Epidemiology and clinical signicance of pLVPK-like virulence plasmid in KPC-2-producing Klebsiella pneumoniae infections in eastern China: a preliminary exploration

Background: By acquiring a pLVPK-like virulence plasmid (pVir), Klebsiella pneumoniae carbapenemase (KPC)-producing K. pneumoniae (KPC-kp) evolves to a hypervirulent variant, with increasing cases reported worldwide. However, little is known about the epidemiological trend of pVir in KPC-kp, as well as clinical characteristics of infections caused by this novel strain (pVir + -KPC-kp). Methods: From 2014-2018, 662 carbapenem-resistant K. pneumoniae (CRKP) were consecutively collected from a tertiary hospital of eastern China. The conrmed KPC-kp were subjected to antimicrobial susceptibility testing, multilocus sequence typing and detection of pLVPK-related genetic loci (rmpA, rmpA2, iucA and iroN) to identify pVir + -KPC-kp strains. Then, the clinical characteristics and outcomes of KPC-kp infection patients with and without pVir were compared. Moreover, the risk factors for pVir + -KPC-kp infections also determined by a multivariable logistic regression analysis. Results: of the 662 CRKP, 86.6% (573/662) were KPC-kp including 285 (49.7%) pVir + -KPC-kp and 288 (50.3%) pVir - -KPC-kp. Notably, the prevalence of pVir + -KPC-kp by year increased remarkably from 2014 (19.5%, 8/41) to 2018 (60.0%, 90/150). Sequence type (ST) 11 was the most predominant ST, accounting for 88.9% of all pVir + -KPC-kp. For the 352 KPC-kp infection cases (186 with pVir + -KPC-kp and 166 with pVir - -KPC-kp), multivariable analysis indicated neurosurgery (Odds ratio [OR], 2.92; 95% condence interval [CI], 1.48-5.75; P =0.002) was independently associated with pVir + -KPC-kp infections. Although patients with pVir + -KPC-kp infections had higher incidence of septic shock (31.2% vs. 21.1%, P =0.03), the two groups showed no signicant differences in 7-day mortality (23.1% vs. 18.1%, P =0.24) or 28-day mortality (45.7% vs. 44.0%, P =0.75). virulence


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Contrary to hvKP, carbapenem-resistant K. pneumoniae (CRKP) is recognized as an important pathogen associated with nosocomial infections in immunocompromised patients [7]. Since rst reported in China in 2007 [8], Klebsiella pneumoniae carbapenemase (KPC) has become the most predominant contributor to carbapenem resistance in CRKP of China [9]. It is a common sense that KPC-producing K. pneumoniae (KPC-kp) is absent of pVir and nearly avirulent experimentally [10]. However, convergent K. pneumoniae strains of widely distributed ST11 carrying both pVir and KPC (pVir + -KPC-kp) have emerged, especially in China [11][12][13]. So far, most cases of pVir + -KPC-kp infections were still isolated or sporadic. However, recently, new evidence alerted that a rapidly increasing prevalence of pVir + -KPC-kp might occur due to the potential spread of a novel conjugative IncFIB plasmid containing bla KPC and key virulence-encoding region of pVir simultaneously [14,15]. Therefore, there is an urgent need to evaluate the current epidemiological trend of pVir in KPC-kp.
Besides displaying high levels of resistance to commonly used antibiotics, pVir + -KPC-kp strains in general exhibited enhanced anti-phagocytic and serum resistance properties in vitro and were associated with extremely high mortality both in affected patients and experimental models [11,12]. However, the association of virulence traits and pVir in multidrug-resistant K. pneumoniae remains controversial and the clinical features of patients infected with pVir + -KPC-kp strains are less revealed [16,17].
To address these problems, in present study, we rst investigated the prevalence of pVir among KPC-kp collected from a tertiary hospital in eastern China over four consecutive years. Then, we characterized the clinical features and outcomes of pVir + -KPC-kp infections by comparing with pVir --KPC-kp strains. Also, the risk factors of pVir + -KPC-kp infections were determined.

Design, Setting and Ethics
This retrospective cohort study was performed in The First A liated Hospital, College of Medicine, Zhejiang University, from July 2014 to August 2018. It is one of the largest hospitals in eastern China, having approximate 2500 beds and 131,000 annual admissions. The rst report of KPC-kp in China came from here in 2007 [8]. Since then, KPC-kp has been endemic in the hospital [18].
The present study was conducted according to the principles stated in the Declaration of Helsinki and approved by the ethical review board of The First A liated Hospital, College of Medicine, Zhejiang University.

Patient Cohort
The source population consisted of all patients admitted to the hospital during the study period, whose bacterial cultures were identi ed as CRKP. The clinical specimens mainly included blood, sputum/tracheal aspirate, pleural uid and ascites. Further detailed collection of clinical information was performed for the patients whose isolates were veri ed as KPC-kp. Patients aged <18 years and those with missing or incomplete clinical records were excluded. Patients with polymicrobial infections and those with KPC-kp colonization were excluded as well. Infection or colonization was de ned according to the criteria of the Center for Disease Control and Prevention of America [19]. Only the rst episode for each patient was included in our analysis.

Clinical Data Collection
For all eligible KPC-kp infection cases, chart review was performed to obtain the clinical data. Infectious disease specialists independently collected the following clinical information using the patients' electronic charts and/or paper records: demographic conditions, unit and duration of hospitalization, underlying diseases, Charlson Comorbidity Index, abscess information and previous invasive procedures.
Patients were de ned as immunosuppressed if they had HIV or AIDS, were post-transplant, had received chemotherapy within 4 weeks, had received immunosuppressive agents for more than 2 weeks, or had neutropenia. Previous use of antibiotics was de ned as antimicrobial therapy (intravenous or oral) within 4 weeks before infections. Invasive procedures included surgery and mechanical ventilation performed within 4 weeks prior to the infections. Appropriate empirical or de nitive antimicrobial therapy was de ned as previously described [18].

Outcome Variables
The primary outcome variables were all-cause mortality at day 7 and day 28. Other secondary outcome variables were infection-related mortality and in-hospital mortality. Septic shock and multiple organ dysfunction syndrome (MODS) at the onset of infections, as well as the length of stay (LOS) from culture to discharge, were also assessed.

Bacterial Identi cation and Antimicrobial Susceptibility Testing
Bacterial isolates identi cation and antimicrobial susceptibility testing were performed using Vitek 2 automated system (bioMérieux, France) and the results were interpreted according to Clinical and Laboratory Standards Institute (CLSI) 2019 criteria [20]. For tigecycline, colistin and ceftazidimeavibactam (CAZ/AVI), minimum inhibitory concentrations (MICs) were determined using broth microdilution method. The susceptibility results were categorized in accordance with the CLSI criteria for CAZ/AVI, while European Committee on Antimicrobial Susceptibility Testing (version 9.0, http://www.eucast.org/clinical_breakpoints/) for colistin and tigecycline. The species identi cation was veri ed by MALDI-TOF MS (Bruker Daltonics Inc., Fremont, CA, USA). Pseudomonas aeruginosa ATCC27853 and Escherichia coli ATCC 25922 were used as the quality control strains of antimicrobial susceptibility testing.

Molecular Analysis
All of the CRKP were subjected to detection of bla KPC by PCR. The isolates carrying bla KPC were further investigated for pLVPK-related genetic loci rmpA, rmpA2, iucA and iroN. The primers used for PCR ampli cation and sequencing were acquired from a previous study [11]. Considering the primers of rmpA and rmpA2 target only the plasmid-borne genes, while iucA and iroN for both plasmid-borne and chromosomally-encoded genes, KPC-kp isolates of positive rmpA or rmpA2 were de ned as pVir + -KPC-kp according to the de nition previously described [11,[21][22][23].
Multilocus sequence typing (MLST) was carried out according to the protocol described on the K. pneumoniae MLST website ((https://bigsdb.pasteur.fr/klebsiella/klebsiella.html), and the results were analysed using the corresponding database.

Statistical Analysis
Data were analysed using SPSS software (version 20; SPSS Inc., Chicago, IL, USA). Categorical variables were presented as absolute numbers and their relative frequencies, and analyzed by chi-square or Fisher exact test. For continuous variables, the Kolmogorov-Smirnov test was applied to distinguish between normal and non-normal distribution. Normally distributed data were expressed as mean and standard deviation (SD) and analysed using Student's t-test, while non-normally distributed data were present with median and interquartile rang (IQR) and compared using Mann-Whitney test. To identify independent predictors for pVir + -KPC-kp infections, we used logistic regression. All variables with P value <0.1 in univariate analysis as well as clinical importance were incorporated into multivariable regression model. P value <0.05 was considered statistically signi cant.

Results
During the study period, a total of 662 CRKP isolates were collected. Of these, 573 were KPC-kp, and further were analysed for antimicrobial testing and pLVPK-derived loci. Two hundred and eighty-ve isolates were identi ed as pVir + -KPC-kp and 288 as pVir --KPC-kp. There were 352 cases met the criteria and included in the nal analysis of clinical characteristics. The causes of exclusion are shown in Figure  1, mainly polymicrobial infections (104 cases) and colonization (86 cases).
Furthermore, multivariate analysis of 130 patients with bloodstream infections and 126 intensive care unit (ICU) patients also indicated neurosurgery was the only independent predictor for pVir + -KPC-kp infections. Besides, among these patients, those infected with pVir + -KPC-kp and pVir --KPC-kp had similar clinical outcomes as well (data not shown).

Discussion
The present study found a high prevalence and increasing trend of pVir in ST11 KPC-kp in our hospital during the past four years. We identi ed previous neurosurgery within 4 weeks as the only clinical factor independently associated with pVir + -KPC-kp infections. Although septic shock was more frequently occurred in patients with pVir + -KPC-kp, the two groups did not differ signi cantly in terms of mortality. Most recently, a multicenter molecular epidemiological study of carbapenem-resistant hypervirulent K. pneumoniae (CR-hvKP) in China was reported, while limited clinical data was available [24]. As our best knowledge, it is so far the largest retrospective cohort study of KPC-kp with and without pVir.
Firstly, we screened the carriage of pVir as previously described [11]. Consistent with previous reports, most pVir + -KPC-kp belonged to the epidemic ST11 [23,25,26]. Notably, the prevalence of pVir in KPC-kp in 2014 had already reached 19.5%, much higher than that reported from Gu et al (3% in 2015) [11], suggesting an earlier transmission timeline in our hospital. Most recently, the research of Yang et al indicated a 100kb fragment of pVir integrated into a transmissible bla KPC -bearing IncFIB plasmid, which would prompt the dissemination of pVir among CRKP [14]. Based on the facts that the detection of pVir + -KPC-kp increased from 2014 to 2018 and ST11 pVir + -KPC-kp gradually becomes to be the predominant clone, it is speculated a transmission of IncFIB plasmid carrying both pVir-fragment and bla KPC among ST11 KPC-kp might have happened in our region. Studies aiming to test this hypothesis would be very imperative. Furthermore, we found nearly half of the pVir + -KPC-kp (44.9%) were absent of one or more pVir-featured genes, suggesting common deletion events in the process of plasmid fragment integration.
As most studies focused on the molecular epidemiological investigation of pVir + -KPC-kp [24,25], little was known about the risk factors of infections caused by this newly emerged variant. Here, we showed that previous neurosurgery within 4 weeks could help clinicians to identify patients of high risk to acquire pVir + -KPC-kp infections (p = 0.002). Our previous study reported that about half of K. pneumoniae meningitis was caused by pVir + -KPC-kp in our hospital from 2011 to 2017 [27]. In order to establish the correlation of neurosurgery and meningitis, we performed subgroup analysis of post-neurosurgery patients. However, it is of note that, most of the post-neurosurgical patients (46.3%, 25/54) were presence of pVir + -KPC-kp pneumonia and 35.1% developed to bacterial meningitis. There was no statistical signi cance of detection sites of pVir + -KPC-kp between post-neurosurgical patients and other patients. Moreover, for the patients of bloodstream infection and patients in ICU, neurosurgery within 4 weeks was also the independent factors of pVir + -KPC-kp infections. Further prospective survey and molecular epidemiology will clarify the correlation between neurosurgery and pVir + -KPC-kp infection, as well as the mode of transmission.
In K. pneumoniae, the terms "hypervirulence" primarily referred to isolates displaying hypermucoviscosity and causing invasive infections. However, most recent studies viewed the pVir + -KPC-kp strains associated with an extremely high infectious mortality as CR-hvKP, even if there was a lack of clinical features due to hvKP infections [11,13]. In present study, we compared some well-recognized parameters that could assist in differentiating infection due to hypervirulent and classical K. pneumoniae strains (such as infection development location, patients' age and abscess formation) [1], there was no difference between two groups. Moreover, our results indicated the 7-day, 28-day, overall and infection-related mortality were similar between patients with pVir + -KPC-kp and pVir --KPC-kp infections. Although the inconsistencies in patient care or variability in types of infections might introduce bias to the analysis, further evaluation of the association between pVir + -KPC-kp infections and mortality for ICU patients and patients with BSIs drew the same conclusion. Furthermore, we also noted 11.6% patients (33/285) isolated with pVir + -KPC-kp were absent of any symptoms of infections, who eventually did not develop to invasive infections and had favorable outcomes. Therefore, it was reasonable to believe not all of the KPC-kp would evolve to hypervirulent variants and resulted in fatalities by acquiring pVir. Study on the comprehensive genome analysis of ST11 pVir + -KPC-kp strains with varied virulence level is underway to provide more clues on the virulence mechanisms of this newly emerged strains.
There are some important limitations of our current study that should be acknowledged. First, the study was a single-center study with KPC-kp infected patients enrolled retrospectively. Our hospital located in Zhejiang province in China, where ST11 KPC-kp was epidemic [18] and a fatal outbreak caused by ST11 CR-hvKP was reported here in 2017 [11], therefore, the generalization of our results should be approached with caution. Second, the virulence of serial pVir + -KPC-kp strains that associated with variable prognosis had not be evaluated experimentally, and we just took the clinical mortality as the virulence measure.
However, the present study rstly compared the clinical outcomes between patients infected with pVir + -KPC-kp and pVir --KPC-kp.

Conclusions
Altogether, we observed a high prevalence of pVir in KPC-kp and ST11 KPC-kp with pVir as the predominant clone in our region. Moreover, we also demonstrated the clinical features and mortality was similar between infections caused by KPC-kp with and without pVir. Our nding indicated that not all of the KPC-kp could evolve to hypervirulent variant by acquiring pVir, therefore, the notion that KPC-kp harboring pVir was equivalent to hypervirulent strain should be strongly considered. Regardless, in order to prevent the dissemination of K pneumoniae strain with hyper-resistance and hypervirulence, active surveillance for these KPC-kp strains carrying pVir alone with virulent evaluation was still necessary.
Additionally, research into exploration of virulence mechanism of pVir + -KPC-kp strains may help to treat these infections.

Declarations
Ethics approval and consent to participate Ethical approval was granted from the Ethics Committees and review board of the First A liated Hospital, College of Medicine, Zhejiang University. As this study used secondary data, informed consent was not obtained from patients.

Consent for publication
Not applicable.

Availability of data and materials
The data that support the ndings of this study are available from the corresponding author on reasonable request.