Characterization of a mcr-1 and CRISPR-Cas System Co-harboring Plasmid in a Carbapenemase-Producing High-Risk ST11 Klebsiella pneumoniae Strain.

We set out to study the prevalence of the mcr-1 gene in carbapenemase-producing Klebsiella pneumoniae (CPKP) strains, and to determine whether its presence is associated with a fitness cost. A total of 234 clinical CPKP isolates were collected from a tertiary medical center in Taiwan from January 2018 to January 2019. The mcr-1 and carbapenemase genes were detected by polymerase chain reaction (PCR) followed by Sanger sequencing. The mcr-1-positive carbapenemase-producing strain was characterized by whole genome sequencing, a plasmid stability test and a conjugation assay. In vitro growth rate and an in vivo virulence test were compared between the parental mcr-1-positive strain and its mcr-1 plasmid-cured strain. We identified only one mcr-1 positive strain (KP2509), co-harboring bla KPC- 2 and bla OXA- 48, among 234 (1/234, 0.43%) CPKP strains. KP2509 and its Escherichia coli mcr-1 transconjugant showed moderate colistin resistance (MIC = 8 mg/L). The mcr-1 is located on a large conjugative plasmid (317 kb), pKP2509-MCR, with three replicons, IncHI, IncFIB, and IncN. Interestingly, a complete Type IV-A3 CRISPR-Cas system was identified in pKP2509-MCR. Plasmid pKP2509-MCR was highly stable in KP2509 after 270 generation of passage, and the pKP2509-MCR cured strain PC-KP2509 showed similar growth rate and in vivo virulence in comparison to KP2509. The prevalence of mcr-1 in CPKP strains remains low in our center. Notably, we identified a large plasmid with multiple replicons containing both the mcr-1 and the Type IV-3A CRISPR-Cas genes. The further spread of this highly stable plasmid raises concern that it may promote the increase of mcr-1 prevalence in CPKP.

Risk factors and mechanisms of in vivo emergence of colistin resistance in carbapenem-resistant Klebsiella pneumoniae.

Colistin is one of the last-resort antibiotics for treating carbapenem-resistant Klebsiella pneumoniae (CRKP). However, colistin resistance in CRKP poses a global antimicrobial crisis, as therapeutic options are limited. We investigated risk factors for in vivo emergence of colistin resistance in CRKP and explored the underlying resistance mechanisms. We conducted this matched case-control study of patients with sequential CRKP clinical strains at a medical centre in Taiwan between October 2016 and June 2019. The case group included patients with an index colistin-resistant CRKP (ColR-CRKP) strain and a previous colistin-susceptible CRKP (ColS-CRKP) counterpart. The control group encompassed patients with both an index and previous ColS-CRKP strains. Cases and controls were matched according to the time at risk, and conditional logistic regression was used to evaluate potential risk factors. Alterations in genes associated with resistance were compared between ColR-CRKP and ColS-CRKP strains. We identified 24 CRKP cases with in vivo-emergent colistin resistance, matched in a 1:2 ratio with controls. Multivariate analysis showed that colistin exposure is the only independent risk factor predisposing to colistin resistance (adjusted odds ratio = 19.09, 95% confidence interval 1.26-290.45; P = 0.034). Alteration in the mgrB gene was the predominant mechanism for emergent colistin resistance (17/24; 71%). In conclusion, colistin use is a risk factor for in vivo emergence of colistin resistance in CRKP. Given the lack of a rapid and reliable method to detect colistin resistance in daily practice, physicians should be vigilant for the emergence of resistance during colistin treatment.

Molecular and Clinical Characterization of Multidrug-Resistant and Hypervirulent Klebsiella pneumoniae Strains from Liver Abscess in Taiwan.

Hypervirulent Klebsiella pneumoniae strains are the major cause of liver abscesses throughout East Asia, and these strains are usually antibiotic susceptible. Recently, multidrug-resistant and hypervirulent (MDR-HV) K. pneumoniae strains have emerged due to hypervirulent strains acquiring antimicrobial resistance determinants or the transfer of a virulence plasmid into a classic MDR strain. In this study, we characterized the clinical and microbiological properties of K. pneumoniae liver abscess (KPLA) caused by MDR-HV strains in Taiwan. Patients with community onset KPLA were retrospectively identified at Taipei Veterans General Hospital during January 2013 to May 2018. Antimicrobial resistance mechanisms, capsular types, and sequence types were determined. MDR-HV strains and their parental antimicrobial-susceptible strains further underwent whole-genome sequencing (WGS) and in vivo mice lethality tests. Thirteen MDR-HV strains were identified from a total of 218 KPLA episodes. MDR-HV strains resulted in similar outcomes to antimicrobial-susceptible strains. All MDR-HV strains were traditional hypervirulent clones carrying virulence capsular types. The major resistance mechanisms were the overexpression of efflux pumps and/or the acquisition of ESBL or AmpC ß-lactamase genes. WGS revealed that two hypervirulent strains had evolved to an MDR phenotype due to mutation in the ramR gene and the acquisition of an SHV-12-bearing plasmid, respectively. Both these MDR-HV strains retained high virulence compared to their parental strains. The spread of MDR-HV K. pneumoniae strains in the community raises significant public concerns, and measures should be taken to prevent the further acquisition of carbapenemase and other resistance genes among these strains in order to avoid the occurrence of untreatable KPLA.

Tigecycline-non-susceptible hypervirulent Klebsiella pneumoniae strains in Taiwan.

OBJECTIVES: Emergent antimicrobial-resistant hypervirulent Klebsiella pneumoniae (hvKp) is an important public health issue. We aimed to investigate resistance mechanisms and hypervirulent traits among tigecycline-non-susceptible (TNS) K. pneumoniae clinical strains, focusing on one hvKp strain with in vivo evolution of tigecycline resistance. METHODS: TNS K. pneumoniae strains causing invasive diseases in a medical centre in Taiwan between July 2015 and April 2018 were collected. Resistance mechanisms were determined and hvKp strains were defined as rmpA/rmpA2-carrying strains. Isogenic strains with and without tigecycline resistance were subjected to WGS and in vivo virulence testing. Further, site-directed mutagenesis was used to confirm the resistance mechanism. RESULTS: In total, 31 TNS K. pneumoniae strains were isolated, including six hypervirulent strains. Tigecycline resistance mechanisms were mostly caused by overexpression of AcrAB and OqxAB together with up-regulation of RamA or RarA, respectively. One TNS hypervirulent strain (KP1692; MIC=6 mg/L) derived from its tigecycline-susceptible counterpart (KP1677; MIC=0.75 mg/L) showed acrAB overexpression. WGS revealed four genetic variations between KP1677 and KP1692. In addition, using site-directed mutagenesis, we confirmed that a 1 bp insertion in the ramA upstream region (RamR-binding site), leading to ramA and acrAB overexpression in KP1692, was responsible for tigecycline resistance. The in vivo virulence experiment showed that the TNS hvKp strain KP1692 still retained its high virulence compared with KP1677. CONCLUSIONS: hvKp strains accounted for 19.4% among TNS strains. We identified alterations in the ramA upstream region as a mechanism of in vivo tigecycline resistance development in an hvKp strain.

High mortality among patients infected with hypervirulent antimicrobial-resistant capsular type K1 Klebsiella pneumoniae strains in Taiwan.

Capsular type K1 Klebsiella pneumoniae, highly virulent strains which are common in Asian countries, can cause pyogenic infections. These hypervirulent strains are usually susceptible to most antimicrobials, except for ampicillin. Little is known regarding the clinical and molecular characteristics of antimicrobial-resistant K1 K. pneumoniae strains. This retrospective study evaluated patients infected with capsular type K1 K. pneumoniae strains in a Taiwanese medical centre between April 2013 and March 2016. Antimicrobial-resistant strains were defined based on non-susceptibility to antimicrobial agents except ampicillin. We compared the clinical outcome of patients infected with and without antimicrobial-resistant strains. The in vivo virulence, genetic relatedness, and resistance mechanisms of these hypervirulent antimicrobial-resistant strains were also investigated. A total of 182 capsular type K1 K. pneumoniae strains were identified, including 18 antimicrobial-resistant strains. The 28-day mortality rate among the 18 cases caused by antimicrobial-resistant strains was significantly higher than that among 164 cases caused by antimicrobial-sensitive strains (50% vs. 10.4%, P < 0.001). Infection with antimicrobial-resistant strain independently increased the 28-day mortality risk. Most antimicrobial-resistant strains were not clonally related, and they exhibited high in vivo virulence in a mouse lethality experiment. The major resistance mechanisms involved the presence of ß-lactamases and the overexpression of efflux pumps. In conclusion, hypervirulent antimicrobial-resistant capsular type K1 K. pneumoniae strains can predispose to a fatal outcome. These strains may represent an emerging threat to public health in Taiwan.

A putative RND-type efflux pump, H239_3064, contributes to colistin resistance through CrrB in Klebsiella pneumoniae.

Background: Colistin is one of the last-resort antibiotics used to treat carbapenem-resistant Klebsiella pneumoniae infection. Our previous studies indicated that clinical strains encoding CrrB with amino acid substitutions exhibited higher colistin resistance (MICs ≥512 mg/L) than did colistin-resistant strains encoding mutant MgrB, PmrB or PhoQ. Objectives: CrrAB may regulate another unknown mechanism(s) contributing to colistin resistance, besides modifications of LPS with 4-amino-4-deoxy-l-arabinose and phosphoethanolamine. Methods: To identify these potential unknown mechanism(s), a transposon mutant library of A4528 crrB(N141I) was constructed. Loci that might contribute to colistin resistance and were regulated by crrB were confirmed by deletion and complementation experiments. Results: Screening of 2976 transposon mutants identified 47 mutants in which the MICs of colistin were significantly decreased compared with that for the parent. Besides crrAB, crrC and pmrHFIJKLM operons, these 47 transposon insertion mutants included another 13 loci. Notably, transcript levels of one of these insertion targets, H239_3064 (encoding a putative RND-type efflux pump), were significantly increased in A4528 crrB(N141I) compared with the A4528 parent strain. Deletion of H239_3064 in the A4528 crrB(N141I) background resulted in an 8-fold decrease in the MIC of colistin; complementation of the deletion mutant with H239_3064 restored resistance to colistin. Susceptibilities of A4528-derived strains to other antibiotics were also tested. Mutations of crrB resulted in decreased susceptibility to tetracycline and tigecycline, and deletion of H239_3064 in A4528 crrB(N141I) attenuated this phenomenon. Conclusions: This study demonstrated that missense mutations of K. pneumoniae crrB lead to increased expression of H239_3064, leading in turn to decreased susceptibility to colistin, tetracycline and tigecycline.

Klebsiella Phage ΦK64-1 Encodes Multiple Depolymerases for Multiple Host Capsular Types.

The genome of the multihost bacteriophage ΦK64-1, capable of infecting Klebsiella capsular types K1, K11, K21, K25, K30, K35, K64, and K69, as well as new capsular types KN4 and KN5, was analyzed and revealed that 11 genes (S1-1, S1-2, S1-3, S2-1, S2-2, S2-3, S2-4, S2-5, S2-6, S2-7, and S2-8) encode proteins with amino acid sequence similarity to tail fibers/spikes or lyases. S2-5 previously was shown to encode a K64 capsule depolymerase (K64dep). Specific capsule-degrading activities of an additional eight putative capsule depolymerases (S2-4 against K1, S1-1 against K11, S1-3 against K21, S2-2 against K25, S2-6 against K30/K69, S2-3 against K35, S1-2 against KN4, and S2-1 against KN5) was demonstrated by expression and purification of the recombinant proteins. Consistent with the capsular type-specific depolymerization activity of these gene products, phage mutants of S1-2, S2-2, S2-3, or S2-6 lost infectivity for KN4, K25, K35, or K30/K69, respectively, indicating that capsule depolymerase is crucial for infecting specific hosts. In conclusion, we identified nine functional capsule depolymerase-encoding genes in a bacteriophage and correlated activities of the gene products to all ten hosts of this phage, providing an example of type-specific host infection mechanisms in a multihost bacteriophage.IMPORTANCE We currently identified eight novel capsule depolymerases in a multihost Klebsiella bacteriophage and correlated the activities of the gene products to all hosts of this phage, providing an example of carriage of multiple depolymerases in a phage with a wide capsular type host spectrum. Moreover, we also established a recombineering system for modification of Klebsiella bacteriophage genomes and demonstrated the importance of capsule depolymerase for infecting specific hosts. Based on the powerful tool for modification of phage genome, further studies can be conducted to improve the understanding of mechanistic details of Klebsiella phage infection. Furthermore, the newly identified capsule depolymerases will be of great value for applications in capsular typing.

Amino Acid Substitutions of CrrB Responsible for Resistance to Colistin through CrrC in Klebsiella pneumoniae.

Colistin is a last-resort antibiotic for treatment of carbapenem-resistant Klebsiella pneumoniae A recent study indicated that missense mutations in the CrrB protein contribute to colistin resistance. In our previous study, mechanisms of colistin resistance were defined in 17 of 26 colistin-resistant K. pneumoniae clinical isolates. Of the remaining nine strains, eight were highly resistant to colistin. In the present study, crrAB sequences were determined for these eight strains. Six separate amino acid substitutions in CrrB (Q10L, Y31H, W140R, N141I, P151S, and S195N) were detected. Site-directed mutagenesis was used to generate crrB loci harboring individual missense mutations; introduction of the mutated genes into a susceptible strain, A4528, resulted in 64- to 1,024-fold increases in colistin MICs. These crrB mutants showed increased accumulation of H239_3062, H239_3059, pmrA, pmrC, and pmrH transcripts by quantitative reverse transcription (qRT)-PCR. Deletion of H239_3062 (but not that of H239_3059) in the A4528 crrB(N141I) strain attenuated resistance to colistin, and H239_3062 was accordingly named crrC Similarly, accumulation of pmrA, pmrC, and pmrH transcripts induced by crrB(N141I) was significantly attenuated upon deletion of crrC Complementation of crrC restored resistance to colistin and accumulation of pmrA, pmrC, and pmrH transcripts in a crrB(N141I) ΔcrrC strain. In conclusion, novel individual CrrB amino acid substitutions (Y31H, W140R, N141I, P151S, and S195N) were shown to be responsible for colistin resistance. We hypothesize that CrrB mutations induce CrrC expression, thereby inducing elevated expression of the pmrHFIJKLM operon and pmrC (an effect mediated via the PmrAB two-component system) and yielding increased colistin resistance.

Colistin resistance mechanisms in Klebsiella pneumoniae strains from Taiwan.

Colistin is one of the antibiotics of last resort for the treatment of carbapenem-resistant Klebsiella pneumoniae infection. This study showed that capsular type K64 (50%) and ST11 (53.9%) are the prevalent capsular and sequence types in the colistin-resistant strains in Taiwan. The interruption of transcripts (38.5%) and amino acid mutation (15.4%) in mgrB are the major mechanisms contributing to colistin resistance. In addition, novel single amino acid changes in MgrB (Stop48Tyr) and PhoQ (Leu26Pro) were observed to contribute to colistin resistance.

Vancomycin promotes the bacterial autolysis, release of extracellular DNA, and biofilm formation in vancomycin-non-susceptible Staphylococcus aureus.

Staphylococcus aureus, an important human pathogen, is particularly adept at producing biofilms on implanted medical devices. Although antibiotic treatment of nonsusceptible bacteria will not kill these strains, the consequences should be studied. The present study focuses on investigating the effect of vancomycin on biofilm formation by vancomycin-non-susceptible S. aureus. Biofilm adherence assays and scanning electron microscopy demonstrated that biofilm formation was significantly enhanced following vancomycin treatment. Bacterial autolysis of some subpopulations was observed and was confirmed by the live/dead staining and confocal laser scanning microscopy. A significant increase in polysaccharide intercellular adhesin (PIA) production was observed by measuring icaA transcript levels and in a semi-quantitative PIA assay in one resistant strain. We show that the release of extracellular DNA (eDNA) via cidA-mediated autolysis is a major contributor to vancomycin-enhanced biofilm formation. The addition of xenogeneic DNA could also significantly enhance biofilm formation by a PIA-overproducing S. aureus strain. The magnitude of the development of the biofilm depends on a balance between the amounts of eDNA and PIA. In conclusion, sublethal doses of cell wall-active antibiotics like vancomycin induce biofilm formation through an autolysis-dependent mechanism in vancomycin-non-susceptible S. aureus.