Mechanisms and fitness of ceftazidime/avibactam-resistant Klebsiella pneumoniae clinical strains in Taiwan.

BACKGROUND: Carbapenem-resistant Klebsiella pneumoniae (CRKP) infection is a global public health issue, and ceftazidime/avibactam is recommended by international guidelines as the preferred treatment for KPC- and OXA-48-producing CRKP. Since its introduction in Taiwan in 2019, ceftazidime/avibactam-resistant strains have emerged. Our aim is to investigate the mechanisms of ceftazidime/avibactam resistance in CRKP in Taiwan and study their associated fitness costs. METHODS: Ceftazidime/avibactam-resistant CRKP strains with exposure to ceftazidime/avibactam isolated from clinical specimens were consecutively collected at Taipei Veterans General Hospital in 2020. The serial strains exhibiting ceftazidime/avibactam-susceptible and ceftazidime/avibactam-resistant phenotypes isolated from the same patient were characterized using whole-genome sequencing and tested for their growth rates and competitive abilities. RESULTS: A total of 35 ceftazidime/avibactam-resistant CRKP strains were identified, with 20 being metallo-ß-lactamase producers. Ten strains harboured KPC variants, exhibiting MIC for ceftazidime/avibactam ranging from 64 to ≥256 mg/L. The 10 strains demonstrating high-level ceftazidime/avibactam resistance possessed mutated KPC variants: KPC-33 (n = 3), KPC-31 (n = 1), KPC-39 (n = 1), KPC-44 (n = 1), KPC-58 (n = 1), KPC-90 (n = 1), and two novel KPC variants. Ceftazidime/avibactam-resistant strains with KPC-33 and KPC-39 showed a significant fitness cost and lower growth rate compared to their parental strains. In contrast, ceftazidime/avibactam-resistant strains with KPC-58 and KPC-58 plus D179Y showed similar growth rates and competitive abilities compared to their parental strains. CONCLUSIONS: Mutated KPC variants conferred high-level ceftazidime/avibactam resistance in Taiwan. Significant fitness costs were observed in both the ceftazidime/avibactam-resistant KPC-33 and KPC-39 strains. Despite conferring a similar level of ceftazidime/avibactam resistance, different KPC variants could entail varying degrees of fitness costs.

Emergence of OXA-48-producing hypervirulent Klebsiella pneumoniae strains in Taiwan.

The OXA-48-producing hypervirulent Klebsiella pneumoniae (hvKP) strains were rarely reported. In this study, we characterized three carbapenem-resistant hvKP strains (KP2185, NCRE61, and KP2683-1) isolated from renal abscess, scrotal abscess, and blood samples in a Taiwan hospital. The three strains belonged to two different clones: ST23 K1 (KP2683-1) and ST11 KL64 (KP2185 and NCRE61). KP2683-1 exhibited the highest virulence in an in vivo model. Whole-genome sequencing analysis showed that KP2185 and NCRE61 acquired IncFIB type plasmids containing a set of virulence genes (iroBCDN, iucABCD, rmpA, rmpA2, and iutA), while KP2683-1 acquired an IncL type plasmid harboring blaOXA-48.

Clinical and Microbiological Characteristics of Bacteremic Pneumonia Caused by Klebsiella pneumoniae.

Klebsiella pneumoniae is a common pathogen of nosocomial pneumonia worldwide and community-acquired pneumonia (CAP) in Asia. Previous studies have shown that K. pneumoniae bacteremic CAP is associated with high mortality. We aimed to revisit K. pneumoniae bacteremic pneumonia in the current era and determine the risk factors associated with 28-day mortality. Between January 2014 and August 2020, adult patients with K. pneumoniae bacteremic pneumonia in a medical center in Taiwan were identified. Clinical and microbiological characteristics were compared between CAP and nosocomial pneumonia. Risk factors for 28-day mortality were analyzed using multivariate logistic regression. Among 150 patients with K. pneumoniae bacteremic pneumonia, 52 had CAP and 98 had nosocomial pneumonia. The 28-day mortality was 52% for all patients, 36.5% for CAP, and 60.2% for nosocomial pneumonia. Hypervirulent K. pneumoniae was more prevalent in CAP (61.5%) than in nosocomial pneumonia (16.3%). Carbapenem-resistant K. pneumoniae was more prevalent in nosocomial pneumonia (58.2%) than in CAP (5.8%). Nosocomial pneumonia, a higher Severe Organ Failure Assessment score, and not receiving appropriate definitive therapy were independent risk factors for 28-day mortality. In conclusion, revisiting K. pneumoniae bacteremic pneumonia in the current era showed a high mortality rate. Host factors, disease severity, and timely effective therapy affect the treatment outcomes of these patients.

Risk Factors for the Development of Colistin Resistance during Colistin Treatment of Carbapenem-Resistant Klebsiella pneumoniae Infections.

Colistin is one of the last-resort options for carbapenem-resistant Klebsiella pneumoniae (CRKP) infections if novel antibiotics are unavailable, where the development of colistin resistance during treatment represents a major challenge for clinicians. We aimed to investigate the risk factors associated with the development of colistin resistance in patients with CRKP infections following colistin treatment. We conducted a retrospective case-control study of patients with CRKP strains available before and after colistin treatment at a medical center in Taiwan, between October 2016 and November 2020. Cases (n = 35) included patients with an initial colistin-susceptible CRKP (ColS-CRKP) strain and a subsequent colistin-resistant CRKP (ColR-CRKP) strain. Controls (n = 18) included patients with ColS-CRKP as both the initial and subsequent strains. The 30-day mortality rate after the subsequent CRKP isolation was not different between cases and controls (12/35 [34%] versus 5/18 [28%] [P = 0.631]). blaKPC (n = 38) and blaOXA-48 (n = 11) accounted for the major mechanisms of carbapenem resistance. Alterations in mgrB were found in 18/35 (51%) ColR-CRKP strains, and mcr-1 was not detected in any of the strains. More patients received combination therapy in the control group than in the case group (17/18 versus 21/35 [P = 0.008]). The logistic regression model indicated that combination therapy with tigecycline was protective against the acquisition of colistin resistance (odds ratio, 0.17; 95% confidence interval, 0.05 to 0.62 [P = 0.008]). We observed that the inclusion of tigecycline in colistin treatment mitigated the risk of acquiring colistin resistance. These results offer insight into using the combination of tigecycline and colistin for the treatment of CRKP infections in antimicrobial stewardship. IMPORTANCE Treatment of carbapenem-resistant Klebsiella pneumoniae (CRKP) infections is challenging due to the limited options of antibiotics. Colistin is one of the last-resort antibiotics if novel antimicrobial agents are not available. It is crucial to identify modifiable clinical factors associated with the emergence of resistance during colistin treatment. Here, we found that the addition of tigecycline to colistin treatment prevented the acquisition of colistin resistance. Colistin-tigecycline combination therapy is therefore considered a hopeful option in antimicrobial stewardship to treat CRKP infections.

Intestinal colonisation with hypervirulent or third-generation cephalosporin-resistant Klebsiella pneumoniae strains upon hospital admission in a general ward in Taiwan.

We aimed to investigate the clinical characteristics of patients with hypervirulent and/or third-generation cephalosporin-resistant (3GCR) Klebsiella pneumoniae intestinal colonisation upon admission to a general ward at Taipei Veterans General Hospital in Taiwan in 2017. Stool surveillance cultures were obtained from patients and the clinical characteristics of the patients were studied retrospectively. Klebsiella pneumoniae strains were characterised for antimicrobial susceptibility, mechanisms of the 3GCR phenotype, and the presence of rmpA/A2 genes, which are markers of hypervirulent strains. Whole-genome sequencing was used to identify the relationship between the colonising strain and subsequent infection strain. Of the 408 patients admitted to the general ward, 87 patients with K. pneumoniae intestinal colonisation were identified. Of the 87 colonised patients, 28 (32.2%) and 9 (10.3%) carried 3GCR and hypervirulent K. pneumoniae strains, respectively. Stay in a long-term care facility and diabetes mellitus were more common in patients colonised with 3GCR strains than those with hypervirulent strains. The 28-day mortality rate was similar between the two groups. Major resistance mechanisms among the 3GCR strains involved the presence of extended-spectrum ß-lactamase (ESBL) genes (67.9%) and blaDHA-1 (64.3%). One patient colonised with K. pneumoniae developed subsequent bacteraemia caused by the same strain. In conclusion, 3GCR strains were more common than hypervirulent strains in colonised patients, but clinical outcomes were similar. Future studies to elucidate risk factors for intestinal carriage of hypervirulent and 3GCR K. pneumoniae strains are needed for early identification and better management of these patients.

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.

Fluoroquinolones as an alternative treatment for Klebsiella pneumoniae liver abscess and impact on hospital length of stay.

Klebsiella pneumoniae liver abscess (KPLA) is an endemic disease in East Asia. Patients with KPLA usually require prolonged intravenous (i.v.) ß-lactam therapy and hospitalisation. Fluoroquinolones have high oral bioavailability and the potential to shorten the duration of i.v. therapy. The aim of this study was to investigate the feasibility of fluoroquinolones as an alternative treatment for KPLA in Taiwan. Consecutive patients with KPLA in a medical centre in Taiwan between July 2012 and August 2019 were retrospectively enrolled. Clinical characteristics and outcomes were compared between cases treated with ß-lactams and fluoroquinolones. A multivariate logistic regression model and propensity-score adjusted analysis were performed to identify independent risk factors for prolonged hospitalisation. A total of 234 patients with KPLA were identified during the study period. Most patients received ß-lactams (n = 199; 85.0%), whilst only 35 (15.0%) received fluoroquinolones as the major therapy. Fluoroquinolones had similar clinical efficacy to ß-lactams even in critically ill patients. Patients treated with fluoroquinolones had a shorter i.v. antibiotics duration (18.9 ± 7.6 days vs. 28.5 ± 14.7 days; P < 0.001) and hospital length of stay (LOS) (20.9 ± 8.3 days vs. 29.5 ± 16.2 days; P < 0.001) than patients treated with ß-lactams. Major therapy with fluoroquinolones was an independent protective factor for hospital LOS > 14 days in all patients and for hospital LOS > 21 days in critically ill patients. In conclusion, fluoroquinolones were an effective alternative treatment for KPLA that resulted in a shorter duration of i.v. therapy and hospital LOS.

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.

Emergence of an XDR and carbapenemase-producing hypervirulent Klebsiella pneumoniae strain in Taiwan.

Background: Carbapenemase-producing Klebsiella pneumoniae causes high mortality owing to the limited therapeutic options available. Here, we investigated an emergent carbapenem-resistant K. pneumoniae strain with hypervirulence found among KPC-2-producing strains in Taiwan. Methods: KPC-producing K. pneumoniae strains were collected consecutively from clinical specimens at the Taipei Veterans General Hospital between January 2012 and December 2014. Capsular types and the presence of rmpA/rmpA2 were analysed, and PFGE and MLST performed using these strains. The strain positive for rmpA/rmpA2 was tested in an in vivo mouse lethality study to verify its virulence and subjected to WGS to delineate its genomic features. Results: A total of 62 KPC-2-producing K. pneumoniae strains were identified; all of these belonged to ST11 and capsular genotype K47. One strain isolated from a fatal case with intra-abdominal abscess (TVGHCRE225) harboured rmpA and rmpA2 genes. This strain was resistant to tigecycline and colistin, in addition to carbapenems, and did not belong to the major cluster in PFGE. TVGHCRE225 exhibited high in vivo virulence in the mouse lethality experiment. WGS showed that TVGHCRE225 acquired a novel hybrid virulence plasmid harbouring a set of virulence genes (iroBCDN, iucABCD, rmpA and rmpA2, and iutA) compared with the classic ST11 KPC-2-producing strain. Conclusions: We identified an XDR ST11 KPC-2-producing K. pneumoniae strain carrying a hybrid virulent plasmid in Taiwan. Active surveillance focusing on carbapenem-resistant hypervirulent K. pneumoniae strains is necessary, as the threat to human health is imminent.