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Background: The aim of this study was to investigate the characteristics of carbapenem-resistant Klebsiella pneumoniae (CRKP) ST76 isolates collected during an outbreak in a hospital's intensive ca...

Background Carbapenem-resistant hypervirulent Klebsiella pneumoniae strains have recently come into existence worldwide; however, researchers in northeast China are not aware of their clinical features and molecular characteristics. Methods Here, the molecular and virulent characteristics of 44 carbapenem-resistant K. pneumoniae (CRKP) isolates collected from January 2015 to December 2017 were studied. Multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE) were carried out to define the clonal relatedness among the isolates. PCR and capsular serotyping of the virulence-associated genes, as well as biofilm formation and serum complement-mediated killing assays, were employed to determine the virulent potential. The genomic features and associated mobile genetic elements of JmsCRE57 were detected by whole genome sequencing. Results The only positive isolate was JmsCRE57, which belonged to the ST375 serotype K2 that expressed uge, mrkD, fimH, kpn, aerobactin and rmpA virulence-associated genes and showed strong biofilm formation and serum sensitivity. Sequencing results showed that the JmsCRE57 genome mainly consisted of a circular chromosome, three antibiotic resistance plasmids and a virulent plasmid. The antibiotic resistance plasmid expressing blaKPC-2, blaCTX-M-15, aph(3'')-Ib, aph(6)-Id, qnrB1, aac(3)-IIa, aac(6')-Ib-cr, blaOXA-1, blaTEM-1B, catB4, sul2, dfrA14 and blaSHV-99. The virulent plasmid belonged to the IncHI1B group, which is mainly composed of capsular polysaccharide genes and siderophore-associated genes. The remaining CRKP strains that expressed uge, fimH, mrkD and kpn virulence-associated genes were not successfully typed. We also identified an isolate that expressed the alls gene. Conclusion Our results provide new insights on the epidemiology of carbapenem-resistant K2 hypervirulent K. pneumoniae ST375 and CRKP ST76 strains in northeast China, which may help control their future outbreaks.

This study aimed to investigate the efficacy of colistin both alone and in combination with either meropenem or levofloxacin against CRAB clinical isolates. The OXA carbapenemase genes and multiloc...

Background Enterobacter cloacae complex (ECC) is one of the most common extended-spectrum β-lactamase and carbapenemase-producing pathogen that threatens millions of the elderly and vulnerable sick persons. The objective of this study was to perform the molecular characteristics of the carbapenem-resistant E. cloacae complex (CREC) emerged in Heilongjiang Province of China. Methods Six CREC strains were isolated from the patients with infectious diseases. The identities of ECC isolates were confirmed by sequencing the polymerase chain reaction (PCR) products of 16S rRNA gene. The characterization of the CREC isolates were analyzed by sequencing PCR products of the carbapenemase, ampC and fluoroquinolone resistance genes and performing multilocus sequence typing (MLST), pulsed-field gel electrophoresis (PFGE) and whole genome sequencing. Results All 6 isolates harbored multiple resistance genes. Of them, 5 carried metallo-β-lactamases and one was blaKPC-2-positive. The levofloxacin and ciprofloxacin-resistant strains had substitutions of gyrA83, gyrA87, and parC80 in the quinolone-resistance determining regions. The MLST analyses revealed that 6 isolates belonged to five sequence types (ST520, ST528, ST1119, ST1120, and ST93) while the PFGE patterns of the isolates fallen into four clusters. The strain ST1120 was found to carry two separated plasmids that encode blaNDM-1 and blaIMP-4. Conclusions Our study, for the first time, identified a CREC strain that co-produces blaNDM-1 and blaIMP-4 in the Northeast China. Our finding emphasizes an urgent need for more intensive surveillance and precaution measures to prevent the CERC spread.

Background Enterobacter cloacae is an emerging opportunistic pathogen. We retrospectively conducted a study to assess antimicrobial susceptibility and investigated the Molecular characteristics of carbapenem-resistant Enterobacter cloacae (CREL) isolates. Methods Three hundred forty-two isolates of Enterobacter cloacae were collected from January 2014 to December 2018. Ten strains of CREL were collected for further research. The species identifications and minimum inhibitory concentrations (MICs) of all antibiotics tested were analyzed using the Vitek 2 Compact system (BioMerieux, France) and supplemented by the disk diffusion method. Polymerase chain reaction (PCR) was performed to detect extended-spectrum β-lactamase (ESBL) and carbapenemase resistance genes. Results The results showed that most of the isolates remained susceptible to tested antibiotics; however, the resistance rate of Cefepime has been increasing in recent years. One strain co-producing New Delhi Metallo-β-lactamase NDM-1 and Imipenem hydrolase IMP-4. NDM-1 and IMP-4-producing isolates highlight that active surveillance is necessary to prevent the further spread of the bacteria. Multilocus sequence typing (MLST) showed that two KPC-producing isolates assigned to ST93, two isolates carrying NDM-1 assigned to ST1120. Moreover, the MEGA analysis showed that ST93, ST256, and ST1120 have homology, showing that CREL in our area has a potential spread risk. Conclusions These findings indicating that CREL clonal dissemination may occurred in this region and should be taken seriously concern. Our study highlights an urgent need to monitor these isolates to prevent their further spread.

Background There is increasing resistance to carbapenems among Klebsiella pneumoniae,and fluoroquinolones (FQ) are increasingly used to treat infections from extended-spectrum β- lactamase(ESBLs) and carbapenemase-producing Klebsiella pneumoniae. However, the acquisition of plasmid-mediated quinolone resistance (PMQR) or the spontaneous mutation of the quinolone resistance-determining regions (QRDR) of the gyrA and parC genes can severely affect the therapeutic effect of quinolones. The goal of this study was to investigate the molecular determinants of FQ resistance(FQ-R) in carbapenem-resistant Klebsiella pneumoniae (CRKP) isolates from Heilongjiang Province,China. Materials and methods We isolated 40 strains of CRKP from a treatment center in the eastern part of Heilongjiang Province from January 2016 to December 2018. The VITEK2 Compact analyzer was used to identify and detect drug sensitivity. Different types of drug resistance genes were detected by polymerase chain reaction (PCR). PCR and DNA sequencing were used to assess the presence of qnrA, qnrB, qnrS,qepA and acc(6′) Ib-cr genes,which are plasmid-encode genes that can contribute to resistance. The sequences of gyrA and parC genes were sequenced and compared with the sequences of standard strains to determine if mutations were present.Multi-site sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE) were performed on the strains to assess homology. Results The isolated CRKP strains showed rates of resistance to fluoroquinolones of 22.5% to 42.5%. The resistance rate of ciprofloxacin was significantly higher than that of levofloxacin.Nine CRKP strains (22.5%) showed co-resistance to ciprofloxacin and levofloxacin.The quinolone resistant strains were screened for plasmid-encoded genes that can contribute to resistance (PMQR genes).Among the 17 quinolone resistant strains,one strain contained no PMQR genes,twelve strains contained two PMQR genes,and four strains contained four PMQR genes.Acc (6′) Ib-cr was the most frequently detected PMQR gene, detected in 95% of strains tested (38 of 40) and in 94.1% of the quinolone-resistant strains (16 of 17). The qepA gene encoding an efflux pump was not detected in any strains.No isolate carried five different PMQRs simultaneously.Changes of S83I and D87G changes in gyrA, and the S80I change in parC,which were mediated by QRDR,were identified in two isolates,which showed resistance to both ciprofloxacin and levofloxacin.Most of the FQ-R strains(58.8%,10/17) belong to ST(sequence type) 76, which is dominant in the local area, while all the mutant strains (100%,2/2),that differ in at least one site from standard bacteria, belong to the ST11 group. The strains were isolated from a hospital where there had been a recent outbreak of ST76 type CRKP in the neurosurgery ward and intensive care unit. Conclusion CRKP strains were identified that were insensitive or even resistant to quinolones,and this resistance is common in Heilongjiang Province of eastern China;fluoroquinolone-resistance in these clinical CRKP strains is a complex interplay between PMQR determinants and mutations in gyrA and parC.The resistance level caused by QRDR mutation is higher than that caused by PMQR, however, the high frequency of PMQR genes in the isolated CRKP strains suggests the potential for impact of these genes.PMQR determinants are often found in carbapenemase-producing or ESBLs-producing Klebsiella pneumoniae,and some resistance genes,such as:SHV,TEM, CTX-M-15,and OXA-1 are closely associated with FQ-R. Finally, geographical factors can affect the emergence and spread of PMQR and QRDR.Some genetic lineages have higher potential risks, and continuous close monitoring is required.

The resistance of carbapenem-resistant Klebsiella pneumoniae (CRKP) to colistin has become a major challenge in clinics. The objective of this study was to investigate the synergistic and bactericidal effects of combinations of colistin with meropenem and amikacin in vitro and provide laboratory data required for the treatment of CRKP infections in the Eastern Heilongjiang Province. The minimum inhibitory concentrations of antibiotics were determined by micro-broth dilution method against 40 CRKP isolates. Ten CRKP isolates were selected to determine the minimum bactericidal concentration. The checkerboard microdilution method was performed to determine the synergic effects of colistin combinations with either meropenem or amikacin on CRKP isolates. Time kill assays were used to evaluate the effectiveness of monotherapies and colistin- meropenem or colistin-amikacin combined therapies against 6 distinct sequence types of the CRKP isolates. The minimum inhibitory concentrations of colistin, meropenem and amikacin were 2~32, 4~256, and 1~16 μg/ml, respectively. The minimum bactericidal concentration of the antibiotics was either 1 time or 2 times of the minimum inhibitory concentration. Treatments of 6 CRKP isolates at 16 μg/ml meropenem or amikacin in addition to 2 μg/ml colistin completely killed 4 of them and suppressed the growth of 2 others (sequence type 76 and 11) while none of them showed bactericidal effects in the monotherapy. Our data suggest that colistin would be a valid therapeutic option against colistin-resistant CRKP isolates when it is used in combination with either meropenem or amikacin for treatment of CRKP infections in the Eastern Heilongjiang Province.

Background: The emergence of carbapenem-resistant Enterobacteriaceae (CRE) has become a significant problem for global public health. Currently, treatments program is minimal. This study aimed to evaluate the molecular mechanisms of carbapenem-resistant Enterobacter cloacae complex isolates (CREC) infections. Methods: Resistance genes were detected using PCR with specific primers. Multilocus sequence typing (MLST) was also performed. Furthermore, we evaluated the effects of polymyxin B (PMB) and tigecycline (TGC) antibiotics (Abs) alone and in combination with meropenem (MEM), amikacin (AMK), and levofloxacin (LEV) against CREC isolates. The results were then compared with in vitro synergy testing results obtained from time-kill assays (TKAs), and the microdilution checkerboard method. Results: The synergistic efficiency of PMB + TGC was also evaluated. Abs use clinically achievable concentrations to determine the antibacterial effects of the Ab. Similar sequence type (ST) classifications had a comparably resistant phenotype; PMB-based combination therapy is better than TGC-based combination therapy. Conclusions: we found that the combination of PMB + AMK is promising for the treatment of AMK- sensitive CREC. The high-risk ST93 carrying the blaKPC-2 gene should be monitored.

Background: To investigate the activity of 5 antibiotic monotherapies, including colistin (COL), meropenem (MEM), amikacin (AMK), levofloxacin (LEV), and tigecycline (TGC), when combined with 4 other antibiotics against clinical isolates of carbapenem-resistant Klebsiella pneumoniae (CRKP) in vitro . Methods: The minimum inhibitory concentrations (MICs) of 5 antibiotics against 40 CRKP isolates were determined by micro-broth dilution method. There were synergistic effects between TGC combinations in the 10 CRKP isolates detected with checkerboard microdilution method. Time-kill assay was used to assess the monotherapies and the TGC combinations against 4 distinct sequence typing (STs) CRKP isolates. Polymerase chain reaction (PCR) tests were used to detect the carbapenemase genes, extended-spectrum beta lactamase ( ESBL ) genes, colistin resistance gene, and quinolone resistance genes, while multilocus sequence typing (MLST) was performed for 10 CRKP isolates. Results: The MICs of TGC, COL, MEM, AMK, and LEV were 0.5–2, 2–32, 4–256, 1–16,384, and 0.5–64 μg/mL, respectively. The combinations exerted a significant synergism or additive effect via the checkerboard technique for most tested CRKP isolates, but a portion of the CRKP isolates had an indifferent effect except for the TGC-AMK combination. In addition, time-kill assays revealed that TGC enhanced the bactericidal activity of the 4 other antibiotics. Among 10 CRKP isolates, blaKPC-2 (90%), blaSHV (100%), and blaacc(6’)-Ib (100%) were the most common carbapenemase genes, ESBL genes, and quinolone resistance genes, respectively. ST76 (70%) was the most predominant clone, followed by ST11 (10%), ST375 (10%), and ST530 (10%). Conclusions: In contrast to the currently recommended TGC therapy, our in vitro data suggest that TGC combinations may be a valid therapeutic option against CRKP, even in the presence of 1 antibiotic resistant isolate in TGC combination therapy. TGC-AMK combination is a cost-effective option for treating CRKP in the eastern region of Heilongjiang Province. In addition, TGC combinations might circumvent the overuse of carbapenems during the era of multi-drug resistance in Klebsiella pneumoniae (KP).

A virus with isometric virus particles (ca. 25 nm) was isolated from an apple tree and named Apple latent spherical virus (ALSV). Virus particles purified from infected Chenopodium quinoa formed two bands with densities of 1·41 and 1·43 g/cm3 in CsCl equilibrium density-gradient centrifugation, indicating that the virus is composed of two components. The virus had two ssRNA species (RNA1 and RNA2) and three capsid proteins (Vp25, Vp24 and Vp20). The complete nucleotide sequences of RNA1 and RNA2 were determined to be 6815 nt and 3384 nt excluding the 3′ poly(A) tail, respectively. RNA1 contains two partially overlapping ORFs encoding polypeptides of molecular mass 23 kDa (‘23K’; ORF1) and 235 kDa (‘235K’; ORF2); RNA2 has a single ORF encoding a polypeptide of 108 kDa (‘108K’). The 235K protein has, in order, consensus motifs of the protease cofactor, the NTP-binding helicase, the cysteine protease and the RNA polymerase, in good agreement with the gene arrangement of viruses in the Comoviridae. The 108K protein contains an LPL movement protein (MP) motif near the N terminus. Direct sequencing of the N-terminal amino acids of the three capsid proteins showed that Vp25, Vp20 and Vp24 are located in this order in the C-terminal region of the 108K protein. The cleavage sites of the 108K polyprotein were Q/G (MP/Vp25 and Vp25/Vp20) and E/G (Vp20/Vp24). Phylogenetic analysis of the ALSV RNA polymerase domain showed that ALSV falls into a cluster different from the nepo-, como- and fabavirus lineages.