Accepted Manuscript

Title: Microbiological Characterization of Klebsiella

pneumoniae isolates causing Bloodstream Infection from Five
Tertiary Hospitals in Beijing, China

Authors: Yanning Ma, Chunmei Bao, Jie Liu, Xiuhong Hao,

Jingui Cao, Liyan Ye, Jiyong Yang

PII: S2213-7165(17)30189-3
DOI: https://doi.org/10.1016/j.jgar.2017.10.002
Reference: JGAR 512

To appear in:

Received date: 10-5-2017

Revised date: 12-9-2017
Accepted date: 4-10-2017

Please cite this article as: Yanning Ma, Chunmei Bao, Jie Liu, Xiuhong Hao, Jingui Cao,
Liyan Ye, Jiyong Yang, Microbiological Characterization of Klebsiella pneumoniae
isolates causing Bloodstream Infection from Five Tertiary Hospitals in Beijing, China
(2010), https://doi.org/10.1016/j.jgar.2017.10.002

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Microbiological Characterization of Klebsiella pneumoniae isolates causing

Bloodstream Infection from Five Tertiary Hospitals in Beijing, China

Yanning Ma1, Chunmei Bao2, Jie Liu3, Xiuhong Hao4, Jingui Cao5, Liyan Ye1 and Jiyong Yang1*

1. Department of Microbiology, Chinese PLA General Hospital, Beijing, China 100853

2. Clinical Diagnostic Center, 302nd Hospital of China 100039

3. Department of Laboratory, PLA Army General Hospital, Beijing, China 100700

4. Clinical Laboratory, Navy General Hospital, PLA, Beijing, China 100048

5. Department of Infection Control, Air Force General Hospital, PLA, Beijing, China 100142

*Corresponding author:

Dr. Jiyong Yang

Email: yangjy301@hotmail.com

Tel: +86 10 66936575

Fax: +86 10 68232198

Mailing address: Microbiology Department, 301 Hospital, 28# Fuxing Road, Beijing 100853, China

Highlights
 Diverse sequence types have been identified among BSI-causing K. pneumoniae.
 ST11 and ST23 were the predominant clones.
 The majority (70.4%, 143/203) of BSIKpn isolates were associated with

non-K1/K2/K5/K20/K54/K57 serotypes.

 The rmpA was a predominant factor in determining whether or not a BSIKpn strain

had HV phenotype.
 The majority of BSIKpn strains exhibited low resistance to commonly used

antimicrobial agents.

ABSTRACT

Background: Klebsiella pneumoniae exhibits prevalence in China. Little is known about the

microbiological characterization of clinical K. pneumoniae isolates causing bloodstream infections

(BSIs).

Methods: BSI-causing K. pneumoniae (BSIKpn) isolates were collected from five tertiary-care

hospitals in Beijing, China. Their genetic relatedness was analyzed by pulsed-field gel

electrophoresis (PFGE), antimicrobial susceptibility was determined by agar dilution method, and

sequence type (ST) was evaluated by multilocus sequence typing (MLST). The hypermucoviscosity

(HV) phenotype was identified by a positive string test. Carbapenemase, capsular serotypes and

HV-associated genes were detected by PCR amplification.

Results: A total of 219 non-duplicated BSIKpn isolates were collected from Dec. 2013 to Dec. 2014

and categorized into 203 types (strains) which had a unique PFGE pattern. Among 203 BSIKpn

strains, 105 different STs were identified. Overall, ST11 and ST23 were the predominant clones and

detected from 14 strains (6.9%), respectively, followed by ST412 (n = 13), ST37 (n = 9), ST65 (n =

7), ST15 (n = 6), ST86 (n = 6), ST592 (n = 5) and ST29 (n = 4). There were 74 STs containing only

a single strain. Approximately 8.4% (17/203) of BSIKpn strains exhibited resistance to carbapenem

and most of them produced K. pneumoniae carbapenemase (KPC). The majority (70.4%, 143/203) of

BSIKpn isolates were associated with non-K1/K2/K5/K20/K54/K57 serotypes. Only 16.7% (34/203)

of BSIKpn strains had K1/K2 serotypes. A total of 66 (32.5%) of the BSIKpn strains exhibited HV

phenotype. The rmpA was a predominant factor in determining whether or not a BSIKpn strain had

HV phenotype.

Conclusions: The majority of BSIKpn strains exhibited high level of genetic diversity and low

resistance to commonly used antimicrobial agents. The specific capsular serotype and HV phenotype
were not the major features of BSIKpn strains from China.

KEY WORDS: Bloodstream infection; K. pneumoniae; capsular serotype; hypermucoviscosity.

INTRODUCTION

Bloodstream infection (BSI) is one of the most common infections for hospitalized patients. For

patients in intensive care unit, BSIs are the leading healthcare-associated infections which have been

linked to major morbidity and mortality [1]. With the growing incidence of BSIs caused by various

multi-drug resistant or even pan-drug resistant Enterobacteriaceae isolates, BSIs have been

considered as a public health problem worldwide [2].

Klebsiella pneumoniae is a common cause of a wide range of infections, including pneumonia,

urinary tract infection, bacteremia, and liver abscess. Among Gram-negative pathogens, K.

pneumoniae is second only to E. coli as the causative agent of various bacteremias [3]. The K.

pneumoniae-causing bacteremia can lead to higher mortality (ranged from 27.4 to 37%), prolonged

hospital stay and increased medical costs [3].

Newly described hypervirulent K. pneumoniae (hvKP) strains, which have an easily

recognizable hypermucoviscosity (HV), have been identified 20 years ago, causing great concern

worldwide [3]. Recently, hvKP-causing bacteremias have also been reported [4-7]. The HV

phenotype in K. pneumoniae has been found to be related to the presence of the chromosomally

encoded mucoviscosity-associated gene A (magA) [8]. Further studies have revealed that magA is

responsible for capsular serotype K1 of K. pneumoniae, and it is clarified as the capsular polymerase

gene (renamed as wzy_K1 or wzyKpK1) specific to K. pneumoniae serotype K1 [9-11]. Furthermore,

magA is not detected in any type of other serotypes[12]. Another HV-associated gene, the plasmid

gene regulator of the mucoid phenotype A (rmpA), has been confirmed as a gene regulating capsular

polysaccharide synthesis that confers a mucoid phenotype of clinical K. pneumoniae isolates [13].

However, BSI-causing K. pneumoniae (BSIKpn) isolates are highly diverse, suggesting that HV is

not a reliable indicator of the presence of virulent clones carrying magA and/or rmpA genes [6, 7].
 The emergence and prevalence of carbapenem-resistant K. pneumoniae is another concern for K.

pneumoniae-causing BSI which has an impact on increased infection-related mortality and has

become the most important epidemiologic and therapeutic challenge [2, 14]. Various and

overlapping carbapenemases (including KPC, OXA-48 and NDM) have been involved in the

carbapenem-resistant phenotype in BSIKpn from different areas [2].

Although some clones (such ST23) account for the majority of BSIKpn, the BSIKpn isolates

exhibit highly sequence type (ST) diversity [4, 6, 7, 15]. The high level of clonal diversity of BSIKpn

indicates the widespread diversification of strains due to spread of heterogeneous K. pneumoniae

clones in various medical establishments.

Recently, a retrospective multicenter study was conducted to describe phenotypic and genotypic

characteristics of clinical BSIKpn isolates. In total, 219 BSIKpn isolates were collected from five

tertiary-care hospitals in Beijing, China, and were analyzed for their phenotypic and genotypic

characteristics. It was the first multicenter study about clinical BSIKpn isolates in Beijing.

MATERIALS AND METHODS

Bacterial isolates. A retrospective multicenter study focusing on the prevalence of BSI-causing

Gram-negative pathogens was performed in five tertiary-care hospitals located in Beijing, China,

including Chinese PLA General Hospital (hospital A), 302nd Hospital of China (hospital B), PLA

Army General Hospital (hospital C), Navy General Hospital, PLA (hospital D) and Air Force

General hospital, PLA (hospital E). A total of 219 non-repetitive clinical BSIKpn isolates were

collected from Dec. 2013 to Dec. 2014. All clinical isolates were identified by VITEK® MS

(bioMérieux SA, Marcy-l'Etoile, France). Escherichia coli ATCC 25922 was used as the quality

control strain for antimicrobial susceptibility test. The Salmonella ser. Braenderup strain H9812 was

used as a reference standard for pulsed-field gel electrophoresis (PFGE). No ethical approval was

obtained for using the clinical samples, because these samples were collected during routine

bacteriologic analyses in public hospitals, and the data were anonymously analyzed.
 Antimicrobial susceptibility test. The minimum inhibitory concentrations (MICs) of commonly

used antimicrobial agents (listed in Table 1) were measured by the agar dilution method. All

susceptibility results were interpreted according to the performance standards of 2016 CLSI [16].

PFGE and MLST analyses. PFGE with XbaI was performed for all clinical BSIKpn isolates as

previously described [17]. The PFGE patterns were analyzed by BIONUMERICS software (Applied

Maths NV, Sint-Martens-Latem, Belgium) using the dice similarity coefficient. Isolates were

considered as the same strain (PFGE type) if they possessed a genetic similarity of ≥95%. MLST was

carried out for all BSIKpn strains according to protocols available on the MLST websites

(http://www.pasteur.fr/recherche/genopole/PF8/mlst/Kpneumoniae). STs were clustered into groups

by eBURST v3.0 software (http://eburst.mlst.net/) to determine the clonal relationship of the isolates.

Determination of the HV phenotype. The HV phenotype was identified by a positive string test,

which was defined as the formation of a viscous string >5 mm in length when a colony grown

overnight on a blood agar plate at 37°C was stretched by a bacteriology inoculating loop [4].

Detection of capsular serotypes and HV-associated genes. Capsular serotypes K1, K2, K5, K20,

K54, K57, which have been described as the capsular serotypes of hvKP strains [10], were

determined by PCR detection of K serotype-specific alleles at wzx and wzy loci as described

previously [18]. magA is the serotype K1 wzy allele in BSIKpn [9-11, 18]. rmpA and rmpA2 genes

were detected by PCR with specific primer sets [18].

Molecular detection of carbapenemase genes. Carbapenemase genes, including blaKPC, blaIMP,

blaAIM, blaDIM, blaGIM, blaSIM, blaVIM, blaSPM, blaNDM and blaOX-48, were screened for

carbapenem-non-susceptible BSIKpn as previously described [19].

RESULTS

Prevalence of BSIKpn. A total of 857 BSI-causing Gram-negative bacilli were collected from

Dec. 2013 to Dec. 2014 from five tertiary-care hospitals located in Beijing, China. E. coli, K.

pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacter cloacae and other

Gram-negative bacilli accounted for 40.72% (349/857), 25.55% (219/857), 11.32% (97/857), 9.45%

(81/857), 5.02% (43/857) and 7.93% (68/857) of all the Gram-negative bacilli BSI episodes,

respectively. Among 219 non-repetitive BSIKpn isolates, 69 of them were obtained from hospital A,

while 65, 28, 28 and 29 isolates were collected from hospital B, C, D and E, respectively.

Genetic relatedness of BSIKpn. The 219 non-repetitive BSIKpn isolates were categorized into

203 types (strains) which had a unique PFGE pattern. Twelve types contained two BSIKpn isolates,

two types were composed of three isolates, while other 189 types contained only one isolate. Isolates

with same PFGE type were considered as the same strain. Therefore, a total of 203 BSIKpn strains

with no genetic relationship were further analyzed.

MLST. A total of 105 different STs were identified from 203 BSIKpn strains. Figure 1shows that

the currently known 89 STs were clustered into 8 clonal complexes (CC) and 66 singletons. Overall,

ST11 and ST23 were the predominant clones, and they were detected from 14 strains (6.9%),

respectively, followed by ST412 (n = 13), ST37 (n = 9), ST65 (n = 7), ST15 (n = 6), ST86 (n = 6),

ST592 (n = 5) and ST29 (n = 4). Six STs (ST36, ST48, ST147, ST375, ST395 and ST462) contained

three strains, and 16 STs (ST12, ST17, ST25, ST35, ST60, ST101, ST200, ST231, ST290, ST367,

ST420, ST722, ST896, ST927, ST1373 and ST2374) were responsible for only two strains. The rest

of the 74 STs including 16 novel STs which have not been submitted to the MLST databases

contained only a single strain.

Antimicrobial susceptibility and prevalence of carbapenemase genes. Table 1 lists the

antimicrobial susceptibility patterns of the BSIKpn strains. Approximately 8.4% (17/203) of BSIKpn

strains exhibited carbapenem-resistant phenotype. Fourteen of them produced KPC, while three

carbapenem-resistant BSIKpn strains did not produced any commonly known carbapenemase genes

(Table 2).

Capsular serotypes and HV-associated genes. The majority (70.4%, 143/203) of BSIKpn strains

were associated with non-K1/K2/K5/K20/K54/K57 serotypes. Only 16.7% (34/203) of BSIKpn

strains had K1/K2 serotypes, followed by K54 (2.96%, 6/203), K57 (2.46%, 5/203), K20 (1.97%,
4/203) and K5 (0.49%, 1/203). About one-third (32.5%, 66/203) of the BSIKpn strains exhibited HV

phenotype. Table 3 lists the prevalence of capsular serotypes and HV-associated genes among

BSIKpn strains with and without HV phenotype. The HV positive BSIKpn isolates exhibited high

level of clonal diversity, and only two, five and eight isolates belonged to ST23, ST65 and ST412,

respectively. The majority (10 of 12) of BSIKpn isolates with K1 serotype belonged ST23, and only

seven (33.3%) K2 BSIKpn isolates were ST65.

DISCUSSION

In this multi-center study, we described molecular characterization of BSIKpn strains collected

from five tertiary-care hospitals in Beijing, China. PFGE results showed that 86.3% (189/203) of

clinical BSIKpn isolates had a unique pattern, indicating widespread diversification of BSIKpn

strains in this area. This high level of genetic diversity of clinical BSIKpn isolates also suggested that

no outbreak of BSIKpn strain occurred, and no BSIKpn, including carbapenemase-producing strain,

possessed the advantage of transmission in the process of causing BSI. Substantial differences in

clonal structure of BSIKpn strains were also confirmed in this study. The predominant clones (ST11

and ST23) accounted for only 6.9% of all BSIKpn strains, respectively, while a large proportion of

strains with other STs were detected (Figure 1). This high level of clonal diversity has also been

found among BSIKpn strains from other areas [4, 7, 15], suggesting the common epidemiological

characteristics of the BSIKpn strains across different region. Therefore, further studies on a larger

scale are necessary to better understand the prevalence and clonal structure of clinical BSIKpn

isolates.

The BSIKpn strains exhibited low resistance to commonly used antimicrobial agents (Table 1).

In this study, the third generation cephalosporin resistance in BSIKpn was close to that of the United

States (about 29%), but it was lower than that of other studies in China (about 34%) [2]. About 8.4%

(17/203) of BSIKpn strains exhibited carbapenem-resistant phenotype, and the majority of them

produced KPC (Table 2). About half (8/17) of them belonged to ST11, which emerged as the major
clone responsible for the prevalence of carbapenemase-producing K. pneumoniae in this area [17, 20].

However, we also detected several non-ST11 BSIKpn clones (Table 2), suggesting that the high level

of clonal diversity of carbapenem-resistant BSIKpn was significantly different from the previous

molecular epidemiological characteristics of carbapenemase-producing K. pneumoniae [17, 20]. As a

heterogeneous bacterial pathogen causing various infections, clones of K. pneumoniae are adept at

acquiring additional genetic traits and becoming either hypervirulent or antibiotic resistant. A few

studies have demonstrated that both carbapenem resistance and severity of illnesses are associated

with increased mortality [14, 21], adding urgency to better understand and to prevent the spread of

carbapenem-resistant BSIKpn.

In this study, we detected six serotypes (K1, K2, K5, K20 K54 and K57), which were

considered as highly virulent phenotypes associated with severe infections in humans among the 77

described capsular (K) types of the serotyping scheme [10, 22]. The majority (70.4%) of BSIKpn

strains was associated with non-K1/K2/K5/K20/K54/K57 serotypes, and only 16.7% of BSIKpn

strains had K1/K2 serotypes. Meanwhile, the hypervirulent clones (such as CC23K1 and CC65K2)

were infrequent among BSIKpn strains. Although the K1/K2 capsular serotypes are very common in

some hvKP strains, some studies have revealed that a considerable proportion of the hvKP strains

may have a non-K1/K2 serotype [10, 18, 23, 24], and the majority of non-K1/K2 serotypes are K5,

K20, K54 and K57 [18, 24]. However, it has been confirmed from cases of respiratory infections and

septicemia that pyogenic liver abscess-causing K1 isolates are genetically distinct from other K1

isolates, and the pathogenic potential of liver abscess K1 isolates resides in their genomic

background and surface antigens, but not in capsular serotype [22, 25, 26]. our results also indicated

that serotypes other than K1/K2/K5/K20/K54/K57 also played important roles in the BSI.

The HV phenotype of K. pneumoniae may be associated with the development of a distinctive

invasive syndrome, such as liver abscess, meningitis or endophthalmitis [27-29]. However, only

one-third (32.5%, 66/203) of the BSIKpn strains exhibited HV phenotype in this study, which was

similar to the data from other studies in the same region (31.4-42.4%) [4, 6], but much higher than
the data of other areas (5.4-12.9%) [5, 7]. The prevalence of each serotype exhibited no significant

difference between groups with and without HV phenotype (Table 3). The virulent serotype K1 even

presented lower prevalence among hypermucoviscous strains (1.5% vs. 8%). All these data suggested

that there was no relationship between the capsular type and HV phenotype among clinical BSIKpn

strains. It has been confirmed that an HV phenotype, which has been used as phenotypic

characteristic for identification of hvKP strain, is not enough to recognize those virulent clones [7, 13,

29, 30]. Our results also suggested that factors other than HV phenotype may be required for the

systemic dissemination of K. pneumoniae. Furthermore, the phenotypic string test has poor positive

predictive value and test specificity for hvKP identification [31]. Altogether, it is very unreliable to

determine hvKP strains only by detecting HV phenotype. In other words, the phenotype should not

be used as the unique feature for the identification of hvKP. The prevalence of rmpA gene showed

significant difference between two groups (Table 3), suggesting that the rmpA was a predominant

factor in determining whether or not a BSIKpn strain had an HV phenotype. However, some strains

were positive for rmpA but negative for HV phenotype (Table 3). It has been pointed out that the role

of RmpA/RmpA2 and/or the HV phenotype are difficult to interpret [13, 29, 30]. Further efforts are

required to understand the underlying mechanisms of differential prevalence of BSIKpn and related

determinants.

However, the present study had the following limitations. First, the main drawback of the study

is the paucity of information on the clinical characterization of the patients. Second, other resistant

and virulent determinants of BSIKpn (such as AllS, Kfu, Aerobactin and RepA) did not been

unexplored.

In conclusion, the molecular characterizations of 219 non-duplicated BSIKpn isolates were

analyzed in this study. The BSIKpn isolates exhibited high level of genetic diversity and low

resistance to commonly used antimicrobial agents. Approximately 8.4% (17/203) of BSIKpn strains

exhibited resistance to carbapenems. ST11 and ST23 were the predominant clones, but only 13.8%.

There were 74 STs containing only a single strain. Only 16.7% of BSIKpn strains had K1/K2
serotypes, while 32.5% of the BSIKpn strains exhibited HV phenotype. Therefore, The specific

capsular serotype and HV phenotype were not the major features of BSIKpn strains from China.

DECLARATIONS

Funding: This study was financially supported by the Special Key Project of Biosafety Technologies

for the National Major Research & Development Program of China (2017YFC1200800).

Ethical Approval: No ethical approval was needed.

Competing Interests: None declared

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Figure 1. The eBURST diagram of MLST. Lines connect single locus variants. The relative size of

the circles indicates the abundance of each ST.

 Table 1. Susceptibility to common-used antimicrobial agents of BSIKpn.

Antimicrobial agents Resistance (%) MIC50 (mg/L) MIC90 (mg/L)

Ampicillin 98.03 128 >256
Cefotaxime 29.68 1 128
Ceftazidime 25.12 0.5 64
Cefepime 21.18 <0.125 32
Cefoperazone/sulbactam N/A 8/4 128/8
Piperacillin/tazobactam 11.82 8/4 128/4
Imipenem 7.39 0.25 0.5
Meropenem 8.37 <0.06 0.25
Amikacin 10.84 2 64
Ciprofloxacin 26.6 <0.06 64
Levofloxzcin 22.67 0.5 32
N/A: Not Applicable

Table 2. Characteristics of carbapenem-resistant BSIKpn.

Capsu MIC (mg/L)
Stri
le K Carbapene rm ma
No. ST ng A F S
Seroty mases pA gA CT C PT IP ME A CI LE
test M E C
pe X AZ Z M M K P V
P P F
K301 ST13 >25
Other* KPC + + - 6
128 128 32 512 512 8 8 4 4 0.5
004 73
K301 Oth >25
ST37 KPC - - - 6
256 256 64 512 512 8 8 4 2 1
007 er
K301 ST39 Oth >25 >25 >51 >51 25
KPC + + - 6 6
128 64
2 2
32 128 4
6
256
012 5 er
K301 Oth >25 >25 >25 >51 >51
ST11 KPC - + - 6 6 6
128
2 2
32 128 4 64 32
035 er
K301 ST92 Oth >25 >51 >51
Undetected - - - 6
32 32 32
2 2
4 8 4 64 64
036 7 er
K301 ST13 Oth >25 >25 >25 >51 >51
KPC - - - 6 6 6
64
2 2
4 32 4 0.5 1
038 06 er
K301 Oth >25
ST37 KPC + - - 6
32 8 4 256 256 2 4 2 0.5 1
040 er
K302 ST14 Oth - >25
KPC - - 6
64 32 16 256 512 8 8 8 32 32
028 7 er
K302
ST8
Oth
Undetected - - -
>25
256 256 256 128 32 0.5 4 2 1 4
059 er 6
K303 Oth - >25 >25 >5 51
ST11 KPC - - 6 6
256 256 128 64 16 16
12 2
256
008 er
K303 Oth - >25 >25 >51 >51
ST11 KPC - - 6 6
256 128
2 2
32 128 32 32 32
010 er
K303
ST23
Oth
Undetected - - -
>25
32 32 16 32 256 16 64
>5 >3
16
016 er 6 12 2
K308 Oth - >25 >25
ST11 KPC - - 6 6
128 64 512 512 8 64 4 32 32
014 er
K311 Oth >25 >51 >5
ST11 KPC + + - 6
128 32 128
2
512 16 128
12
32 32
001 er
K311 Oth >25 >5
ST11 KPC - - - 6
64 32 128 512 512 4 128
12
32 32
003 er
K311 Oth >25 >51 51
ST11 KPC - - - 6
32 32 128
2
512 16 128
2
32 32
006 er
K311 Oth >25 >25 >51 >5
ST11
er
KPC - - - 6 6
64 128
2
512 16 128
12
32 32
007
*: non-K1/K2/K5/K20/K54/K57
 Table 3. Characteristics of BSIKpn with different HV phenotypes

No. of positive isolates (%)

String test

magA+ rmpA+ K1/K2 K1 K2 K5 K20 K54 K57 Other

Positive
1 (1.5) 56 (84.8) 12 (18.2) 1 (1.5) 11 (16.7) 0 (0) 2 (3) 2 (3) 3 (4.5) 47 (71.2)
(n=66)

Negative
11 (8) 11 (8.03) 22 (16.1) 11 (8) 10 (7.3) 1 (0.7) 2 (1.5) 4 (2.9) 2 (1.5) 96 (70.1)
(n=137)