International Journal of Infectious Diseases 37 (2015) 107–112
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International Journal of Infectious Diseases journal homepage: www.elsevier.com/locate/ijid
Clinical and molecular characteristics of multi-clone carbapenem-resistant hypervirulent (hypermucoviscous) Klebsiella pneumoniae isolates in a tertiary hospital in Beijing, China Bei Yao a, Xiumei Xiao a, Fei Wang b, Lei Zhou a, Xiaowei Zhang a, Jie Zhang a,* a b
Department of Laboratory Medicine, Peking University Third Hospital, 49 North Garden Road, Haidian District, Beijing, 100191, China Department of Respiratory Medicine, Peking University Third Hospital, Haidian District, Beijing, China
A R T I C L E I N F O
Article history: Received 1 April 2015 Received in revised form 24 June 2015 Accepted 25 June 2015 Corresponding Editor: Eskild Petersen, Aarhus, Denmark. Keywords: Klebsiella pneumoniae Hypervirulent Hypermucoviscous Carbapenem-resistant
S U M M A R Y
Objectives: To provide the clinical and molecular characteristics of carbapenem-resistant hypervirulent (hypermucoviscous) Klebsiella pneumoniae (cr-hvKP) in a tertiary hospital in Beijing, China. Methods: The clinical characteristics of four patients with cr-hvKP isolates and 29 patients with carbapenem-resistant classic K. pneumoniae (cr-cKP) infections were analyzed retrospectively. The molecular characteristics of cr-hvKP and cr-cKP isolates were compared. Results: The KPC-2 gene was detected in all cr-hvKPs except for cr-hvKP6. The cr-hvKPs belonged to three sequence types (STs; ST25, ST65, and ST11), with three pulsed-field gel electrophoresis patterns (I, II, and III) and two capsular serotypes (K2 and non-typeable). Although cr-hvKP1–7 did not cause invasive clinical syndromes such as community-acquired liver abscess with or without extrahepatic complications, they were all nosocomially acquired; cr-hvKP1–5 were clones disseminated between patients A and B. Compared with cr-cKPs, pLVPK-related loci, repA, iroN, and K2 capsular serotype were more prevalent in cr-hvKPs, although no significant difference was found in clinical characteristics between patients with cr-hvKP and cr-cKP infection. Conclusions: The hypervirulent ST65 and ST25 K. pneumoniae, along with carbapenem-resistant clonal populations ST11, appear to have evolved into cr-hvKP strains. The evidence of bi-directional evolution and emergence of hospital-acquired multi-clone cr-hvKP indicates a confluence of virulence and carbapenem resistance, which might pose major problems in the management of K. pneumoniae infection. ß 2015 The Authors. Published by Elsevier Ltd on behalf of International Society for Infectious Diseases. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/bync-nd/4.0/).
1. Introduction Classic Klebsiella pneumoniae (cKP) is an important pathogen, causing various infections including pneumonia, bacteremia, septicemia, purulent infections, and urinary tract infections, all of which may occur in either a community or hospital setting.1 In 1986, a new and hypervirulent (hypermucoviscous) clinical variant of K. pneumoniae (hvKP) was identified in Taiwan, and hvKP strains are increasingly being reported in the USA, Argentina, Scandinavia, Korea, and Australia.2–7 hvKP may cause an invasive clinical syndrome, which has been defined as an infection presenting as community-acquired liver abscess with or without extrahepatic complications, such as endophthalmitis * Corresponding author. Tel.: +86 1082265719; fax: +86 1082265719. E-mail address: [email protected] (J. Zhang).
and necrotizing fasciitis.8 Further, hvKP infection has the ability to spread metastatically in the absence of host compromise.8 With regard to the bacterial phenotypic features, hvKP isolates differ from the classic strains in that colonies grown on agar plates appear hypermucoviscous, which contributes significantly to the virulence of K. pneumoniae in invasive infections and appears to be a surrogate marker for the presence of hvKP.9–11 Different to hvKP strains, most of which are resistant only to ampicillin,12 increasing resistance to carbapenems among cKP strains represents a real threat worldwide.13–16 Infections caused by carbapenem-resistant K. pneumoniae pose an increasing therapeutic dilemma because of their extended antibiotic resistance phenotypes and ability to disseminate rapidly. As a result, there are few available therapeutic options, and these strains are associated with high mortality rates.17,18
http://dx.doi.org/10.1016/j.ijid.2015.06.023 1201-9712/ß 2015 The Authors. Published by Elsevier Ltd on behalf of International Society for Infectious Diseases. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
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A more complicated clinical situation concerns the emergence of carbapenem-resistant hypervirulent (hypermucoviscous) K. pneumoniae (cr-hvKP) in America and Brazil, and the increasing antimicrobial resistance of hvKP in China, which are a cause for concern.12,19,20 To better understand the incidence and characteristics of cr-hvKP strains in China, multi-clone cr-hvKP isolates from a single medical center in China were characterized in the present study. 2. Materials and methods 2.1. Study setting and design Peking University Third Hospital (PUTH) is a universityaffiliated medical center with a 1498-bed capacity and 79 000 hospital admissions per year. It provides both primary and tertiary referral care to patients from Beijing, and to patients referred from various outside institutions throughout China. From 2010, the identification and antimicrobial susceptibility testing of all K. pneumoniae isolates from patients admitted to PUTH was performed using Vitek panels (bioMe´rieux, Marcy l’Etoile, France); isolates were stored at 80 8C. To identify cr-hvKP isolates for the current study, the computerized databases of the microbiology laboratories were searched retrospectively. All K. pneumoniae isolates exhibiting an imipenem or meropenem minimum inhibitory concentration (MIC) 4 mg/ml by Vitek 2 were examined for the hypermucoviscosity phenotype (described below). Non-repetitive putative cr-hvKP isolates were selected, with only the first isolate from each different anatomical site for each patient included in the study. If frozen samples contained both a cKP and an hvKP isolate, then the cKP isolate was also included in the study. For comparison of clinical and molecular characteristics, all the carbapenem-resistant classic K. pneumoniae (cr-cKP) strains isolated during the same study period were included. Different from hvKP, only the first cr-cKP strain from the first anatomical site of each patient was included in the study. The medical records of all patients colonized or infected with cr-hvKP and cr-cKP were reviewed retrospectively by an independent physician. For the purposes of this study, colonization implied that the patient had a sufficiently high concentration of cr-hvKP and cr-cKP at a site to allow detection, yet the organism was not causing any symptoms. The standard US Centers for Disease Control and Prevention definitions were used to define infections at different anatomical sites.21 For cr-hvKP infection cases, the treatment outcome was evaluated on day 7. Cure was defined as no clinical or laboratory evidence of infection. Improvement was defined as partial resolution of signs, symptoms, and laboratory parameters of infection. Cure and improvement were characterized as a successful outcome; all other outcomes were characterized as failures. For colonization cases, treatment outcomes were defined as ‘not assessable’. 2.2. Detection of the hypermucoviscosity phenotype The frozen carbapenem-resistant isolates were subcultured overnight on blood agar at 37 8C. Isolates were considered positive for the hypermucoviscosity phenotype if an inoculation loop touched to the surface of the colony generated a viscous string of 5 mm in length when pulled away from the colony.22 Carbapenemresistant K. pneumoniae strains with a positive string test were designated cr-hvKP. 2.3. Antimicrobial susceptibility testing Susceptibility of the isolates to 18 antimicrobial agents (ampicillin, ampicillin/sulbactam, piperacillin, piperacillin/
tazobactam, cefazolin, cefuroxime, cefuroxime axetil, cefotetan, ceftriaxone, cefepime, aztreonam, gentamicin, tobramycin, nitrofurantoin, trimethoprim/sulfamethoxazole, cefoxitin, ertapenem, and tigecycline) was examined using Vitek panels (bioMe´rieux), in accordance with the manufacturer’s instructions. For cr-hvKP, the Etest method was used for further determination of MICs for minocycline, amikacin, cefotaxime, ceftazidime, moxifloxacin, ciprofloxacin, levofloxacin, meropenem, and imipenem on Mueller–Hinton agar plates, in accordance with manufacturer’s instructions (bioMe´rieux). Results were interpreted according to the interpretive standards of the Clinical and Laboratory Standards Institute (CLSI).23 Breakpoints for tigecycline were as defined by the US Food and Drug Administration (susceptible, 2 mg/ml; resistant, 8 mg/ml).24 Escherichia coli ATCC 25922 was used as a quality control strain for susceptibility testing. 2.4. Modified Hodge test and PCR detection of carbapenemase genes The modified Hodge test (MHT) was performed for the detection of carbapenemases, as described previously.23 PCR detection and sequencing of carbapenemase genes (blaIMP, blaVIM, blaSPM, blaNDM, blaKPC, blaBIC, blaAIM, blaGIM, blaSIM, and blaDIM) were performed as described previously.25 For the blaKPC-positive strain, the subtype of KPC was identified.13 2.5. Capsular polysaccharide (CPS) typing and detection of virulence factors K1, K2, K5, K20, K54, and K57 capsular serotypes were identified by PCR, as described previously.26 The presence of genes encoding virulence factors (mrkD, kfuBC, fimH, uge, wabG, ureA, ycfM, entB, ybtS, iroN, and allS), pLVPK-related loci (terW– iutA–rmpA–silS), and repA was determined by PCR using primers documented previously.27–29 The first strain with a DNA sequence of the PCR product that was identical to the published sequence was selected as a positive control for the subsequent PCR experiments. 2.6. Multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE) analysis of cr-hvKP MLST was performed by amplifying and sequencing seven housekeeping genes according to protocols provided on the MLST website for K. pneumoniae (http://bigsdb.web.pasteur.fr/klebsiella/ klebsiella.html). Isolates were typed by PFGE of XbaI-digested total genomic DNA, and DNA patterns were interpreted according to Tenover et al.30,31 Strains were considered to be the same clone (type) if they showed 85% genetic identity, or fewer than four fragment differences in the PFGE profiles. 2.7. Statistical analysis Data were analyzed using the statistical package SPSS for Windows version 17.0 (SPSS Inc., Chicago, IL, USA). The Chi-square test or Fisher’s exact test was used for categorical variables. All statistical tests were two-tailed and a p-value of 0.05 was considered to be statistically significant. 3. Results 3.1. Clinical characteristics of patients with cr-hvKP infection From January 2010 to August 2014, a total of four patients admitted to PUTH tested positive for cr-hvKP and 53 tested positive for cr-cKP. Seven non-replicate cr-hvKP isolates were obtained from patients A (cr-hvKP1, cr-hvKP4), B
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(cr-hvKP2, cr-hvKP3, cr-hvKP5), C (cr-hvKP6), and D (cr-hvKP7). Clinical characteristics of these four patients are shown in Table 1. A non-hypermucoviscous K. pneumoniae was also isolated from the sample containing cr-hvKP6. The four patients had multiple co-morbidities and had required the use of invasive devices. For patient A, cr-hvKP isolates 1 and 4 were isolated from tracheal secretion and urine, respectively, and were not the cause of clinical disease. As a result, patient A was not treated with antimicrobials. Isolates cr-hvKP2 and cr-hvKP3 were the cause of a urinary tract infection and secondary bloodstream infection, respectively, in patient B. In addition, crhvKP5 was isolated from the tracheal secretion of patient B, but was considered to be a colonizer only. cr-hvKP 6 and 7 were the cause of pneumonia in patients C and D, respectively. While patients B and C were treated successfully with antimicrobial drugs, patient D died from heart failure. Patient A remained persistently colonized with cr-hvKP, with positive results obtained from both urine and tracheal secretion specimens. All isolates were acquired nosocomially, with a median duration of hospitalization of 769 days (range 11–1171 days) prior to testing positive for the organism. 3.2. Antimicrobial susceptibility and the presence of carbapenemases of cr-hvKP The antimicrobial susceptibility testing results for cr-hvKP isolates 1–7 and cKP1 are summarized in Table 2. cr-hvKP isolates 1–5 had very similar susceptibility profiles for the 27 antimicrobials tested, with all five isolates showing sensitivity to cefepime, trimethoprim/sulfamethoxazole, gentamicin, amikacin, minocycline, tigecycline, and quinolones, while cr-hvKP7 was sensitive only to trimethoprim/sulfame. Although cr-hvKP6 and cKP1 were isolated from the same tracheal secretion sample, they exhibited different sensitivity profiles to piperacillin/tazobactam, ceftriaxone, cefepime, aztreonam, ceftazidime, trimethoprim/sulfame, minocycline, and carbapenems. Results of the carbapenemase assays showed that crhvKP isolates 1–5 and cr-hvKP7 were MHT-positive, with only the blaKPC-2 gene being detected.
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3.3. Molecular characteristics of cr-hvKP Capsular serotyping showed that cr-hvKP isolates 1–6 and cKP1 were serotype K2. cr-hvKP7 was non-typeable. Isolates cr-hvKP1– 5 from patients A and B produced identical PFGE profiles and were typed as ST65, suggesting a clonal origin. Isolates cr-hvKP6 and cKP1 from patient C also showed identical PFGE types, and both belonged to the clonal group ST25. Isolate cr-hvKP7 had a distinct PFGE profile and belonged to clonal group ST11 (Figure 1, Table 3). The presence/absence of virulence factors in each isolate is outlined in Table 3. Of the virulence genes tested, wabG, fimH, entB, uge, and ureA were present in all of the isolates. ycfM and mrkD were present in all cr-hvKP isolates, but were not amplified from cKP1. pLVPK-related loci and iroN were amplified from all isolates apart from cr-hvKP7. None of the isolates contained kfuBC, while ybtS was only detected in cr-hvKP7, and allS was only found in crhvKP6 and cKP1. 3.4. Comparison of the clinical and molecular characteristics between the cr-hvKP infection group and cr-cKP infection group Among the four cr-hvKPs, three strains were considered to be infection, and among the 53 cr-cKPs, 29 were considered to be infection. There were no differences in clinical characteristics between the cr-cKP infection group (n = 29) and the cr-hvKP infection group (n = 3) (Table 4). The virulence genes uge, wabG, fimH, entB, ybtS, kfuBC, mrkD, ycfM, ureA, and allS were distributed equally in the two groups, whereas pLVPK-related loci, repA, and iroN differed significantly between the two groups (p = 0.006). For the capsular serotypes, significant differences were found in K2 and non-typeable serotypes between the two groups (p 0.05). There were no differences in the carbapenemase genes between the groups, and only the KPC-2 gene was positive in both groups (Table 5). 4. Discussion In this study, ST65 K2, ST25 K2, and ST11 non-typeable cr-hvKP isolates from patients at PUTH were characterized; this appears to
Table 1 Clinical characteristics of the four patients with cr-hvKP-positive samples Patient
A
B
Age/sex
78/M
85/M
Comorbidities
RF, PKD, CVA, DU, HTN, CHC, UTI, PAF
COPD, RF, UTI, HTN, SE, CVA
Use of invasive devices
Bc, CVC, MV, TPN, Trac, NFT
Bc, CVC, MV, TPN, Trac, NFT
C
68/M
RF, CVA, HTN, SE
Bc, NFT
D
91/F
RF, COPD, colon CA, PE,CAD
Bc, TPN, MV, Trac, CVC, NFT
Date of first isolation at the different anatomical sites (infection/ colonization)
Antimicrobial therapy
Treatment outcome (final outcome)
Before ID/AST
After ID/AST
2/4/2013, urine (colonization); cr-hvKP1
None
None
1/20/2014, tracheal secretion (colonization); cr-hvKP4 1/7/2014, urine (UTI); cr-hvKP2
None
None
IPM
BIN, IPM, ISP
1/8/2014, blood (secondary BSI); cr-hvKP3 2/17/2014, tracheal secretion (colonization); cr-hvKP5 3/26/2014, tracheal secretion (pneumonia); cr-hvKP6; cKP1 8/28/2014, tracheal secretion (pneumonia); cr-hvKP7
IPM, ISP
BIN, IPM, ISP
None
None
IPM, VA
SCF, ISP
Success (discharge)
SCF
ETP
Failure (death); died from heart failure
Not assessable
Success for UTI and BSI, not assessable for tracheal secretion isolate
Bc, bladder catheter; BIN, bladder irrigation with nitrofurazone solution (0.02%); BSI, bloodstream infection; CA, carcinoma; CAD, coronary artery disease; CHC, chronic hepatitis C; cKP, classic Klebsiella pneumoniae; COPD, chronic obstructive pulmonary disease; cr-hvKP, carbapenem-resistant hypervirulent (hypermucoviscous) Klebsiella pneumoniae; CVA, cerebrovascular accident; CVC, central vascular catheter; DU, duodenal ulcer; ETP, ertapenem; F, female; HTN, hypertension; ID/AST, identification/ antimicrobial susceptibility; IPM, imipenem; ISP, isepamicin; M, male; MV, mechanical ventilation; NFT, nasogastric feeding tube; PAF, paroxysmal atrial fibrillation; PE, pleural effusion; PKD, Parkinson’s disease; RF, respiratory failure; SCF, cefoperazone–sulbactam; SE, secondary epilepsy; TPN, total parenteral nutrition; Trac, tracheostomy; UTI, urinary tract infection; VA, vancomycin.
AK, amikacin; AMP, ampicillin; ATM, aztreonam; CI, ciprofloxacin; cKP, classic Klebsiella pneumoniae; cr-hvKP, carbapenem-resistant hypervirulent (hypermucoviscous) Klebsiella pneumoniae; CRO, ceftriaxone; CT, cefotaxime; CTT, cefotetan; CXA, cefuroxime axetil; CXM, cefuroxime; CZO, cefazolin; ETP, ertapenem; FEP, cefepime; FOX, cefoxitin; GEN, gentamicin; IP, imipenem; LE, levofloxacin; MC, minocycline; MIC, minimum inhibitory concentration; MP, meropenem; MX, moxifloxacin; NIT, nitrofurantoin; PIP, piperacillin; SAM, ampicillin/sulbactam; SXT, trimethoprim/sulfamethoxazole; TGC, tigecycline; TOB, tobramycin; TZ, ceftazidime; TZP, piperacillin/tazobactam.
>32 >32 >32 >32 >32 >32 >32 3 >32 >32 >32 >32 >32 >32 >32 0.75 0.5 3 24 >32 192 3 3 2 0.5 3 24 >32 32 4 3 2 0.5 3 24 >32 128 3 3 2 0.5 3 24 >32 192 3 3 2 0.5 3 24 >32 96 4 3 2 0.5 4 4 >32 >256 0.25 0.25 0.19 8 >32 >256 >32 >256 >32 >32 32 0.5 12 4 >32 48 0.75 0.75 0.75 1/19 8 8 1/19 8 8 1/19 8 8 1/19 8 8 1/19 8 8 1/19 8 8 2/38 64 8 4/76 8 0.25 16 64 16 64 16 64 16 64 16 64 1 256 16 512 1 512 1 1 1 1 1 1 16 1 64 64 64 64 64 64 64 4 64 16 2 64 16 2 64 16 2 64 16 2 64 16 2 64 64 16 64 64 64 64 16 1 64 64 64 64 64 64 64 64 64 64 64 64 64 64 64 64 64 64 64 64 64 64 64 64 128/4 128/4 128/4 128/4 128/4 128/4 128/4 4/4 128 128 128 128 128 128 128 32 32 32 32 32 32 32 32 32 32 32 32 32 32 32 32 32 cr-hvKP1 cr-hvKP2 cr-hvKP3 cr-hvKP4 cr-hvKP5 cr-hvKP6 cr-hvKP7 cKP1
AMP SAM PIP
TZP
CZO CXM CXA CTT
CRO FEP
ATM GEN TOB NIT
SXT
FOX ETP
TGC
MC
AK
CT
TZ
MX
CI
LE
MP
IP
KPC KPC KPC KPC KPC KPC -
+ + + + +
Carbapenemase MHT gene Etest MIC (mg/l) Vitek 2 MIC (mg/l) Isolate
Table 2 Antimicrobial susceptibility, presence of carbapenemase genes, and modified Hodge test (MHT) results for cr-hvKP1–7 and cKP1
+
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Figure 1. Pulsed-field gel electrophoresis profiles of Klebsiella pneumoniae isolates following digestion with XbaI. Lanes 1–3, lane 6, and lane 8 correspond to cr-hvKP1– 5. Lanes 4 and 5 correspond to cr-hvKP6 and cKP1. Lane 7 corresponds to cr-hvKP7.
be the first report of multi-clonal cr-hvKP isolates from a single medical center in China. The first reported cr-hvKP isolate belonged to clonal complex 65 (CC65), which includes ST65 and ST25, and has been identified as a virulent clone that mostly corresponds to capsular serotype K2.27 In the current study, crhvKP isolates 1–5 were classified as K2/ST65, and were shown to have the same PFGE type, demonstrating the potential spread of crhvKP1 from patient A to patient B. At the end of the study period, patient A, who was a long-term urinary catheterization patient, was still colonized with cr-hvKP1. This may explain the persistent colonization and potential for dissemination to patient B. The process of colonization and dissemination can be lengthy, yet isolates cr-hvKP1–5 all contained the same virulence factors, showing the stability of the virulence genes and the possibility of continued evolution. cr-hvKP1 had identical virulence factors to a previously reported non-cr-hvKP isolate, demonstrating that acquisition of KPCs does not result in a loss of virulence.27,29,32,33 The KPC plasmid can be conjugated and retained by virulent K2/ ST65 K. pneumoniae while still maintaining a high serum resistance and murine lethality.34 The present study has confirmed for the first time that a KPC gene has disseminated into hypervirulent K. pneumoniae K2/ST65, and that the drug resistance and virulence can be maintained long-term. Isolate cr-hvKP6 was also found to belong to CC65 and to have virulence factors consistent with the previous report discussed above.27,29,32 However, it was determined that cKP1, also K2/ST25 with the same PFGE profile as cr-hvKP6, was isolated from the same sample. Both isolates were negative for carbapenemase genes and the MHT. Based on these data, it is speculated that crhvKP6 may have evolved from cKP1, although a whole genome comparative analysis is required to further evaluate this possibility.
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Table 3 Molecular characteristics of cr-hvKP1–7 and cKP1 Isolate
K type/ST
PFGE type
pLVPK-related loci terW–iutA–rmpA–silS
repA
uge
wabG
fimH
entB
iroN
cr-hvKP1 cr-hvKP2 cr-hvKP3 cr-hvKP4 cr-hvKP5 cr-hvKP6 cr-hvKP7 cKP1
K2/ST65 K2/ST65 K2/ST65 K2/ST65 K2/ST65 K2/ST25 N/ST11 K2/ST25
I I I I I II III II
+ + + + + +
+ + + + + +
+ + + + + + + +
+ + + + + + + +
+ + + + + + + +
+ + + + + + + +
+ + + + + +
+ + + + + +
+ + + + + +
+ + + + + +
++++
+
ybtS
kfuBC
+
mrkD
ycfM
ureA
+ + + + + + +
+ + + + + + +
+ + + + + + + +
+
allS
+ +
cKP, classic Klebsiella pneumoniae; cr-hvKP, carbapenem-resistant hypervirulent (hypermucoviscous) Klebsiella pneumoniae; PFGE, pulsed-field gel electrophoresis; ST, sequence type.
Although ST11, associated with multi-drug resistance, has been demonstrated to be the prevalent clone associated with the spread of KPC-producing K. pneumoniae in China, there are no previous reports of hvKP belonging to ST11, let alone cr-hvKP.35 In China, isolate cr-hvKP7 identified in this study is the first ST11 clone that combines a KPC gene with the hyperviscous phenotype, and this was shown to be pan drug-resistant to all antimicrobials tested except trimethoprim/sulfame. Although the patient was never treated with tigecycline, cr-hvKP7 showed resistance to tigecycline in vitro, and this mechanism of resistance needs further investigation. The virulence of K. pneumoniae has been associated with clone rather than with serotype, which could explain the
Table 4 Comparison between the cr-hvKP group and cr-cKP group for clinical characteristics Characteristics
cr-cKP infection group (n = 29)
cr-hvKP infection group (n = 3)
p-Valuea
Age, >60 years Sex, n Male Female Hepatitis B virus Hepatitis C virus Alcoholic hepatitis Fatty liver Gallbladder stone Liver abscess Respiratory tract disorder Cardiovascular disease Diabetes Tuberculosis Pancreatitis Nephritis Non-hepatic malignancy Non-hepatic abscess Duodenal ulcer Parkinson’s disease Cerebrovascular accident Secondary epilepsy Hypertension Respiratory failure Thyroid disorder Use of invasive devices Nasogastric feeding tube Total parenteral nutrition Bladder catheter Endotracheal intubation Central vascular catheter Pleural effusion Mortality
25
3
1.000 0.552
11 18 1 0 0 1 3 0 6 21 7 1 4 3 4 1 0 2 9 2 17 12 1
2 1 0 0 0 0 0 0 2 1 0 0 0 0 1 0 0 0 2 2 2 3 0
1.000 NA NA 1.000 1.000 NA 0.147 0.224 1.000 1.000 1.000 1.000 0.410 1.000 NA 1.000 0.266 0.035 1.000 0.092 1.000
27 20 27 16 12 2 15
3 2 2 2 2 1 1
1.000 1.000 0.263 1.000 0.568 0.263 1.000
cr-cKP, carbapenem-resistant classic Klebsiella pneumoniae; cr-hvKP, carbapenemresistant hypervirulent (hypermucoviscous) Klebsiella pneumoniae; NA, not assessable. a cr-cKP vs. cr-hvKP.
difference in virulence factors between ST11 and ST25/ST65.27 The pLVPK-derived terW–iutA–rmpA–silS loci are significantly correlated with abscess formation and prevalence of K1 and K2 serotype strains,29,32 but these virulence loci have not previously been detected in ST11 K. pneumoniae. Compared with previously reported ST11 strains,20 cr-hvKP7 has more virulence factors, including the hypermucoviscosity phenotype.10 In contrast to a crhvKP isolate reported in Brazil, which did not contain either magA or rmpA,20 the hypermucoviscous phenotype of cr-hvKP7 persisted without decrease following several rounds of subculturing. Several reports have shown that aminoglycosides are effective for the clearance of urinary tract infections caused by aminoglycoside-susceptible carbapenem-resistant K. pneumoniae.36 In the present study, cr-hvKP isolates 2, 3, and 6 were treated successfully with a regimen that included isepamicin, indicating that aminoglycosides may also be effective in treating cr-hvKP. Although the four patients included in this study had a variety of underlying diseases and use of invasive devices, none had invasive liver abscess syndrome or extra-hepatic metastatic infections.8 Further
Table 5 Comparison between the cr-hvKP group and cr-cKP group for molecular characteristics
K serotype K1 K2 K5 K20 K54 K57 K non-typeable Virulence gene pLVPK-related loci repA uge wabG fimH entB iroN ybtS kfuBC mrkD ycfM ureA allS blaKPC Other carbapenemase genes
cr-cKP infection group (n = 29)
cr-hvKP infection group (n = 3)
p-Value
0 0 0 0 0 0 29
0 3 0 0 0 0 1
NA 0.000a NA NA NA NA 0.006a
0 0 29 29 29 29 0 21 0 25 29 29 0 28 0
2 2 3 3 3 3 2 1 0 3 3 3 1 3 0
0.006a 0.006a NA NA NA NA 0.006a 0.224 NA 1.000 NA NA 0.094 1.000 NA
cr-cKP, carbapenem-resistant classic Klebsiella pneumoniae; cr-hvKP, carbapenemresistant hypervirulent (hypermucoviscous) Klebsiella pneumoniae; NA, not assessable. a A p-value 0.05 was considered to be statistically significant.
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to these clinical observations, the ability of cr-hvKP to cause invasive infection should be evaluated further in vitro, as well as in phagocytosis assays and murine lethality tests.37 The length of stay prior to isolation of cr-hvKP in the current study differed markedly, with the shortest stay being 11 days (cr-hvKP6) and the longest being 1171 days (cr-hvKP5). Combined with differences in molecular characteristics and resistance patterns, it is concluded that cr-hvKP isolates 1, 6, and 7 represent three separate evolutionary lineages. The emergence of regional polyclonal crhvKP isolates is of significant concern and may herald the cr-hvKP era. It has been reported that patients with hvKP infection tend to have different underlying illnesses and more invasive infections compared to those with cKP.38 However, in the present study, no differences in clinical characteristics were found between cr-hvKP and cr-cKP infected patients. Reports on the virulence genes and K serotype of cr-cKP, let alone cr-hvKP, are scarce. The present study showed that pLVPK-related loci, iroN, and K2 serotype were more prevalent in cr-hvKP than in cr-cKP, but the significance of these differences needs to be studied further. In summary, this study provides the first clinical and molecular characterization of ST65, ST25, and ST11 cr-hvKP isolates in China. The clonal populations of invasive infection (ST65 and ST25) have evolved to become multidrug-resistant, while a related multidrugresistant clonal population (ST11) has evolved to become hypermucoviscous. The confluence of virulence and carbapenem resistance and the bi-directional evolution might pose major problems in the future for the management of K. pneumoniae infections. Conflict of interest/funding: None. References 1. Podschun R, Ullmann U. Klebsiella spp. as nosocomial pathogens: epidemiology, taxonomy, typing methods, and pathogenicity factors. Clin Microbiol Rev 1998;11:589–603. 2. Liu YC, Cheng DL, Lin CL. Klebsiella pneumoniae liver abscess associated with septic endophthalmitis. Arch Intern Med 1986;146:1913–6. 3. Buppajarntham S, Shah M, Junpaparp P. Tumor-like pyogenic liver abscess caused by Klebsiella pneumoniae in diabetes. Endocrine 2014;47:656–7. 4. Vila A, Cassata A, Pagella H, Amadio C, Yeh KM, Chang FY, et al. Appearance of Klebsiella pneumoniae liver abscess syndrome in Argentina: case report and review of molecular mechanisms of pathogenesis. Open Microbiol J 2011;5:107–13. 5. Gundestrup S, Struve C, Stahlhut SG, Hansen DS. First case of liver abscess in Scandinavia due to the international hypervirulent Klebsiella pneumoniae clone ST23. Open Microbiol J 2014;8:22–4. 6. Hyun JI, Kim YJ, Jeon YH, Kim SI, Park YJ, Kang MW, et al. A case of ventriculitis associated with renal abscess caused by serotype K1 Klebsiella pneumoniae. Infect Chemother 2014;46:120–4. 7. Vandevelde A, Stepanovic B. On a boat: a case in Australia of endophthalmitis and pyogenic liver, prostatic, and lung abscesses in a previously well patient due to Klebsiella pneumoniae. Case Rep Infect Dis 2014;2014:137248. 8. Siu LK, Yeh KM, Lin JC, Fung CP, Chang FY. Klebsiella pneumoniae liver abscess: a new invasive syndrome. Lancet Infect Dis 2012;12:881–7. 9. Fang CT, Chuang YP, Shun CT, Chang SC, Wang JT. A novel virulence gene in Klebsiella pneumoniae strains causing primary liver abscess and septic metastatic complications. J Exp Med 2004;199:697–705. 10. Kawai T. Hypermucoviscosity: an extremely sticky phenotype of Klebsiella pneumoniae associated with emerging destructive tissue abscess syndrome. Clin Infect Dis 2006;42:1359–61. 11. Pomakova DK, Hsiao CB, Beanan JM, Olson R, MacDonald U, Keynan Y, et al. Clinical and phenotypic differences between classic and hypervirulent Klebsiella pneumonia: an emerging and under-recognized pathogenic variant. Eur J Clin Microbiol Infect Dis 2012;31:981–9. 12. Li W, Sun G, Yu Y, Li N, Chen M, Jin R, et al. Increasing occurrence of antimicrobial-resistant hypervirulent (hypermucoviscous) Klebsiella pneumoniae isolates in China. Clin Infect Dis 2014;58:225–32. 13. Yigit H, Queenan AM, Anderson GJ, Domenech-Sanchez A, Biddle JW, Steward CD, et al. Novel carbapenem-hydrolyzing beta-lactamase, KPC-1, from a carbapenem-resistant strain of Klebsiella pneumoniae. Antimicrob Agents Chemother 2001;45:1151–61.
14. Villegas MV, Lolans K, Correa A, Suarez CJ, Lopez JA, Vallejo M, Quinn JP, Colombian Nosocomial Resistance Study Group. First detection of the plasmid-mediated class A carbapenemase KPC-2 in clinical isolates of Klebsiella pneumoniae from South America. Antimicrob Agents Chemother 2006;50: 2880–2. 15. Leavitt A, Navon-Venezia S, Chmelnitsky I, Schwaber MJ, Carmeli Y. Emergence of KPC-2 and KPC-3 in carbapenem-resistant Klebsiella pneumoniae strains in an Israeli hospital. Antimicrob Agents Chemother 2007;51:3026–9. 16. Wei ZQ, Du XX, Yu YS, Shen P, Chen YG, Li LJ. Plasmid-mediated KPC-2 in a Klebsiella pneumoniae isolate from China. Antimicrob Agents Chemother 2007;51:763–5. 17. Carmeli Y, Akova M, Cornaglia G, Daikos GL, Garau J, Harbarth S, et al. Controlling the spread of carbapenemase-producing Gram-negatives: therapeutic approach and infection control. Clin Microbiol Infect 2010;16:102–11. 18. Zarkotou O, Pournaras S, Tselioti P, Dragoumanos V, Pitiriga V, Ranellou K, et al. Predictors of mortality in patients with bloodstream infections caused by KPCproducing Klebsiella pneumoniae and impact of appropriate antimicrobial treatment. Clin Microbiol Infect 2011;17:1798–803. 19. Cejas D, Fernandez Canigia L, Rincon Cruz G, Elena AX, Maldonado I, Gutkind GO, et al. First isolate of KPC-2-producing Klebsiella pneumoniae sequence type 23 from the Americas. J Clin Microbiol 2014;52:3483–5. 20. Andrade LN, Vitali L, Gaspar GG, Bellissimo-Rodrigues F, Martinez R, Darini AL. Expansion and evolution of a virulent, extensively drug-resistant (polymyxin Bresistant), QnrS1-, CTX-M-2-, and KPC-2-producing Klebsiella pneumoniae ST11 international high-risk clone. J Clin Microbiol 2014;52:2530–5. 21. Horan TC, Andrus M, Dudeck MA. CDC/NHSN surveillance definition of health care-associated infection and criteria for specific types of infections in the acute care setting. Am J Infect Control 2008;36:309–32. 22. Lee HC, Chuang YC, Yu WL, Lee NY, Chang CM, Ko NY, et al. Clinical implications of hypermucoviscosity phenotype in Klebsiella pneumoniae isolates: association with invasive syndrome in patients with community-acquired bacteraemia. J Intern Med 2006;259:606–14. 23. Clinical and Laboratory Standards Institute. Performance standards for antimicrobial susceptibility testing: twenty-second informational supplement. Document M100-S22. Wayne, PA: CLSI; 2012. 24. Pillar CM, Draghi DC, Dowzicky MJ, Sahm DF. In vitro activity of tigecycline against Gram-positive and Gram-negative pathogens as evaluated by broth microdilution and Etest. J Clin Microbiol 2008;46:2862–7. 25. Poirel L, Walsh TR, Cuvillier V, Nordmann P. Multiplex PCR for detection of acquired carbapenemase genes. Diagn Microbiol Infect Dis 2011;70:119–23. 26. Fang CT, Lai SY, Yi WC, Hsueh PR, Liu KL, Chang SC. Klebsiella pneumoniae genotype K1: an emerging pathogen that causes septic ocular or central nervous system complications from pyogenic liver abscess. Clin Infect Dis 2007;45:284–93. 27. Brisse S, Fevre C, Passet V, Issenhuth-Jeanjean S, Tournebize R, Diancourt L, et al. Virulent clones of Klebsiella pneumoniae: identification and evolutionary scenario based on genomic and phenotypic characterization. PLoS One 2009;4:e4982. 28. Lafeuille E, Decre D, Mahjoub-Messai F, Bidet P, Arlet G, Bingen E. OXA-48 carbapenemase-producing Klebsiella pneumoniae isolated from Libyan patients. Microb Drug Resist 2013;19:491–7. 29. Tang HL, Chiang MK, Liou WJ, Chen YT, Peng HL, Chiou CS, et al. Correlation between Klebsiella pneumoniae carrying pLVPK-derived loci and abscess formation. Eur J Clin Microbiol Infect Dis 2010;29:689–98. 30. Han H, Zhou H, Li H, Gao Y, Lu Z, Hu K, et al. Optimization of pulse-field gel electrophoresis for subtyping of Klebsiella pneumoniae. Int J Environ Res Public Health 2013;10:2720–31. 31. Tenover FC, Arbeit RD, Goering RV, Mickelsen PA, Murray BE, Persing DH, et al. Interpreting chromosomal DNA restriction patterns produced by pulsed-field gel electrophoresis: criteria for bacterial strain typing. J Clin Microbiol 1995;33:2233–9. 32. Luo Y, Wang Y, Ye L, Yang J. Molecular epidemiology and virulence factors of pyogenic liver abscess causing Klebsiella pneumoniae in China. Clin Microbiol Infect 2014;20:O818–24. 33. Jung SW, Chae HJ, Park YJ, Yu JK, Kim SY, Lee HK, et al. Microbiological and clinical characteristics of bacteraemia caused by the hypermucoviscosity phenotype of Klebsiella pneumoniae in Korea. Epidemiol Infect 2013;141:334–40. 34. Siu LK, Huang DB, Chiang T. Plasmid transferability of KPC into a virulent K2 serotype Klebsiella pneumoniae. BMC Infect Dis 2014;14:176. 35. Qi Y, Wei Z, Ji S, Du X, Shen P, Yu Y. ST11, the dominant clone of KPC-producing Klebsiella pneumoniae in China. J Antimicrob Chemother 2011;66:307–12. 36. Satlin MJ, Kubin CJ, Blumenthal JS, Cohen AB, Furuya EY, Wilson SJ, et al. Comparative effectiveness of aminoglycosides, polymyxin B, and tigecycline for clearance of carbapenem-resistant Klebsiella pneumoniae from urine. Antimicrob Agents Chemother 2011;55:5893–9. 37. Lin JC, Koh TH, Lee N, Fung CP, Chang FY, Tsai YK, et al. Genotypes and virulence in serotype K2 Klebsiella pneumoniae from liver abscess and non-infectious carriers in Hong Kong, Singapore and Taiwan. Gut Pathog 2014;6:21. 38. Liu YM, Li BB, Zhang YY, Zhang W, Shen H, Li H, et al. Clinical and molecular characteristics of emerging hypervirulent Klebsiella pneumoniae bloodstream infections in mainland China. Antimicrob Agents Chemother 2014;58:5379–85.
Contents lists available at ScienceDirect
International Journal of Infectious Diseases journal homepage: www.elsevier.com/locate/ijid
Clinical and molecular characteristics of multi-clone carbapenem-resistant hypervirulent (hypermucoviscous) Klebsiella pneumoniae isolates in a tertiary hospital in Beijing, China Bei Yao a, Xiumei Xiao a, Fei Wang b, Lei Zhou a, Xiaowei Zhang a, Jie Zhang a,* a b
Department of Laboratory Medicine, Peking University Third Hospital, 49 North Garden Road, Haidian District, Beijing, 100191, China Department of Respiratory Medicine, Peking University Third Hospital, Haidian District, Beijing, China
A R T I C L E I N F O
Article history: Received 1 April 2015 Received in revised form 24 June 2015 Accepted 25 June 2015 Corresponding Editor: Eskild Petersen, Aarhus, Denmark. Keywords: Klebsiella pneumoniae Hypervirulent Hypermucoviscous Carbapenem-resistant
S U M M A R Y
Objectives: To provide the clinical and molecular characteristics of carbapenem-resistant hypervirulent (hypermucoviscous) Klebsiella pneumoniae (cr-hvKP) in a tertiary hospital in Beijing, China. Methods: The clinical characteristics of four patients with cr-hvKP isolates and 29 patients with carbapenem-resistant classic K. pneumoniae (cr-cKP) infections were analyzed retrospectively. The molecular characteristics of cr-hvKP and cr-cKP isolates were compared. Results: The KPC-2 gene was detected in all cr-hvKPs except for cr-hvKP6. The cr-hvKPs belonged to three sequence types (STs; ST25, ST65, and ST11), with three pulsed-field gel electrophoresis patterns (I, II, and III) and two capsular serotypes (K2 and non-typeable). Although cr-hvKP1–7 did not cause invasive clinical syndromes such as community-acquired liver abscess with or without extrahepatic complications, they were all nosocomially acquired; cr-hvKP1–5 were clones disseminated between patients A and B. Compared with cr-cKPs, pLVPK-related loci, repA, iroN, and K2 capsular serotype were more prevalent in cr-hvKPs, although no significant difference was found in clinical characteristics between patients with cr-hvKP and cr-cKP infection. Conclusions: The hypervirulent ST65 and ST25 K. pneumoniae, along with carbapenem-resistant clonal populations ST11, appear to have evolved into cr-hvKP strains. The evidence of bi-directional evolution and emergence of hospital-acquired multi-clone cr-hvKP indicates a confluence of virulence and carbapenem resistance, which might pose major problems in the management of K. pneumoniae infection. ß 2015 The Authors. Published by Elsevier Ltd on behalf of International Society for Infectious Diseases. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/bync-nd/4.0/).
1. Introduction Classic Klebsiella pneumoniae (cKP) is an important pathogen, causing various infections including pneumonia, bacteremia, septicemia, purulent infections, and urinary tract infections, all of which may occur in either a community or hospital setting.1 In 1986, a new and hypervirulent (hypermucoviscous) clinical variant of K. pneumoniae (hvKP) was identified in Taiwan, and hvKP strains are increasingly being reported in the USA, Argentina, Scandinavia, Korea, and Australia.2–7 hvKP may cause an invasive clinical syndrome, which has been defined as an infection presenting as community-acquired liver abscess with or without extrahepatic complications, such as endophthalmitis * Corresponding author. Tel.: +86 1082265719; fax: +86 1082265719. E-mail address: [email protected] (J. Zhang).
and necrotizing fasciitis.8 Further, hvKP infection has the ability to spread metastatically in the absence of host compromise.8 With regard to the bacterial phenotypic features, hvKP isolates differ from the classic strains in that colonies grown on agar plates appear hypermucoviscous, which contributes significantly to the virulence of K. pneumoniae in invasive infections and appears to be a surrogate marker for the presence of hvKP.9–11 Different to hvKP strains, most of which are resistant only to ampicillin,12 increasing resistance to carbapenems among cKP strains represents a real threat worldwide.13–16 Infections caused by carbapenem-resistant K. pneumoniae pose an increasing therapeutic dilemma because of their extended antibiotic resistance phenotypes and ability to disseminate rapidly. As a result, there are few available therapeutic options, and these strains are associated with high mortality rates.17,18
http://dx.doi.org/10.1016/j.ijid.2015.06.023 1201-9712/ß 2015 The Authors. Published by Elsevier Ltd on behalf of International Society for Infectious Diseases. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
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A more complicated clinical situation concerns the emergence of carbapenem-resistant hypervirulent (hypermucoviscous) K. pneumoniae (cr-hvKP) in America and Brazil, and the increasing antimicrobial resistance of hvKP in China, which are a cause for concern.12,19,20 To better understand the incidence and characteristics of cr-hvKP strains in China, multi-clone cr-hvKP isolates from a single medical center in China were characterized in the present study. 2. Materials and methods 2.1. Study setting and design Peking University Third Hospital (PUTH) is a universityaffiliated medical center with a 1498-bed capacity and 79 000 hospital admissions per year. It provides both primary and tertiary referral care to patients from Beijing, and to patients referred from various outside institutions throughout China. From 2010, the identification and antimicrobial susceptibility testing of all K. pneumoniae isolates from patients admitted to PUTH was performed using Vitek panels (bioMe´rieux, Marcy l’Etoile, France); isolates were stored at 80 8C. To identify cr-hvKP isolates for the current study, the computerized databases of the microbiology laboratories were searched retrospectively. All K. pneumoniae isolates exhibiting an imipenem or meropenem minimum inhibitory concentration (MIC) 4 mg/ml by Vitek 2 were examined for the hypermucoviscosity phenotype (described below). Non-repetitive putative cr-hvKP isolates were selected, with only the first isolate from each different anatomical site for each patient included in the study. If frozen samples contained both a cKP and an hvKP isolate, then the cKP isolate was also included in the study. For comparison of clinical and molecular characteristics, all the carbapenem-resistant classic K. pneumoniae (cr-cKP) strains isolated during the same study period were included. Different from hvKP, only the first cr-cKP strain from the first anatomical site of each patient was included in the study. The medical records of all patients colonized or infected with cr-hvKP and cr-cKP were reviewed retrospectively by an independent physician. For the purposes of this study, colonization implied that the patient had a sufficiently high concentration of cr-hvKP and cr-cKP at a site to allow detection, yet the organism was not causing any symptoms. The standard US Centers for Disease Control and Prevention definitions were used to define infections at different anatomical sites.21 For cr-hvKP infection cases, the treatment outcome was evaluated on day 7. Cure was defined as no clinical or laboratory evidence of infection. Improvement was defined as partial resolution of signs, symptoms, and laboratory parameters of infection. Cure and improvement were characterized as a successful outcome; all other outcomes were characterized as failures. For colonization cases, treatment outcomes were defined as ‘not assessable’. 2.2. Detection of the hypermucoviscosity phenotype The frozen carbapenem-resistant isolates were subcultured overnight on blood agar at 37 8C. Isolates were considered positive for the hypermucoviscosity phenotype if an inoculation loop touched to the surface of the colony generated a viscous string of 5 mm in length when pulled away from the colony.22 Carbapenemresistant K. pneumoniae strains with a positive string test were designated cr-hvKP. 2.3. Antimicrobial susceptibility testing Susceptibility of the isolates to 18 antimicrobial agents (ampicillin, ampicillin/sulbactam, piperacillin, piperacillin/
tazobactam, cefazolin, cefuroxime, cefuroxime axetil, cefotetan, ceftriaxone, cefepime, aztreonam, gentamicin, tobramycin, nitrofurantoin, trimethoprim/sulfamethoxazole, cefoxitin, ertapenem, and tigecycline) was examined using Vitek panels (bioMe´rieux), in accordance with the manufacturer’s instructions. For cr-hvKP, the Etest method was used for further determination of MICs for minocycline, amikacin, cefotaxime, ceftazidime, moxifloxacin, ciprofloxacin, levofloxacin, meropenem, and imipenem on Mueller–Hinton agar plates, in accordance with manufacturer’s instructions (bioMe´rieux). Results were interpreted according to the interpretive standards of the Clinical and Laboratory Standards Institute (CLSI).23 Breakpoints for tigecycline were as defined by the US Food and Drug Administration (susceptible, 2 mg/ml; resistant, 8 mg/ml).24 Escherichia coli ATCC 25922 was used as a quality control strain for susceptibility testing. 2.4. Modified Hodge test and PCR detection of carbapenemase genes The modified Hodge test (MHT) was performed for the detection of carbapenemases, as described previously.23 PCR detection and sequencing of carbapenemase genes (blaIMP, blaVIM, blaSPM, blaNDM, blaKPC, blaBIC, blaAIM, blaGIM, blaSIM, and blaDIM) were performed as described previously.25 For the blaKPC-positive strain, the subtype of KPC was identified.13 2.5. Capsular polysaccharide (CPS) typing and detection of virulence factors K1, K2, K5, K20, K54, and K57 capsular serotypes were identified by PCR, as described previously.26 The presence of genes encoding virulence factors (mrkD, kfuBC, fimH, uge, wabG, ureA, ycfM, entB, ybtS, iroN, and allS), pLVPK-related loci (terW– iutA–rmpA–silS), and repA was determined by PCR using primers documented previously.27–29 The first strain with a DNA sequence of the PCR product that was identical to the published sequence was selected as a positive control for the subsequent PCR experiments. 2.6. Multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE) analysis of cr-hvKP MLST was performed by amplifying and sequencing seven housekeeping genes according to protocols provided on the MLST website for K. pneumoniae (http://bigsdb.web.pasteur.fr/klebsiella/ klebsiella.html). Isolates were typed by PFGE of XbaI-digested total genomic DNA, and DNA patterns were interpreted according to Tenover et al.30,31 Strains were considered to be the same clone (type) if they showed 85% genetic identity, or fewer than four fragment differences in the PFGE profiles. 2.7. Statistical analysis Data were analyzed using the statistical package SPSS for Windows version 17.0 (SPSS Inc., Chicago, IL, USA). The Chi-square test or Fisher’s exact test was used for categorical variables. All statistical tests were two-tailed and a p-value of 0.05 was considered to be statistically significant. 3. Results 3.1. Clinical characteristics of patients with cr-hvKP infection From January 2010 to August 2014, a total of four patients admitted to PUTH tested positive for cr-hvKP and 53 tested positive for cr-cKP. Seven non-replicate cr-hvKP isolates were obtained from patients A (cr-hvKP1, cr-hvKP4), B
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(cr-hvKP2, cr-hvKP3, cr-hvKP5), C (cr-hvKP6), and D (cr-hvKP7). Clinical characteristics of these four patients are shown in Table 1. A non-hypermucoviscous K. pneumoniae was also isolated from the sample containing cr-hvKP6. The four patients had multiple co-morbidities and had required the use of invasive devices. For patient A, cr-hvKP isolates 1 and 4 were isolated from tracheal secretion and urine, respectively, and were not the cause of clinical disease. As a result, patient A was not treated with antimicrobials. Isolates cr-hvKP2 and cr-hvKP3 were the cause of a urinary tract infection and secondary bloodstream infection, respectively, in patient B. In addition, crhvKP5 was isolated from the tracheal secretion of patient B, but was considered to be a colonizer only. cr-hvKP 6 and 7 were the cause of pneumonia in patients C and D, respectively. While patients B and C were treated successfully with antimicrobial drugs, patient D died from heart failure. Patient A remained persistently colonized with cr-hvKP, with positive results obtained from both urine and tracheal secretion specimens. All isolates were acquired nosocomially, with a median duration of hospitalization of 769 days (range 11–1171 days) prior to testing positive for the organism. 3.2. Antimicrobial susceptibility and the presence of carbapenemases of cr-hvKP The antimicrobial susceptibility testing results for cr-hvKP isolates 1–7 and cKP1 are summarized in Table 2. cr-hvKP isolates 1–5 had very similar susceptibility profiles for the 27 antimicrobials tested, with all five isolates showing sensitivity to cefepime, trimethoprim/sulfamethoxazole, gentamicin, amikacin, minocycline, tigecycline, and quinolones, while cr-hvKP7 was sensitive only to trimethoprim/sulfame. Although cr-hvKP6 and cKP1 were isolated from the same tracheal secretion sample, they exhibited different sensitivity profiles to piperacillin/tazobactam, ceftriaxone, cefepime, aztreonam, ceftazidime, trimethoprim/sulfame, minocycline, and carbapenems. Results of the carbapenemase assays showed that crhvKP isolates 1–5 and cr-hvKP7 were MHT-positive, with only the blaKPC-2 gene being detected.
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3.3. Molecular characteristics of cr-hvKP Capsular serotyping showed that cr-hvKP isolates 1–6 and cKP1 were serotype K2. cr-hvKP7 was non-typeable. Isolates cr-hvKP1– 5 from patients A and B produced identical PFGE profiles and were typed as ST65, suggesting a clonal origin. Isolates cr-hvKP6 and cKP1 from patient C also showed identical PFGE types, and both belonged to the clonal group ST25. Isolate cr-hvKP7 had a distinct PFGE profile and belonged to clonal group ST11 (Figure 1, Table 3). The presence/absence of virulence factors in each isolate is outlined in Table 3. Of the virulence genes tested, wabG, fimH, entB, uge, and ureA were present in all of the isolates. ycfM and mrkD were present in all cr-hvKP isolates, but were not amplified from cKP1. pLVPK-related loci and iroN were amplified from all isolates apart from cr-hvKP7. None of the isolates contained kfuBC, while ybtS was only detected in cr-hvKP7, and allS was only found in crhvKP6 and cKP1. 3.4. Comparison of the clinical and molecular characteristics between the cr-hvKP infection group and cr-cKP infection group Among the four cr-hvKPs, three strains were considered to be infection, and among the 53 cr-cKPs, 29 were considered to be infection. There were no differences in clinical characteristics between the cr-cKP infection group (n = 29) and the cr-hvKP infection group (n = 3) (Table 4). The virulence genes uge, wabG, fimH, entB, ybtS, kfuBC, mrkD, ycfM, ureA, and allS were distributed equally in the two groups, whereas pLVPK-related loci, repA, and iroN differed significantly between the two groups (p = 0.006). For the capsular serotypes, significant differences were found in K2 and non-typeable serotypes between the two groups (p 0.05). There were no differences in the carbapenemase genes between the groups, and only the KPC-2 gene was positive in both groups (Table 5). 4. Discussion In this study, ST65 K2, ST25 K2, and ST11 non-typeable cr-hvKP isolates from patients at PUTH were characterized; this appears to
Table 1 Clinical characteristics of the four patients with cr-hvKP-positive samples Patient
A
B
Age/sex
78/M
85/M
Comorbidities
RF, PKD, CVA, DU, HTN, CHC, UTI, PAF
COPD, RF, UTI, HTN, SE, CVA
Use of invasive devices
Bc, CVC, MV, TPN, Trac, NFT
Bc, CVC, MV, TPN, Trac, NFT
C
68/M
RF, CVA, HTN, SE
Bc, NFT
D
91/F
RF, COPD, colon CA, PE,CAD
Bc, TPN, MV, Trac, CVC, NFT
Date of first isolation at the different anatomical sites (infection/ colonization)
Antimicrobial therapy
Treatment outcome (final outcome)
Before ID/AST
After ID/AST
2/4/2013, urine (colonization); cr-hvKP1
None
None
1/20/2014, tracheal secretion (colonization); cr-hvKP4 1/7/2014, urine (UTI); cr-hvKP2
None
None
IPM
BIN, IPM, ISP
1/8/2014, blood (secondary BSI); cr-hvKP3 2/17/2014, tracheal secretion (colonization); cr-hvKP5 3/26/2014, tracheal secretion (pneumonia); cr-hvKP6; cKP1 8/28/2014, tracheal secretion (pneumonia); cr-hvKP7
IPM, ISP
BIN, IPM, ISP
None
None
IPM, VA
SCF, ISP
Success (discharge)
SCF
ETP
Failure (death); died from heart failure
Not assessable
Success for UTI and BSI, not assessable for tracheal secretion isolate
Bc, bladder catheter; BIN, bladder irrigation with nitrofurazone solution (0.02%); BSI, bloodstream infection; CA, carcinoma; CAD, coronary artery disease; CHC, chronic hepatitis C; cKP, classic Klebsiella pneumoniae; COPD, chronic obstructive pulmonary disease; cr-hvKP, carbapenem-resistant hypervirulent (hypermucoviscous) Klebsiella pneumoniae; CVA, cerebrovascular accident; CVC, central vascular catheter; DU, duodenal ulcer; ETP, ertapenem; F, female; HTN, hypertension; ID/AST, identification/ antimicrobial susceptibility; IPM, imipenem; ISP, isepamicin; M, male; MV, mechanical ventilation; NFT, nasogastric feeding tube; PAF, paroxysmal atrial fibrillation; PE, pleural effusion; PKD, Parkinson’s disease; RF, respiratory failure; SCF, cefoperazone–sulbactam; SE, secondary epilepsy; TPN, total parenteral nutrition; Trac, tracheostomy; UTI, urinary tract infection; VA, vancomycin.
AK, amikacin; AMP, ampicillin; ATM, aztreonam; CI, ciprofloxacin; cKP, classic Klebsiella pneumoniae; cr-hvKP, carbapenem-resistant hypervirulent (hypermucoviscous) Klebsiella pneumoniae; CRO, ceftriaxone; CT, cefotaxime; CTT, cefotetan; CXA, cefuroxime axetil; CXM, cefuroxime; CZO, cefazolin; ETP, ertapenem; FEP, cefepime; FOX, cefoxitin; GEN, gentamicin; IP, imipenem; LE, levofloxacin; MC, minocycline; MIC, minimum inhibitory concentration; MP, meropenem; MX, moxifloxacin; NIT, nitrofurantoin; PIP, piperacillin; SAM, ampicillin/sulbactam; SXT, trimethoprim/sulfamethoxazole; TGC, tigecycline; TOB, tobramycin; TZ, ceftazidime; TZP, piperacillin/tazobactam.
>32 >32 >32 >32 >32 >32 >32 3 >32 >32 >32 >32 >32 >32 >32 0.75 0.5 3 24 >32 192 3 3 2 0.5 3 24 >32 32 4 3 2 0.5 3 24 >32 128 3 3 2 0.5 3 24 >32 192 3 3 2 0.5 3 24 >32 96 4 3 2 0.5 4 4 >32 >256 0.25 0.25 0.19 8 >32 >256 >32 >256 >32 >32 32 0.5 12 4 >32 48 0.75 0.75 0.75 1/19 8 8 1/19 8 8 1/19 8 8 1/19 8 8 1/19 8 8 1/19 8 8 2/38 64 8 4/76 8 0.25 16 64 16 64 16 64 16 64 16 64 1 256 16 512 1 512 1 1 1 1 1 1 16 1 64 64 64 64 64 64 64 4 64 16 2 64 16 2 64 16 2 64 16 2 64 16 2 64 64 16 64 64 64 64 16 1 64 64 64 64 64 64 64 64 64 64 64 64 64 64 64 64 64 64 64 64 64 64 64 64 128/4 128/4 128/4 128/4 128/4 128/4 128/4 4/4 128 128 128 128 128 128 128 32 32 32 32 32 32 32 32 32 32 32 32 32 32 32 32 32 cr-hvKP1 cr-hvKP2 cr-hvKP3 cr-hvKP4 cr-hvKP5 cr-hvKP6 cr-hvKP7 cKP1
AMP SAM PIP
TZP
CZO CXM CXA CTT
CRO FEP
ATM GEN TOB NIT
SXT
FOX ETP
TGC
MC
AK
CT
TZ
MX
CI
LE
MP
IP
KPC KPC KPC KPC KPC KPC -
+ + + + +
Carbapenemase MHT gene Etest MIC (mg/l) Vitek 2 MIC (mg/l) Isolate
Table 2 Antimicrobial susceptibility, presence of carbapenemase genes, and modified Hodge test (MHT) results for cr-hvKP1–7 and cKP1
+
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110
Figure 1. Pulsed-field gel electrophoresis profiles of Klebsiella pneumoniae isolates following digestion with XbaI. Lanes 1–3, lane 6, and lane 8 correspond to cr-hvKP1– 5. Lanes 4 and 5 correspond to cr-hvKP6 and cKP1. Lane 7 corresponds to cr-hvKP7.
be the first report of multi-clonal cr-hvKP isolates from a single medical center in China. The first reported cr-hvKP isolate belonged to clonal complex 65 (CC65), which includes ST65 and ST25, and has been identified as a virulent clone that mostly corresponds to capsular serotype K2.27 In the current study, crhvKP isolates 1–5 were classified as K2/ST65, and were shown to have the same PFGE type, demonstrating the potential spread of crhvKP1 from patient A to patient B. At the end of the study period, patient A, who was a long-term urinary catheterization patient, was still colonized with cr-hvKP1. This may explain the persistent colonization and potential for dissemination to patient B. The process of colonization and dissemination can be lengthy, yet isolates cr-hvKP1–5 all contained the same virulence factors, showing the stability of the virulence genes and the possibility of continued evolution. cr-hvKP1 had identical virulence factors to a previously reported non-cr-hvKP isolate, demonstrating that acquisition of KPCs does not result in a loss of virulence.27,29,32,33 The KPC plasmid can be conjugated and retained by virulent K2/ ST65 K. pneumoniae while still maintaining a high serum resistance and murine lethality.34 The present study has confirmed for the first time that a KPC gene has disseminated into hypervirulent K. pneumoniae K2/ST65, and that the drug resistance and virulence can be maintained long-term. Isolate cr-hvKP6 was also found to belong to CC65 and to have virulence factors consistent with the previous report discussed above.27,29,32 However, it was determined that cKP1, also K2/ST25 with the same PFGE profile as cr-hvKP6, was isolated from the same sample. Both isolates were negative for carbapenemase genes and the MHT. Based on these data, it is speculated that crhvKP6 may have evolved from cKP1, although a whole genome comparative analysis is required to further evaluate this possibility.
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Table 3 Molecular characteristics of cr-hvKP1–7 and cKP1 Isolate
K type/ST
PFGE type
pLVPK-related loci terW–iutA–rmpA–silS
repA
uge
wabG
fimH
entB
iroN
cr-hvKP1 cr-hvKP2 cr-hvKP3 cr-hvKP4 cr-hvKP5 cr-hvKP6 cr-hvKP7 cKP1
K2/ST65 K2/ST65 K2/ST65 K2/ST65 K2/ST65 K2/ST25 N/ST11 K2/ST25
I I I I I II III II
+ + + + + +
+ + + + + +
+ + + + + + + +
+ + + + + + + +
+ + + + + + + +
+ + + + + + + +
+ + + + + +
+ + + + + +
+ + + + + +
+ + + + + +
++++
+
ybtS
kfuBC
+
mrkD
ycfM
ureA
+ + + + + + +
+ + + + + + +
+ + + + + + + +
+
allS
+ +
cKP, classic Klebsiella pneumoniae; cr-hvKP, carbapenem-resistant hypervirulent (hypermucoviscous) Klebsiella pneumoniae; PFGE, pulsed-field gel electrophoresis; ST, sequence type.
Although ST11, associated with multi-drug resistance, has been demonstrated to be the prevalent clone associated with the spread of KPC-producing K. pneumoniae in China, there are no previous reports of hvKP belonging to ST11, let alone cr-hvKP.35 In China, isolate cr-hvKP7 identified in this study is the first ST11 clone that combines a KPC gene with the hyperviscous phenotype, and this was shown to be pan drug-resistant to all antimicrobials tested except trimethoprim/sulfame. Although the patient was never treated with tigecycline, cr-hvKP7 showed resistance to tigecycline in vitro, and this mechanism of resistance needs further investigation. The virulence of K. pneumoniae has been associated with clone rather than with serotype, which could explain the
Table 4 Comparison between the cr-hvKP group and cr-cKP group for clinical characteristics Characteristics
cr-cKP infection group (n = 29)
cr-hvKP infection group (n = 3)
p-Valuea
Age, >60 years Sex, n Male Female Hepatitis B virus Hepatitis C virus Alcoholic hepatitis Fatty liver Gallbladder stone Liver abscess Respiratory tract disorder Cardiovascular disease Diabetes Tuberculosis Pancreatitis Nephritis Non-hepatic malignancy Non-hepatic abscess Duodenal ulcer Parkinson’s disease Cerebrovascular accident Secondary epilepsy Hypertension Respiratory failure Thyroid disorder Use of invasive devices Nasogastric feeding tube Total parenteral nutrition Bladder catheter Endotracheal intubation Central vascular catheter Pleural effusion Mortality
25
3
1.000 0.552
11 18 1 0 0 1 3 0 6 21 7 1 4 3 4 1 0 2 9 2 17 12 1
2 1 0 0 0 0 0 0 2 1 0 0 0 0 1 0 0 0 2 2 2 3 0
1.000 NA NA 1.000 1.000 NA 0.147 0.224 1.000 1.000 1.000 1.000 0.410 1.000 NA 1.000 0.266 0.035 1.000 0.092 1.000
27 20 27 16 12 2 15
3 2 2 2 2 1 1
1.000 1.000 0.263 1.000 0.568 0.263 1.000
cr-cKP, carbapenem-resistant classic Klebsiella pneumoniae; cr-hvKP, carbapenemresistant hypervirulent (hypermucoviscous) Klebsiella pneumoniae; NA, not assessable. a cr-cKP vs. cr-hvKP.
difference in virulence factors between ST11 and ST25/ST65.27 The pLVPK-derived terW–iutA–rmpA–silS loci are significantly correlated with abscess formation and prevalence of K1 and K2 serotype strains,29,32 but these virulence loci have not previously been detected in ST11 K. pneumoniae. Compared with previously reported ST11 strains,20 cr-hvKP7 has more virulence factors, including the hypermucoviscosity phenotype.10 In contrast to a crhvKP isolate reported in Brazil, which did not contain either magA or rmpA,20 the hypermucoviscous phenotype of cr-hvKP7 persisted without decrease following several rounds of subculturing. Several reports have shown that aminoglycosides are effective for the clearance of urinary tract infections caused by aminoglycoside-susceptible carbapenem-resistant K. pneumoniae.36 In the present study, cr-hvKP isolates 2, 3, and 6 were treated successfully with a regimen that included isepamicin, indicating that aminoglycosides may also be effective in treating cr-hvKP. Although the four patients included in this study had a variety of underlying diseases and use of invasive devices, none had invasive liver abscess syndrome or extra-hepatic metastatic infections.8 Further
Table 5 Comparison between the cr-hvKP group and cr-cKP group for molecular characteristics
K serotype K1 K2 K5 K20 K54 K57 K non-typeable Virulence gene pLVPK-related loci repA uge wabG fimH entB iroN ybtS kfuBC mrkD ycfM ureA allS blaKPC Other carbapenemase genes
cr-cKP infection group (n = 29)
cr-hvKP infection group (n = 3)
p-Value
0 0 0 0 0 0 29
0 3 0 0 0 0 1
NA 0.000a NA NA NA NA 0.006a
0 0 29 29 29 29 0 21 0 25 29 29 0 28 0
2 2 3 3 3 3 2 1 0 3 3 3 1 3 0
0.006a 0.006a NA NA NA NA 0.006a 0.224 NA 1.000 NA NA 0.094 1.000 NA
cr-cKP, carbapenem-resistant classic Klebsiella pneumoniae; cr-hvKP, carbapenemresistant hypervirulent (hypermucoviscous) Klebsiella pneumoniae; NA, not assessable. a A p-value 0.05 was considered to be statistically significant.
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to these clinical observations, the ability of cr-hvKP to cause invasive infection should be evaluated further in vitro, as well as in phagocytosis assays and murine lethality tests.37 The length of stay prior to isolation of cr-hvKP in the current study differed markedly, with the shortest stay being 11 days (cr-hvKP6) and the longest being 1171 days (cr-hvKP5). Combined with differences in molecular characteristics and resistance patterns, it is concluded that cr-hvKP isolates 1, 6, and 7 represent three separate evolutionary lineages. The emergence of regional polyclonal crhvKP isolates is of significant concern and may herald the cr-hvKP era. It has been reported that patients with hvKP infection tend to have different underlying illnesses and more invasive infections compared to those with cKP.38 However, in the present study, no differences in clinical characteristics were found between cr-hvKP and cr-cKP infected patients. Reports on the virulence genes and K serotype of cr-cKP, let alone cr-hvKP, are scarce. The present study showed that pLVPK-related loci, iroN, and K2 serotype were more prevalent in cr-hvKP than in cr-cKP, but the significance of these differences needs to be studied further. In summary, this study provides the first clinical and molecular characterization of ST65, ST25, and ST11 cr-hvKP isolates in China. The clonal populations of invasive infection (ST65 and ST25) have evolved to become multidrug-resistant, while a related multidrugresistant clonal population (ST11) has evolved to become hypermucoviscous. The confluence of virulence and carbapenem resistance and the bi-directional evolution might pose major problems in the future for the management of K. pneumoniae infections. Conflict of interest/funding: None. References 1. Podschun R, Ullmann U. Klebsiella spp. as nosocomial pathogens: epidemiology, taxonomy, typing methods, and pathogenicity factors. Clin Microbiol Rev 1998;11:589–603. 2. Liu YC, Cheng DL, Lin CL. Klebsiella pneumoniae liver abscess associated with septic endophthalmitis. Arch Intern Med 1986;146:1913–6. 3. Buppajarntham S, Shah M, Junpaparp P. Tumor-like pyogenic liver abscess caused by Klebsiella pneumoniae in diabetes. Endocrine 2014;47:656–7. 4. Vila A, Cassata A, Pagella H, Amadio C, Yeh KM, Chang FY, et al. Appearance of Klebsiella pneumoniae liver abscess syndrome in Argentina: case report and review of molecular mechanisms of pathogenesis. Open Microbiol J 2011;5:107–13. 5. Gundestrup S, Struve C, Stahlhut SG, Hansen DS. First case of liver abscess in Scandinavia due to the international hypervirulent Klebsiella pneumoniae clone ST23. Open Microbiol J 2014;8:22–4. 6. Hyun JI, Kim YJ, Jeon YH, Kim SI, Park YJ, Kang MW, et al. A case of ventriculitis associated with renal abscess caused by serotype K1 Klebsiella pneumoniae. Infect Chemother 2014;46:120–4. 7. Vandevelde A, Stepanovic B. On a boat: a case in Australia of endophthalmitis and pyogenic liver, prostatic, and lung abscesses in a previously well patient due to Klebsiella pneumoniae. Case Rep Infect Dis 2014;2014:137248. 8. Siu LK, Yeh KM, Lin JC, Fung CP, Chang FY. Klebsiella pneumoniae liver abscess: a new invasive syndrome. Lancet Infect Dis 2012;12:881–7. 9. Fang CT, Chuang YP, Shun CT, Chang SC, Wang JT. A novel virulence gene in Klebsiella pneumoniae strains causing primary liver abscess and septic metastatic complications. J Exp Med 2004;199:697–705. 10. Kawai T. Hypermucoviscosity: an extremely sticky phenotype of Klebsiella pneumoniae associated with emerging destructive tissue abscess syndrome. Clin Infect Dis 2006;42:1359–61. 11. Pomakova DK, Hsiao CB, Beanan JM, Olson R, MacDonald U, Keynan Y, et al. Clinical and phenotypic differences between classic and hypervirulent Klebsiella pneumonia: an emerging and under-recognized pathogenic variant. Eur J Clin Microbiol Infect Dis 2012;31:981–9. 12. Li W, Sun G, Yu Y, Li N, Chen M, Jin R, et al. Increasing occurrence of antimicrobial-resistant hypervirulent (hypermucoviscous) Klebsiella pneumoniae isolates in China. Clin Infect Dis 2014;58:225–32. 13. Yigit H, Queenan AM, Anderson GJ, Domenech-Sanchez A, Biddle JW, Steward CD, et al. Novel carbapenem-hydrolyzing beta-lactamase, KPC-1, from a carbapenem-resistant strain of Klebsiella pneumoniae. Antimicrob Agents Chemother 2001;45:1151–61.
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