cohabitation of a novel hybrid plasmid co-harbouring the blaNDM-1 and virulence genes, besides a blaKPC-2-carrying plasmid. Herein, the high rate of CRKP might be due to the continuous usage of carbapenems as empirical therapy, besides the failure to implement an antibiotic stewardship program in Egyptian hospitals. Thus, this study serves to alert the contagious disease clinicians to the presence of hypervirulence in CRKP isolates in Egyptian hospitals.

Two multidrug-resistant (MDR) mcr-1 -harboring Klebsiella pneumoniae isolates from patients with urinary tract infections and one MDR Klebsiella quasipneumoniae isolate from a patient with bloodstream infection were identified to carry tmexCD1-toprJ1 . The addition of the efflux pump inhibitor reduced the tigecycline MIC against all three isolates by 8- to 16-fold. pKQBSI104-1 was transferred from K. quasipneumoniae to Escherichia coli J53 via conjugation.