Risk factors for CRKP infection

Factors associated with CRKP infection included prior use of two or more antibiotics (including carbapenems), prior use of carbapenems, hypertension, cancer, neurological disorders, ICU admission, and the coexistence of carbapenem-resistant A. baumannii in clinical specimens (P < 0.05) (Table 2). Among them, hypertension (odds ratio [OR], 1.824; 95% confidence interval [CI], 1.053–3.161; P = 0.032), neurological disorders (OR, 2.890; 95% CI, 1.568–5.326; P < 0.001), ICU admission (OR, 2.099; 95% CI, 1.148–3.836; P = 0.016), prior use of two or more antibiotics (OR, 4.124; 95% CI, 2.142–7.939; P < 0.001) and prior use of carbapenems (OR, 2.613; 95% CI, 1.314–5.196; P = 0.006) were independent risk factors for CRKP infection (Table 2).

Table 2 Clinical characteristics of patients infected with K. pneumoniaeClinical cohort study of mortality: survival versus death in patients with K. pneumoniae infection

Among the 311 patients with K. pneumoniae infection, 57 (18.3%) patients died. Univariate analysis showed that respiratory failure, ICU admission, and prior use of carbapenems were factors related to death of in these patients. Multivariate analysis confirmed that respiratory failure (OR, 5.909; 95% CI, 2.911–11.996; P < 0.001) and ICU admission (OR, 12.285; 95% CI, 4.180–36.109; P < 0.001) were independent risk factors for death in these patients (Table 3).

Table 3 Analysis of associated risk factors for death associated with K. pneumoniae infectionAST of the K. pneumoniae isolates

All of the K. pneumoniae isolates were resistant to AM. In total, 140 (45.0%) isolates were categorised as CRKP based on their IPM susceptibility. Compared with the CSKP isolates, the CRKP isolates had higher resistance rates to all antibiotics except ampicillin, to which all isolates were resistant. In addition, the CRKP isolates showed resistance rates of 1.4% and 6.4% for polymyxin B and ceftazidime–avibactam, respectively (Table 4). The detailed results of antibiotic susceptibility testing of CSKP and CRKP isolates are presented in Table 4.

Table 4 Antimicrobial resistance profiles of K. pneumoniaeMLST of the K. pneumoniae isolates

All isolates were successfully typed by MLST and assigned to 102 STs. The most prevalent ST was ST11 (112, 36.0%), followed by ST23 (15, 4.8%), ST37 (9, 2.9%) and ST15 (9, 2.9%). Notably, all of the 112 ST11 isolates were CRKP. Fourteen STs were found among the 140 CRKP isolates. The detailed results of MLST for CRKP isolates are provided in Fig. 1. Among the CSKP isolates, 95 STs were found, with ST23 (13, 7.6%) being the most common ST, followed by ST68 (8, 4.7%), ST37 (6, 3.5%), ST25 (5, 2.9%), ST15 (4, 2.3%), ST412 (4, 2.3%), ST36 (4, 2.3%) and ST307 (4, 2.3%).

Fig. 1

Classification characteristics of MLST of CRKP

Virulence gene and resistance gene profiles

Three hundred and eleven K. pneumoniae isolates were tested for the presence of the virulence genes rmpA, rmpA2, iroN and iucA. Overall, the positive rates of rmpA, rmpA2, iroN and iucA were 58.2% (181), 48.6% (151), 51.1% (159) and 46.6% (145), respectively. Furthermore, 133 (42.8%) of the total isolates contained all of the four virulence genes, 53 (17.0%) isolates contained one to three of the virulence genes, whereas 125 (40.2%) isolates carried none of the virulence genes. Among the 140 CRKP isolates, the positive rates of rmpA, rmpA2, iroN and iucA were 67.1% (94), 62.9% (88), 60.0% (84) and 59.3% (83), respectively; 83 (59.3%) of these isolates contained all of the four virulence genes, 13 (0.9%) isolates contained one to three of the virulence genes, while 44 (31.4%) isolates carried none of the virulence genes. Among the 171 CSKP isolates, the positive rates of rmpA, rmpA2, iroN and iucA were 50.9% (87), 36.8% (63), 43.9% (75) and 36.3% (62) respectively; 50 (29.2%) of these isolates contained all of the four virulence genes, 40 (23.4%) isolates contained one to three of the virulence genes, while 81 (47.4%) isolates carried none of the virulence genes. As shown in Table 5, CRKP was more likely than CSKP to carry all of the four virulence genes.

Table 5 Analysis of K. pneumoniae carrying all of the four virulence genes

Three common carbapenemase-encoding genes—blaKPC-2, blaNDM like and blaOXA-48 like—were detected using a PCR assay. Among the 140 CRKP isolates, the positive rates of blaKPC-2, blaNDM like and blaOXA-48 like were 82.1%, 10.7% and 10.7%, respectively. The distribution of resistance genes in different STs is shown in Fig. 2. Of the 112 ST11 isolates, 105 (93.8%) harboured blaKPC-2; moreover, 78 (74.3%) of these 105 ST11-KPC-2 isolates contained all of the four virulence genes rmpA, rmpA2, iroN and iucA. The proportional relationships between STs and prior antibiotic use, resistance genes, virulence genes, and resistance ratios to different antibiotics are shown in Fig. 3.

Fig. 2

Genotyping characteristics of various drug resistance genes of CRKP

Fig. 3

Heatmap of virulence gene carrier rates, multiple antibiotic use rates, carbapenem antibiotic use rates and antimicrobial resistance rates in different MLST CRKP strains

Comparison of the G. mellonella virulence test

Patients with CRKP infection had a higher mortality rate than those with CSKP infection (23.6% vs 14.0%, P < 0.05) (Table 2); however, co-carrying rmpA, rmpA2, iroN and iucA was not associated with mortality in patients with K. pneumoniae infection (43.9% vs 42.5%, P = 0.854) (Table 3). To further investigate this phenomenon, 24 isolates carrying all of the four virulence genes were randomly selected and subjected to the G. mellonella virulence test. It was found that six of these isolates exhibited high virulence with a 72-h G. mellonella survival rate < 50%, 14 isolates showed low virulence with a 72-h G. mellonella survival rate > 50%, and four isolates were found to be nonvirulent with a 72-h G. mellonella survival rate of 100%. The hypervirulent positive-control K. pneumoniae strain NTUH-K2044 was found to have a 36-h G. mellonella survival rate of 0%, whereas seven K. pneumoniae isolates without these four virulence genes and the negative control strain had a 72-h G. mellonella survival rate of 100% (Fig. 4).

Fig. 4

Experiments with different K. pneumoniae infecting Galleria mellonella. A Survival rates of NTUH-K2044 and 7 strains K. pneumoniae without these four virulence genes, + PBS empty control and blank control. Survival rates of 24 strains containing these four virulence genes; B 6 strains were high virulent, with a survival rate of 50% or less within 72 h; C 14 strains showed low virulence with a 72-h survival rate greater than 50%; D 4 strains were nonvirulent with 100% survival at 72 h. NTUH-K2044 carried these virulence genes: iroB, iroC, iroD, iroN, iucA, iucB, iucC, iucD, iutA, rmpA and rmpA2