Capsular Polysaccharide Types and Virulence-Related Traits of Epidemic KPC-Producing Klebsiella pneumoniae Isolates in a Chinese University Hospital

Abstract

Klebsiella pneumoniae is an important human pathogen associated with a variety of diseases and the prevalence of bla_KPC carrying K. pneumoniae (KPC-Kp) is rapidly increasing. Capsule is an important virulence factor in K. pneumoniae. In this study, we determined to first systematically characterize capsular polysaccharide (CPS) and virulence traits in KPC-Kp strains. A total of 56 KPC-Kp isolates were recovered from clinical samples in a Chinese hospital, which were assigned to clonal lineages by multilocus sequence typing (MLST). Capsule typing (wzi sequencing and wzc polymerase chain reaction [PCR]) and virulence genes were characterized by molecular approaches. The virulence of these strains was determined by biofilm formation, serum killing resistance, phagocytosis, and infection models. Six different STs were found among 56 KPC-Kp isolates: 76.8% (43 of 56 isolates) belonged to ST11, 6 isolates belonged to ST147, 4 isolates belonged to ST15, 1 isolate belonged to ST1456, 1 isolate belonged to ST65, and 1 isolate was ST23. Based on the wzi gene DNA sequences and wzc PCR, these 56 strains were classified as capsular type wzi47-K47 (n = 37), wzi64-K64 (n = 8), wzi8-K8 (n = 4), wzi37-K37 (n = 4), wzi53-K53 (n = 1), wzi125-K2 (n = 1), and wzi1-K1 (n = 1). Heterogeneity was detected in biofilm formation and phagocytosis among different CPS types. ST11 strains were less virulent than other ST strains. KPC-Kp strains exhibit variability of virulence-associated traits. Differences were associated with the ST types and CPS.

Introduction

In the past decades KPC-Kp strains have emerged in the United States since 2001 and worldwide.¹ Currently, the majority of clinical KPC-Kp isolates in China are of multilocus sequence typing (MLST)-defined clonal background ST11 that carry bla_KPC-2.^2,3 KPC-Kp infections have high mortality rates and result in increased treatment and hospitalization costs.⁴ With no novel antimicrobials for emerging KPC-Kp in sight, efforts to explore alternative treatment options and prevention of global dissemination are warranted.

Klebsiella pneumoniae pathogenicity is due to various virulence factors that allow it to overcome innate host immunity and to maintain infection in a mammalian host.⁵ One of the main virulence factors of K. pneumoniae is its capsular polysaccharide (CPS).⁶ Strategies targeting the CPS have been successful both in vaccine development as well as passive immunotherapy for other encapsulated pathogens.⁷ CPS is expressed in vivo, promotes biofilm formation, and exerts an antiopsonic effect, all of which evade the host immune response. CPS genes in K. pneumoniae strains are chromosomally encoded and clustered in the cps genomic locus.⁸ Over 77 capsular (K) serotypes have been described.⁹ The prevalence of capsular types in each K. pneumoniae-related disease could be crucial for disease control and prevention. Previous study showed that carbapenem-resistant K. pneumoniae (CR-Kp) ST258 strains exhibit variability of virulence-associated traits.¹⁰ However, KPC-Kp strains have not been extensively characterized for their K-serotype and virulence-associated traits. In this study, we characterized 56 KPC-Kp strains with respect to their CPS, biofilm formation, resistance to serum, and phagocytosis as well as virulence in infection models. This study was the first to document significant CPS-associated variability, including wzc and wzi alleles among KPC-Kp strains. Significant variability was documented with respect to virulence-associated traits.

Materials and Methods

KPC-Kp strains

The strains studied in this work included 56 nonreplicate KPC-Kp clinical isolates collected from inpatients at the First Affiliated Hospital of Nanchang University (FAHNU, Nanchang, China) between December 2012 and January 2015. Identification of the isolates was completed using the Vitek II system (bioMérieux, Balmes-les-Grottes, France) and manual biochemical identification when necessary. Antimicrobial susceptibility testing was performed on all isolates by the Vitek II system or Etest (AB bioMérieux, Solna, Sweden). Susceptibilities were categorized according to Clinical and Laboratory Standards Institute (CLSI) guidelines.¹¹ The presence of the bla_KPC gene was confirmed by polymerase chain reaction (PCR), and the bla_KPC-2 gene was determined by direct amplicon sequencing, as previously described.¹² Hypermucoviscosity phenotype was determined with the string test as described.¹³

Serotyping and virulence genes detection

Isolates were serotyped by wzi sequencing, which was strongly associated with K-type.⁸ The sequences were compared with those of reference strains¹⁴ by NCBI-nucleotide blast, and the corresponding capsular types were determined according to the criteria of ≥94% DNA identity for the same types and <80% identity for different types, with reconfirmation by wzc PCR for inconclusive results.

For virulence investigation, PCR assays were performed to search for 15 virulence factor-encoding genes (ureA, fimH, kfuBC, uge, wabG, mrkD, allS, rmpA, hlyA, rmpA2, traT, iroN, iutA, aerobactin, and cf29a) that have been associated with the virulence phenotype in K. pneumoniae.¹⁵

Molecular typing

All isolates were subjected to MLST according to the protocol described.¹⁶ In brief, house-keeping genes, including gapA, infB, mdh, pgi, phoE, rpoB, and tonB, were sequenced and compared to the MLST alleles profiles available at www.pasteur.fr/mlst (Genotyping of Pathogens and Public Health, Institut Pasteur, Paris, France).

Biofilm formation assays

Biofilm formation assays were performed at 37°C as described elsewhere.¹⁷ For interpretation of the biofilm results, the isolates were classified as follows: weak, moderate, and strong-producing, based on the following optic density (OD) average values: OD ≤0.4, weak-producing; OD ≤0.6 and >0.4, moderate-producing; and OD >0.6, strong-producing.

Phagocytosis assay

Phagocytosis was measured using a standard assay as published and described.¹⁸ The mean percentage of neutrophils that contained FITC-stained bacteria at 15 minutes in six repeated results was used as the phagocytosis rate.

Serum killing resistance

Human blood was obtained from healthy individuals. Pooled serum was separated and stored in small volumes at −80°C until use. Serum-killing assay was performed as previously described.¹⁹ An inoculums of 25 μl (adjusted to 10⁶ colony-forming units [CFUs]/ml) prepared from the mid-log phase was diluted by 0.9% saline and was added to 75 μl of pooled human sera contained in a 10 × 75 mm Falcon polypropylene tube (BD Biosciences, Franklin Lakes, NJ). Viable counts were checked at 0, 1, 2, and 3 hours of incubation at 37°C.

♦ Grade 1 is viable counts <10% of the inoculum after 1 and 2 hours and <0.1% after 3 hours.

♦ Grade 2 is viable counts 10–100% of the inoculum after 1 hour and <10% after 3 hours.

♦ Grade 3 is viable counts that exceeded those of the inoculum after 1 hour, but <100% after 2 and 3 hours.

♦ Grade 4 is viable counts >100% of the inoculum after both 1 and 2 hours, but <100% after 3 hours.

♦ Grade 5 is viable counts >100% of the inoculums 1, 2, and 3 hours, but that decreased during the third hour.

♦ Grade 6 is viable counts that exceeded those of the inoculum after 1, 2, and 3 hours and increased throughout this time period.

Each strain was tested at least three times, and the mean results were expressed as percent inoculums. Each isolate was classified as “highly sensitive” (Grade 1 or 2), “intermediately sensitive” (Grade 3 or 4), or “resistant” (Grade 5 or 6).

Galleria mellonella and murine infection models

Virulence of KPC-Kp strains was assessed in G. mellonella by injecting 20 larvae with 10⁴ CFU of KPC-Kp in 10 μl phosphate-buffered saline (PBS). Control animals were injected with PBS only. Larvae were kept at 37°C in the dark on sterile Petri plates and survival was assessed for 3 weeks. To compare in vivo replication dynamics of KPC-Kp strains, 5 μl of hemolymph was pooled from 20 larvae at different time points. CFU counts were calculated from 50 μl of hemolymph.¹⁰

Pathogen-free male BALB/c mice (age, 6–8 weeks old; weight, 20–25 g) were obtained from the Laboratory Animal Center of Nanchang University (Nanchang, China). All experiments involving mice were approved by the Committee on Institutional Animal Care and Use of the FAHNU. Mice were injected intraperitoneally with the designated strains.²⁰ Mouse mortality was assessed for up to 14 days. Each experiment with six mice of each strain was performed three times and repeated twice.

Statistical analysis

Data are presented in mean ± standard deviation or median, range. Differences between patient data were analyzed by a Fisher's exact test. Survival data were analyzed with a log-rank test. Statistical tests were performed with GraphPad Prism 6 for Mac.

Results

Patient characteristics

Fifty-six KPC-Kp strains were derived from inpatients, who were hospitalized at FAHNU. The median age of the patients was 65 years (range 32–81 years). All had various underlying diseases with a median Charlson weighted comorbidity score of 7 (range 0–12). The median number of days of hospitalization from admission to identification of KPC-Kp was 45 days (range 7–125 days). All patients had a previous history of carbapenem administration within the 3 months before identification of the microorganism. Ten KPC-Kp-infected patients never received effective treatment before death. Mortality was high (44.6%, 25/56). Antibiotic susceptibility of the strains was performed by the microbiology laboratory, which identified by standard CLSI laboratory practice, 87.5% were also resistant to ciprofloxacin, 35.7% to amikacin, and 53.6% to gentamicin.

Capsule typing and strain typing

The capsular types of 56 KPC-Kp strains were determined by wzi sequencing and reconfirmed by wzc-PCR genotyping. Sequencing of the 56 wzi PCR products of the KPC-Kp strains distinguished 6 wzi alleles (Table 1). Among the 56 strains, 52 strains corresponded to known capsular types (≥94% DNA identity across the wzi), including 37 (66.1%) of K47, 8 (14.3%) of K64, 4 (7.1%) of K8, 1 (1.8%) of K2, 1 (1.8%) of K53, and 1 (1.8%) of K1 (Table 1). All strains with distinct K-types exhibited <80% DNA sequence identity across this region, with the exception of K22/K37. Strains K22 and K37 shared identical CPS genes except for one gene with a difference at a single base, which resulted in frameshift mutation. Four strains exhibit 89% DNA identity to K22 wzi sequences, but it was subsequently confirmed to be not K22 by wzc-genotyping using K22-specific primers. In contrast, this strain was positive for K37 wzc sequencing. Thus, this strain may possess same cps as parental K37 reference strain and the wzc of this capsulated strain may represent K37 type.

Table 1.

Results of Capsular Polysaccharide Typing, Virulence Factor-Encoding Genes and Multilocus Sequence Typing for 56 KPC-Producing Klebsiella pneumoniae Isolates Collected from the Nanchang University Hospital in 2014

CPS type (No. of isolates)	Virulence factor-encoding genes	CG	ST ^a,b (No. of isolates)
wzi47-K47 (37)	fimH-1, mrkD	CG258	ST11 (35)
	fimH-1, mrkD	CG258	ST11 (1)
	fimH-1, mrkD, rmpA	CG258	ST11 (1)
wzi64-K64 (8)	fimH-1, mrkD, rmpA	CG147	ST147 (5)
	fimH-1, mrkD	CG258	ST11 (2)
	fimH-1, mrkD, rmpA2, iutA	CG147	ST147 (1)
wzi37-K37 (4)	fimH-1, mrkD	CG258	ST11 (3)
	fimH-1, mrkD	CG258	ST11 (1)
wzi8-K8 (4)	fimH-1, mrkD, iroN, allS, rmpA	CG15	ST15 (4)
wzi125-K2 (1)	fimH-1, mrkD, rmpA, rmpA2, iroN, allS, aerobactin, iutA	CG65	ST65 (1)
wzi1-K1 (1)	fimH-1, mrkD, magA, rmpA, rmpA2, iroN, allS, aerobactin, traT, iutA	CG23	ST23 (1)
wzi53-K53 (1)	fimH-1	Miscellaneous	ST1456 (1)

CGs or STs that included isolates with different CPS types are in bold.

Novel STs obtained in this study are underlined.

CG, clonal group; CPS, capsular polysaccharide.

MLST typing was performed on the 56 KPC-Kp strains. Most (43/56, 76.8%) of KPC-Kp strains belonged to ST11. Other sequence types were identified, including ST147 (6/56, 10.7%), ST15 (4/56, 7.1%), ST23 (1/56, 1.8%), ST65 (1/56, 1.8%), and ST1456 (1/56, 1.8%) (Table 1). ST11 was also predominant in some capsular types, such as K64, K47, and K37. Four ST15 isolates were included and had an identical wzi sequence (allele wzi-8) to that of the K8 reference strain.

Virulence factors and biofilm formation

The prevalence and distribution of virulence factors are given in Table 1. fimH-1 and mrkD genes, encoding type 1 and type 3 fimbrial adhesins, were present in 100% and 98.2% of isolates, respectively. iroN, alls, rmpA, rmpA2, traT, aerobactin, and iutA genes were detected at prevalences of 10.7%, 10.7%, 21.4%, 5.4%, 1.8%, 3.6%, and 5.4%, respectively. hlyA and cnf-1 genes were not detected. While rmpA (for regulator of mucoid phenotype A) was found in only 1 KPC-producing ST11 isolate, it was present in 11 non-ST11 isolates (p = 0.018). Although rmpA gene was reported to be associated with hypermucoviscosity, five nonhypermucoviscous KPC-Kp strains carried rmpA gene (6/12, 50%), indicating that there is no direct correlation between hypermucoviscosity and rmpA expression.

Biofilm formation of KPC-Kp strains varied considerably (Fig. 1). The highest biofilm producer was KPC-Kp#1 (ST23), which exhibited the wzi1-K1 molecular serotype and hypermucoviscous phenotype, followed by KPC-Kp#8 (ST65). Isolates belonging to the endemic ST11 lineage were found to produce less biofilm compared with other ST isolates (median A540 0.07 vs. 0.15, respectively; p < 0.05) (Fig. 1A). To confirm the differences in biofilm formation between ST11 isolates and other lineages, we tested the lowest and the highest biofilm-producing isolates (two of each group) in 20 biological replicates for their biofilm formation ability. All four isolates showed continuously reproducible biofilm-forming results.

FIG. 1.

(A) Biofilm formation of KPC-Kp isolates. Biofilms (expressed in A540 values) were prepared from each isolate using the standard crystal violet staining method. Biofilms are presented for both ST groups, ST11 (filled circles) and other nonepidemic STs (open circles). Isolates belonging to the ST11 lineage formed significantly less biofilm than the other group (median A540 of 0.47 vs. 0.93, respectively; p < 0.05). (B) Levels of biofilm production onto polystyrene plates measured by crystal violet quantity according to the CPS types. Line represents the average biofilm production, A540 ≅ 0.5. Vertical stripes denote low BF, horizontal stripes high producers, and gray average BF. BF, biofilm; CPS, capsular polysaccharide.

Serum killing resistance, phagocytosis, and virulence in infection models

The complement system is a critical component of the host's innate immune system. Resistance to human serum was compared, as it constitutes an important virulence trait that allows K. pneumoniae to persist in vivo. Again, considerable variability in serum resistance was documented for KPC-Kp strains. Among KPC-Kp strains, 25% were highly resistant, 12.5% moderate, and 62.5% not resistant to serum. The growth/survival of wzi1-K1 strain was significantly greater than other KPC-Kp strains (Fig. 2). These data support the concept that resistance to complement-mediated bactericidal activity in vivo is one mechanism responsible for the increased growth/survival of some KPC-Kp strains in vivo.

FIG. 2.

There is a significant increase in the growth/survival of the KPC-Kp strains in vitro in 90% human serum. Growth/survival of Klebsiella pneumoniae strains #1 (wzi1/K1), #34 (wzi47/K47), #7 (wzi37/K37), #28 (wzi64/K64), #43 (wzi8/K8), #8 (wzi125/K2), and #23 (wzi53/K53) in vitro in 90% human serum. Data are mean ± SEM for n = 3. To normalize data, log₁₀-transformed values were utilized, the area under each curve was calculated and compared using two-tailed unpaired t tests. *#1 (wzi1/K1) compared with: #34 (wzi47/K47), #7 (wzi37/K37), #28 (wzi64/K64), #43 (wzi8/K8), #23 (wzi53/K53), p < 0.01; #8 (wzi125/K2), p < 0.05. SEM, standard error of the mean.

As a next step, we assessed the ability of KPC-Kp to resist killing by human neutrophils, another critical component of the innate immune system. Survival of K. pneumoniae strains with different CPS was measured at 15 minutes in the presence and absence of human neutrophils as described.¹⁹ There was a significantly higher rate of resistance to phagocytosis for wzi1-K1 and wzi125-K2 strains (p < 0.01) (Fig. 3). In contrast, wzi 47-K47, wzi8-K8, wzi64-K64, wzi37-K37, and wzi53-K53 were significantly killed.

FIG. 3.

Effects of all isolates on phagocytosis according to the CPS types. The phagocytosis rate is calculated as the percentage of neutrophils ingesting FITC-labeled bacteria at 15 minutes. Except for wzi125-K2 and wzi1-K1 isolates with a phagocytic rate of below 40% at 15 minutes, all isolates were highly susceptible to phagocytosis by neutrophils.

Infection with 10⁴ CFU from KPC-Kp strains resulted in profound variability of G. mellonella wax worm survival ranging from avirulence to death within days (Fig. 4). Specifically, wzi37-K37 strains were avirulent at that dose in the wax worm model (median survival 13–17 days, PBS control 14 days), whereas wzi125-K2 and wzi1-K1 strains induced rapid death (median survival 1–2 days). Isolates belonging to the endemic ST11 lineage were significantly more avirulent than other ST isolates (Chi-square, p-value 0.0025). Virulence of strain KPC-Kp#34 (wzi47-K47), KPC-Kp#28 (wzi64-K64), KPC-Kp#7 (wzi37-K37), KPC-Kp#43 (wzi8-K8), KPC-Kp#23 (wzi53-K53), KPC-Kp#8 (wzi125-K2), and KPC-Kp#1 (wzi1-K1) was also tested in two murine infection models. Only KPC-Kp#20 and KPC-Kp#40 could kill mice (1-day median survival), consistent with it belonging to virulent clone CC23-K1 and CC65-K2, whereas the other strains were avirulent in mice even when high inocula were used (10⁸ CFU).

FIG. 4.

Virulence of KPC-Kp strains exhibited different CPS types. Survival rates of 20 Galleria mellonella individuals infected with 104 KPC-Kp CFU were assessed. Median days are displayed as a line within the box; maximum and minimum of the boxes display the 25th and 75th percentiles. Horizontal stripes represent avirulence, gray average virulence, and vertical stripes are high virulence. CFU, colony-forming unit.

Discussion

KPC-producing K. pneumoniae can cause hospital-acquired infections as well as community-acquired pneumonia and pyogenic liver abscess (PLA) and are associated with unacceptably high mortality rates.^20,21 Capsule has been regarded as a major virulence factor of K. pneumoniae, and capsular types are related to the severity of infection. The prevalence of capsular types (K1 and K2) in clinical settings of community-acquired infections, such as liver abscess and pneumonia, were relatively clear.²² Nevertheless, capsular types of KPC-Kp were rarely documented because KPC-Kp commonly belonged to the “classic” K. pneumoniae. Recent reports have described that significant CPS-associated variability among CR-Kp strains was documented with respect to virulence-associated traits.¹⁰ In this study, we sought to contribute to the most comprehensive characterization of capsular variability in KPC-Kp strains and the first systematic comparison to our knowledge of virulence-associated traits in these strains.

In principle, molecular typing of K. pneumoniae is easier to perform than conventional serotyping.²³ We currently unveiled the capsular types of KPC-Kp in central China using wzi sequencing and reconfirmed by wzc PCR. K47 which accounted for 37/56 (66.1%) in KPC-Kp strains was the most prevalent capsular type. K64 was the second most common type (8/56, 18.3%), followed by K8 (4/56, 7.1%) and K37 (3/56, 5.4%). The ST11 clone (clonal group [CG]258] exhibiting capsular type K47 was identified for a long period of time and seems to be the predominant lineage among KPC-Kp strains.²⁴ The identification of three distinct ST11 (CG258) lineages in this study (ST11-K47, ST11-K64, and ST11-K37) was in line with recent studies based on wzi-capsule typing unveiling the circulation of distinct lineages within this CG, which might have differences in their relative occurrence, geographical, or niche distribution and/or host susceptibility. The result implicated that KPC-Kp (mostly from nosocomial infections) showed different composition of capsular types from community-acquired isolates.

The virulence factors that are implicated in the virulence of K. pneumoniae strains include the capsular serotype, lipopolysaccharide, iron-scavenging systems, and fimbrial and nonfimbrial adhesins and may be responsible for the enhancement of the virulence potential of KPC-Kp strains, and thus the development of more invasive disease. Intriguingly, two highly virulent KPC-Kp isolates exhibited capsular serotype K1 and K2 were found for the first time in mainland China, which seem to be the most common capsular types causing liver abscess or pneumonia. Moreover, the virulence context found in these bacteria also represents a problem for medical treatment. These virulence factors improve the conditions for bacterial colonization and persistence in these patients and in the hospital.²⁵ However, compared with the genomic analysis, a key limitation to the use of the PCR to identify virulence factors has been apparent false-negative results, possibly due to a small number of relevant virulence genes.

Biofilm-associated infections are very difficult to treat due to high resistance to antibiotics.^26,27 With the increasing worldwide occurrence of KPC-Kp, data on their ability to form biofilms are crucial. In this study, biofilm formation was variable among all tested KPC-Kp strains. We found that all isolates belonging to the endemic lineage ST11 formed even lower biofilm biomass as compared with KPC-Kp isolates belonging to other lineage groups. Biofilm has been studied in hypervirulent Kp strains with a hypermucoviscous K1 or K2 CPS.¹⁷ In this study only two hypermucoviscous strains were identified as ST23-K1 and ST65-K2.

Furthermore, phagocytosis experiments showed that wzi47/K47, wzi64-K64, wzi37-K37, wzi8/K8, and wzi53-K53 strains were internalized at high rates (45–95% within 15 minutes), while wzi1/K1 and wzi125/K2 strains exhibited only marginal phagocytosis (∼30% within 15 minutes) (Fig. 2). Based on the neutrophil phagocytosis reaction, the resistance was greater in wzi1-K1 KPC-Kp strain than in other KPC-Kp strains. This effect seemed to be a consequence of the elaboration of a copious capsule (mucoidy). The antiphagocytic property of capsule was a major virulence factor.

Galleria and mouse models have been used to assess virulence of infectious microorganisms, including K. pneumoniae.²⁸ Strains expressing serotype K1 or K2 are especially virulent. Although most KPC-Kp strains with susceptibility to innate immunity, including serum killing and neutrophil phagocytosis, were confirmed by Galleria and mouse models, our results demonstrated that significant differences in virulence were detected between K1/K2 KPC-Kp isolates and other CPS types KPC-Kp isolates. In the virulence analysis, antiphagocytosis and—serum killing could be used to predict hypervirulence or relative nonvirulent in KPC-Kp isolates. A ST23 isolate with susceptibility to both neutrophil and serum killing had an LD50 equal to 10². This isolate carried nine virulence-encoding genes, but lacked hlyA and cnf-1.

In summary, we disclose a clustering of capsular types of KPC-Kp strains and have been observed variability of virulence-associated traits. One possible vaccination strategy would be to target a wider range of capsule types, similar to the approach currently used in the design of Streptococcus pneumoniae capsular vaccines. However, comprehensive population surveillance would be required. Data presented in this study also confirm that the capsular types do not unambiguously correlate with any particular ST, confirming the notion that capsular gene clusters can be exchanged between different KPC-Kp strains. Furthermore, the clonal populations of invasive infection (ST65 and ST23) have evolved to become multidrug resistant. The confluence of virulence and KPC and the bidirectional evolution might pose major problems in the future for management of K. pneumoniae infections.

Footnotes

Acknowledgments

Financial support was provided by the National Natural Science Foundation of China (81560323), Jiangxi Science and Technology Department in China (20151BAB215028 and 20161BAB205247), and Health and Family Planning Commission of Jiangxi Province (2013A035 and 20155140).

Disclosure Statement

No competing financial interests exist.

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