Molecular Epidemiology of Carbapenem-Resistant Klebsiella pneumoniae Infections in Southwest China

Background: We determined epidemiological characteristics and resistance mechanisms of carbapenem-resistant Klebsiella pneumoniae (CRKP) strains found in Southwest China and assessed disease burden to provide evidence-based strategies for control and treatment of CRKP infection. Methods: A total of 159 strains of CRKP were isolated from sputa, blood, urine, ascites and wound secretions from three tertiary hospitals in Southwest China between August 1 st , 2018 and December 31 st , 2019. The sensitivity of each strain to 12 antibiotic agents was determined by micro-broth dilution. Identication of carbapenemase genes and multi-locus sequence typing (MLST) were performed using polymerase chain reaction (PCR). The disease burdens of patients with CRKP were assessed based on invasive procedures, antibiotic use, laboratory tests and clinical outcomes. Results: Of 159 CRKP strains analyzed, 50.9% were isolated from sputum samples. The percentage of patients who underwent invasive procedures before positive cultures for CRKP were detected was 96.3%. The mortality of blood infection was highest (66.6%) among patients with CRKP infection. All strains were insensitive to carbapenems. The resistance rates to levooxacin and amikacin were 85.5% and 81.8%, respectively. All CRKP strains produced carbapenemases, with a majority of isolates (81.1%) producing KPC-2. The MICs of strains harbouring both KPC-2 and NDM-1 were higher than those of strains with only KPC-2 or NDM-1. ST11 is the most popular clonotype found in Southwest China. Conclusions: CRKP strains in Southwest China are characterized by strong drug resistance and associated with poor clinical prognoses. It is therefore urgent to both strengthen control measures and improve prevention awareness. in this study were reviewed to collect clinical and patient data, including specimen source, demographic characteristics, underlying medical conditions, indwelling devices 30 days prior to culture, infection type(s), antimicrobial therapy, laboratory tests and clinical outcomes. Patients with clinically signicant hospital-acquired infection or healthcare-associated infection due to CRKP were eligible for this study. Exclusion criteria were community-acquired infection, missing key data, isolates with colonization only, screening samples and subsequent episodes in an individual patient.


Background
Carbapenem-resistant Enterobacteriaceae (CRE) have recently emerged as a class of bacterial pathogens that pose a signi cant threat, both to global public health and to high-risk patients undergoing lifethreatening procedures [1]. Although multiple genera of Enterobacteriaceae have been found to carry carbapenemase enzymes, carbapenem-resistant Klebsiella pneumoniae (CRKP)remains the most epidemiologically important on a global scale [2]. The prevalence of healthcare-related CRE infections has increased from 1.2% in 2001 to 4.2% in 2011 according to the National Healthcare Safety Network (NHSN). Similarly, the National Nosocomial Infection Surveillance System (NNIS), which concentrated mainly on Klebsiella species, reported an increase in infection rate from 1.6-10.4% [3]. Clinical studies have revealed high failure rates associated with treatment of CRKP infections, and infection mortality rates range from 22-72% [4,5]. Extensive resistance phenotypes have resulted in a dearth of clinical therapeutic choices for treating nosocomial infections due to CRKP, which has, in turn, posed additional di culties for patient management. Since the initial identi cation of a Klebsiella pneumoniae carbapenemase (KPC)-producing K. pneumoniae isolate in 2007, CRKP has been con rmed in almost every province in China [6,7]. A multicenter study that collected 999 strains of CRE from 27 provinces and regions across China between 2014 and 2015 found that CRKP accounted for the vast majority of strains (70%), far higher than both Escherichia coli (16%) and other Enterobacteriaceae (13%) [7]. There are a few reports regarding the distribution and resistance rate of CRKP in China [7][8][9][10]. However, there remains a lack of information concerning the molecular epidemiology of CRKP in Southwest China. In this study, CRKP strains collected from three tertiary hospitals in Southwest China were analyzed. The clinical characteristics, laboratory tests and outcomes of patients with CRKP infection were also compared. As a whole, this study provides critical information to inform the treatment and prevention of CRKP infections. were characterized using an automatic microbial analysis system (Phoenixtm-100, BD, USA), followed by ampli cation of the RNA polymerase β subunit gene (rpoB) and evaluation with a modi ed Hodge test in accordance with Clinical and Laboratory Standards Institute (CLSI) guidelines [11]. K. pneumoniae ATCC700603 was used as the quality control strain.

Study Population
Inpatients with clinical symptoms and positive CRKP culture from sputa, urine, blood, ascites or wound secretions were evaluated for CRKP infection according to the Centers for Disease Control and Prevention (CDC) and National Healthcare Safety Network (NHSN) criteria [12]. Hospital-acquired CRE infection was de ned as an infection that occurred after more than 48 h of hospitalization. A CRKP case was de ned as the rst clinical culture with carbapenem-nonsusceptible (imipenem, meropenem, or ertapenem) K. pneumoniae for each individual patient.

Data Collection
Medical records of all cases in this study were reviewed to collect clinical and patient data, including specimen source, demographic characteristics, underlying medical conditions, indwelling devices 30 days prior to culture, infection type(s), antimicrobial therapy, laboratory tests and clinical outcomes. Patients with clinically signi cant hospital-acquired infection or healthcare-associated infection due to CRKP were eligible for this study. Exclusion criteria were community-acquired infection, missing key data, isolates with colonization only, screening samples and subsequent episodes in an individual patient.
Primers are shown in Table 1. All ampli ed PCR products were submitted for direct sequencing twice on both strands with an automated DNA sequencer (ABI 3730XL, Weiterstadt, Germany). Nucleotide sequences were analyzed using the online basic local alignment search tool (BLAST) and Clustal W programs (multiple sequences alignment, pairwise comparisons of sequences and dendrograms) against the CRKP carbapenemase gene (GenBank).
DNA for multi-locus sequence typing (MLST) analysis was prepared using a commercial bacterial DNA extraction kit (TIANamp Bacteria DNA Kit, China) according to the manufacturer's instructions. MLST was performed according to the protocol described on the Pasteur Institute MLST website (http://bigsdb.pasteur. fr/klebsiella/klebsiella.html) with ampli cation of seven housekeeping loci (gapA, infB, mdh, pgi, phoE, rpoB and tonB) [12].

Discussion
CRKP presents a particularly critical problem worldwide due to rapidly rising resistance rates and subsequent high mortality. Prior to this study, there was limited understanding of the molecular epidemiology of CRKP in Southwest China. In this study, 159 CRKP strains were isolated from clinical samples taken at three tertiary hospitals across Southwest China. This research serves as a powerful supplement to multicenter studies of CRKP epidemiology in China.
According to this study, in Southwest China, death by CRKP infection was primarily caused by bloodstream infections (66.6%). Meta-analysis shows that the mortality rates of CRKP infections in North America, South America, Europe and Asia were 33.24%, 46.71%, 50.06% and 44.82%, respectively [16]. The mortality rate of bloodstream CRKP infection reaches 54.3%, which is quite high [16]. In this study, most of the patients with bloodstream infections experienced invasive operations such as deep vein catheterization, tracheal intubation and mechanical ventilation before the collection of a positive culture.
One study, which collected 100 CRKP strains from Shanghai, China, reports that mechanical ventilation could lead to a higher CRKP infection rate [17]. In our study, all patients with bloodstream infections experienced antimicrobial therapy before CRKP culture, 81.8% of which were treated with either third-or fourth-generation cephalosporins or carbapenem. Hospitalization and history of antibiotic use -especially of β-lactams and carbapenems --are considered independent risk factors for CRE infection [18,19]. More than half of CRKP strains taken from bloodstream samples in our study produce both KPC-2 and NDM-1, which we found to be associated with higher MICs for a panel of antibiotics.
The distribution of carbapenemases has been shown to regionally different; this study shows that KPC-2 is most prevalent in Southwest China, while NDM is the dominant CRKP carbapenemase in some provinces of China [7,10,20]. In the United States, the most prevalent mechanism of carbapenem resistance among Enterobacteriaceae is production of KPC, even though the combination of extendedspectrum β-lactamase (ESBLs)/AmpC cephalosporinase and membrane permeability can also make Klebsiella pneumoniae resistant to carbapenems [21,22]. Recent reports indicate that KPC-producing, gram-negative isolates are being identi ed throughout the United States, as well as in parts of Europe, Asia and South America. [23][24][25]At present, it is believed that transmission of colonies and speci c elements from existing drug-resistant strains is the main route for the emergence of new CRKP, suggesting that future control strategies should focus on inhibition of this gene transmission from drugresistant strains [10].
In this study, resistance rates to gentamicin, amikacin and levo oxacin were found to be 100%, 81.1% and 85.5%, respectively. These values are higher than those reported from both other provinces of China and other countries [26,27]. A retrospective study in China showed that, in an outbreak of nosocomial neonatal CRKP infection, the MICs of CRKP strains producing KPC-2 are higher than those of strains producing NDM-1 [28]. As mentioned above, a majority of CRKP strains (86.8%) analyzed in this study were found to produce KPC-2. In addition, more than half of the patients in this study were seriously ill in the ICU, were over 65 years old and had previously experienced multiple antibiotics. CRKP treatment is still absolutely di cult, as in vitro antibiotic-sensitivity assays do not fully re ect the response of CRKP to medicines in vivo. Nephrotoxicity is still an issue that requires attention in clinical practice for CRKP infections, particularly with the use of amikacin or polymyxin [29]. For patients with CRKP blood infections, it has been suggested that dual therapy based on high-dose carbapenem may bring clinical bene ts [30]. The use of new antibiotics, such as ceftazidime-avibactam, may be associated with better clinical prognosis and survival [27].
As previously reported, the majority of KPC-producing K. pneumoniae isolates in China belong to a common sequence type, ST11 (26). In contrast, a majority of these isolates in the United States belong to the ST258 type (27). Most of the CRKP strains in Southwest China belong to ST11 (64.2%), consistent with its national prevalence [10]. In our previous studies, we compared the age, sex, infection and antimicrobial use of 37 individual patients with either ST11 or non-ST11 CRKP infections [31]. Although there is no statistically signi cant difference between the two groups, CRKP sequence type still plays a key role in tracing strains at the genotype level, facilitating our understanding of global epidemiology.
To the best of our knowledge, this is the rst report of the molecular epidemiology of CRKP infections in Southwest China. This study is limited by the fact that the infection rate of CRKP in admitted patients could not be fully acquired. However, it is clear that CRKP isolates in Southwest China are strongly drugresistant to multiple antibiotics. In particular, patients with CRKP bloodstream infections have poor prognoses and high mortality. Moving forward, there is an urgent need to strengthen control measures and improve prevention of CRKP infections across Southwest China.

Declarations
Ethics approval and consent to participate The study was reviewed and approved by the research ethics committee of Guizhou Provincial People's Hospital (No. 2015022).

Consent for publication
The study does not contain any individual personal data, therefore, consent for publication is not required Availability of data and materials Data sharing not applicable to this article as no datasets were generated or analysed during the current study.

Competing interests
The authors declare that they have no competing interests