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Xiaoxue Liang,1,* Ping Chen,2,* Baoguo Deng,2,* Feng-Hui Sun,1,* Yongqiang Yang,3– 5 Yanxian Yang,3,4 Ruowen He,3,4 Mingyang Qin,2 Yiping Wu,3,4 Fan Yang,2 Guo-Bao Tian,3,4 Min Dai1 1School of Laboratory Medicine, Chengdu Medical College, Chengdu, Sichuan Province, People’s Republic of China; 2School of Basic Medical Science, Xinxiang Medical University, Xinxiang, Henan Province, People’s Republic of China; 3Department of Microbiology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, Guangdong Province, People’s Republic of China; 4Key Laboratory of Tropical Diseases Control (Sun Yat-sen University), Ministry of Education, Guangzhou, Guangdong Province, People’s Republic of China; 5School of Pharmaceutical Sciences (Shenzhen), Sun Yat-sen University, Guangzhou, Guangdong Province, People’s Republic of China*These authors contributed equally to this workCorrespondence: Min Dai, School of Laboratory Medicine, Chengdu Medical College, Chengdu, Sichuan Province, People’s Republic of China, Tel +86 28-62739128, Email daimin1015@163.comPurpose: To compare antimicrobial resistance, virulence, clinical characteristics, and risk factors between carbapenem-resistant K. pneumoniae (CRKP) and carbapenem-susceptible K. pneumoniae (CSKP) isolates from patients with bloodstream infections (BSIs) in China.Patients and Methods: The clinical data of 103 patients with K. pneumoniae BSI from 10 hospitals were retrospectively analyzed. The minimum inhibitory concentrations of 15 antibiotics against the bacteria were determined. A Galleria mellonella infection model was used to evaluate virulence of the isolates. Kaplan–Meier curves were calculated to evaluate the 28-day and in-hospital survival rates of the isolates. The risk factors for CRKP and CSKP infection and respective mortality rate were evaluated by univariate analysis, and independent risk factors were evaluated using the multivariate logistic regression model.Results: Our results indicated that CRKP isolates were more resistant to most tested antibiotics than CSKP isolates. The G. mellonella infection model was used to demonstrate that CRKP isolates were more virulent than CSKP isolates. We found that in-hospital deaths occurred in 39.3% (22/56) of patients with CRKP BSIs and were significantly higher than those in patients with CSKP infections (19.1%, 9/47). Patients infected with CRKP isolates had poorer outcomes than those infected with the CSKP strains. For in-hospital mortality of CRKP BSIs, the independent risk factors included carbapenem-resistant Enterobacterales bacteremia and length of hospitalization after the onset of BSI.Conclusion: Our findings confirm that CRKP isolates are more drug-resistant than CSKP isolates and are associated with poorer outcomes. To prevent CRKP infection, strict infection control strategies and active surveillance should be implemented in hospitals.Keywords: Klebsiella pneumoniae, bloodstream infections, virulence, antimicrobial resistance

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.

We identified two tet (X4)-positive E. cloacae isolates, which has not been previously reported. We obtained the complete sequence of pTECL_2-190k-tetX4 and found that it was a hybrid plasmid with multiple replicon types, including IncFIA, IncHI1A and IncHI1B.

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.

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.

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 blaNDM-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 blaNDM-1-harboring plasmids, were analyzed. Results Whole-genome sequencing showed that BSI034 harbored mcr-9-carrying IncHI2-type pBSI034-MCR9 and blaNDM-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/blaNDM-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.