ST11 Carbapenem-Resistant Klebsiella pneumoniae Clone Harboring blaNDM Replaced a blaKPC Clone in a Tertiary Hospital in China

The nosocomial spread of carbapenem-resistant Enterobacterales (CRE) is extremely common, resulting in severe burdens on healthcare systems. In particular, the high-risk Klebsiella pneumoniae ST11 strain has a wide endemic area in China. The current study describes the results of continuous monitoring of CRE genotypes and phenotypes in a tertiary hospital in North China from 2012 to 2020. A total of 160 isolates were collected, including 109 Klebsiella. pneumoniae (68.13%), 29 Escherichia coli (26.60%), 12 Enterobacter cloacae (7.50%), and 10 other strains (6.25%). A total of 149 carbapenemase genes were detected, of which blaKPC-2 (51.0%) was the most common, followed by blaNDM-1 (22.82%), and blaNDM-5 (23.49%). Based on multi-locus sequence typing, the ST11 strain (66.1%) dominates K. pneumoniae, followed by ST15 (13.8%). Interestingly, the proportion of blaNDM (22.2%, 16/72) in ST11 K. pneumoniae was significantly increased in 2018–2019. Hence, whole-genome sequencing was performed on ST11 K. pneumoniae. Growth curves and in vitro competition experiments showed that K. pneumoniae carrying blaNDM exhibited a stronger growth rate (p < 0.001) and competition index (p < 0.001) than K. pneumoniae carrying blaKPC. Moreover, K. pneumoniae carrying blaNDM had a stronger biofilm-forming ability than K. pneumoniae carrying blaKPC (t = 6.578; p < 0.001). K. pneumoniae carrying blaKPC exhibited increased defense against bactericidal activity than K. pneumoniae carrying blaNDM. Thus, ST11 K. pneumoniae carrying blaNDM has strong adaptability and can locally replace K. pneumoniae carrying blaKPC to become an epidemic strain. Based on these findings, infection control and preventive measures should focus on the high-risk ST11-K. pneumoniae strain.


Introduction
Enterobacterales are the most common pathogenic gram-negative bacteria responsible for causing myriad community-acquired and healthcare-acquired infections [1,2]. In particular, carbapenem-resistant Enterobacterales (CRE) have been reported worldwide, including in China [3][4][5], and represent a significant public health challenge. In 2017, the World Health Organization (WHO) released a list of drug-resistant bacteria that pose a significant threat to human health, including CRE, and highlighted the urgent need to develop new antibiotics [6].
Carbapenem resistance in Enterobacterales can arise through several molecular mechanisms, the most common of which include carbapenemase production. According to the Ambler classification [7][8][9], class A (mainly Klebsiella pneumoniae carbapenemase [KPC]), class B (mainly New Delhi metallo-β-lactamase [NDM], imipenemase [IMP], and Verona integron-encoded metallo-β-lactamase [VIM]), and class D (mainly oxacillinase [OXA]-48-like) are the primary classes of CRE carbapenemases. The second most common resistance mechanism includes the high production of extended-spectrum β-lactamases tube was labeled for the eCIM test. Next, 20 µL of the 0.5 M EDTA was added to the 2 mL TSB tube to obtain a final concentration of 5 mM EDTA, after which the steps for the mCIM procedure were repeated. The mCIM and eCIM tubes were assessed in parallel by placing the meropenem disks from the mCIM and eCIM tubes on the same MHA plate inoculated with 25922 indicator strain. K. pneumoniae ATCC ® BAA 1705 and K. pneumoniae ATCC ® BAA 1706 served as the positive and negative controls, respectively. As previously described, PCR detection of common carbapenemase genes (bla KPC , bla NDM , and bla IMP ) was performed for mCIM-positive isolates [5]. After Sanger sequenced the amplification results, we compared the sequences on the http://www.bldb.eu/Enzymes.php (accessed on 1 December 2021) website to identify each carbapenemase [18].

Antimicrobial Susceptibility Testing
The agar dilution method determined the minimum inhibitory concentrations (MICs) of all the antimicrobial agents except colistin. MICs of colistin were determined by the broth microdilution method. The results were interpreted according to CLSI M100 S30 guidelines [17]. The breakpoint of tigecycline for Enterobacterales was based on the US Food and Drug Administration standard (≤2 µg/mL is susceptible, ≥8 µg/mL is resistant). Escherichia coli ATCC ® 25922 and Pseudomonas aeruginosa ATCC ® 27853 were used as quality control isolates. The tested antimicrobial agents included imipenem, meropenem, ertapenem, colistin, tigecycline, ceftazidime, cefepime, cefoperazone-sulbactam, amikacin, aztreonam, piperacillin-tazobactam, ciprofloxacin, fosfomycin, levofloxacin, and minocycline.

WGS and Phylogenetic Analysis
WGS and evolutionary correlation analysis were performed for K. pneumoniae ST11 strains isolated throughout the study. Total genomic DNA was extracted using the TIANamp Bacteria DNA Kit DP302 (Tiangen Biotech, Beijing, China), followed by genomic DNA sequencing using the Illumina HiSeq X Ten platform (Illumina Inc., San Diego, CA, USA), which produced 150 bp paired-end reads and at least 100-fold coverage of raw reads. The short-read sequence was assembled de novo using SPAdes v3.10.0. All draft genome sequences were deposited into the National Center for Biotechnology Information's genome database and organized under BioProject ID PRJNA867316. All resistance genes were detected by the Resfinder (https://cge.cbs.dtu.dk/services/ResFinder/, accessed on 1 December 2021) and basic local alignment search tool. K-locus (polysaccharide capsule) typing was identified with the Kaptive software [19] using the whole-genome sequences. The core genome's maximum likelihood phylogenetic analysis was performed using RAxML (version v7.2.8, Alexandros Stamatakis, Karlsruhe, Germany). Visualization of the evolutionary tree was performed using iTOL (https://itol.embl.de/, accessed on 1 December 2021). ) were subjected to growth curve assay and in vitro competition growth experiments, as previously described [20]. Briefly, isolates were cultured overnight in Luria-Bertani (LB) broth diluted to an OD 600 of 0.01 and grown at 37 • C with shaking (200 rpm). Triplicate experiments were run for each isolate. The culture cell density was determined every 0.5 h by measuring the OD at 600 nm (Thermo Fisher Scientific, Shanghai, China).

Serum-Killing Assay
Serum-killing assays were conducted to determine virulence in vitro as previously described [22]. Briefly, human blood sera were obtained from ten healthy individuals. K. pneumoniae isolates were inoculated into LB broth medium and incubated at 37 • C with shaking until the logarithmic phase was reached (OD 600 = 0.6). An inoculum of 50 µL diluted culture (containing 1 × 10 6 CFU) was added into 150 µL of pooled human sera contained in a 10 × 75 mm Falcon polypropylene tube (BD Biosciences, Franklin Lakes, NJ, USA) and incubated at 37 • C with shaking. Viable counts were checked at 0, 1, 2, and 3 h, and aliquots of the bacteria were diluted with 0.9% saline solution and plated on LB agar plates. CFU were counted after 18 h of incubation at 37 • C. Each strain was tested at least three times. The mean results were expressed as inoculation percentages, and six grades were assigned to classify the strains as described previously [23]. K. pneumoniae K2044 and K. pneumoniae ATCC ® 13883, which were identified as serum killing sensitive (grade 1) and resistant (grade 5) in our previous study [23], were used as negative and positive controls, respectively.

Biofilm Formation Assay
Biofilm production was determined as described recently [22]. Cultures that were shaken overnight were diluted 1:1000 in LB broth and a total volume of 200 µL was inoculated into wells of an untreated 96-well microplate, with six wells per strain. After 24 h incubation at 37 • C, the LB broth was removed, the wells were washed four times with deionized water and 200 µL of 0.1% crystal violet was added. After a 15 min incubation, crystal violet was removed, and the wells were washed six times with deionized water. Subsequently, 200 µL of 30% acetic acid was added, and the plate was incubated for 10 min at room temperature before determining the OD 590 with Varioskan Flash (Thermo Fisher Scientific, Shanghai, China).

Extraction and Quantification of Capsules
The capsule was extracted, and uronic acids were quantified to assess capsule production. Overnight shaking cultures grown in LB were adjusted to an OD 600 of 0.6 with LB broth. The capsule was extracted using the modified protocol described previously [24]. Briefly, 500 µL cultures were mixed with 100 µL of 1% Zwittergent 3-14 detergent in 100 mM citric acid (pH 2.0) and heated for 30 min at 50 • C with occasional vortexing. Then, the above mix was centrifuged at 16,000× g for five minutes. After centrifuging, 250 µL of the supernatant was transferred to a new tube and precipitated with 1 mL of 80% ethanol at four • C for 30 min. The precipitate was centrifuged at 16,000× g for 5 min and dried, and uptake was performed in 100 µL of deionized water and 12.5 mM sodium tetraboratesulfuric acid solution. The samples were mixed and boiled at 100 • C for 15 min. A series of tubes containing two-fold serial dilutions of glucuronolactone were standard. The samples were cooled to room temperature for approximately 30 min before adding 10 µL of 0.15% Antibiotics 2022, 11, 1373 5 of 14 m-hydroxybiphenyl and further incubation for 15 min to generate the chromophore. The OD was determined at 520 nm. Uronic acids were quantified by comparing the OD with the glucuronolactone standard curve.

Statistical Analysis
Statistical analysis was performed with the GraphPad Prism version 9 (GraphPad Software, San Diego, CA, USA) using one-way analysis of variance followed by Tukey-Kramer tests. Statistical significance was set at p < 0.05. Data from the antibiotic susceptibility tests were analyzed using WHONET 5.6 software (WHO, Geneva, Switzerland).

Ethics Statement
The Research Ethics Committee of Shanxi Provincial People's Hospital approved this study. Medical records and patient information were retrospectively reviewed and collected. Informed consent was not required because the medical records and patient information were anonymously reviewed and collected in this observational study. Figure 1 shows the number of CRE isolates per year. Respiratory tract infections were the predominant source of infection, accounting for 46.8% (75/160), followed by urinary tract (19.3%; 31/160) and bloodstream (15.6%; 25/160). Among the 160 CRE strains, K. pneumoniae accounted for the highest proportion (68.1%), followed by Escherichia coli (18.1%). In this study, CRE strains were mainly isolated in the neurosurgery (25%; 40/160), intensive care unit (ICU; 16.8%; 27/160), neurology (10%; 16/160), and respiratory (6.9%; 11/160) wards. the supernatant was transferred to a new tube and precipitated with 1 mL of 80% ethanol at four °C for 30 min. The precipitate was centrifuged at 16,000× g for 5 min and dried, and uptake was performed in 100 µ L of deionized water and 12.5 mM sodium tetraboratesulfuric acid solution. The samples were mixed and boiled at 100 °C for 15 min. A series of tubes containing two-fold serial dilutions of glucuronolactone were standard. The samples were cooled to room temperature for approximately 30 min before adding 10 µ L of 0.15% m-hydroxybiphenyl and further incubation for 15 min to generate the chromophore. The OD was determined at 520 nm. Uronic acids were quantified by comparing the OD with the glucuronolactone standard curve.

Statistical Analysis
Statistical analysis was performed with the GraphPad Prism version 9 (GraphPad Software, San Diego, CA, USA) using one-way analysis of variance followed by Tukey-Kramer tests. Statistical significance was set at p < 0.05. Data from the antibiotic susceptibility tests were analyzed using WHONET 5.6 software (WHO, Geneva, Switzerland).

Phylogenetic Analysis of ST11 K. pneumoniae Isolates
We performed WGS of all ST11 K. pneumoniae isolates. The phylogenetic tree revealed that ST11 isolates harboring blaKPC-2 could be assigned to ten clades (Figure 3). CRKP-ST11-blaNDM isolates were in the same branch and had no noticeable differences, while CRKP-  [5].

Serum Killing Assay
Different K. pneumoniae strains were compared in terms of their ability to survive the bactericidal activity of human serum (Table 3). K. pneumoniae-bla KPC had intermediate sensitivity to serum killing (grade 3-6), whereas K. pneumoniae-bla NDM was sensitive (grade 2), thus suggesting that K. pneumoniae-bla KPC exhibited a higher defense against the bactericidal activity of sera than K. pneumoniae-bla NDM . Serum killing results were shown by the percent survival of all isolates after incubation in normal human serum at one, two and three h. The grade and interpretive are classified as previously described [22]. R, resistance; S, susceptible; and I, intermediately sensitive.

Quantification of Capsule Production
Uronic acids were quantified to assess capsule production. We found that capsule production was lower in the K. pneumoniae-blaNDM isolates. However, the two groups had no significant difference ( Figure S1).

Discussion
CRE infection has become a significant global public health issue due to the widespread use and inappropriate application of carbapenems. In our hospital in Shanxi province, China, the prevalence of CRKP increased from 0.8% in 2012 to 5.73% in 2019, thus reflecting an upward trend and revealing that K. pneumoniae is the primary domestic CRE strain [5,26].

Quantification of Capsule Production
Uronic acids were quantified to assess capsule production. We found that capsule production was lower in the K. pneumoniae-bla NDM isolates. However, the two groups had no significant difference ( Figure S1).

Discussion
CRE infection has become a significant global public health issue due to the widespread use and inappropriate application of carbapenems. In our hospital in Shanxi province, China, the prevalence of CRKP increased from 0.8% in 2012 to 5.73% in 2019, thus reflecting an upward trend and revealing that K. pneumoniae is the primary domestic CRE strain [5,26].
Respiratory tract infections were the most common source of isolation, which agrees with the results reported by Chen et al. in China [27]. In this study, CRE strains were mainly isolated in the neurosurgery (25%), intensive care unit (ICU; 16.8%), neurology (10%), and respiratory (6.9%) wards. This was likely due to the common use of mechanical ventilation in these departments, which is closely related to the intensity of antibiotic use, invasive operations, and low immunities of patients [28]. The ICU is the department with the most cases of CRE infection. Importantly, the in-hospital transport of patients in the ICU and other departments increases the risk of CRE in-hospital transmission [29]. Therefore, infection control in these departments may be beneficial in preventing the spread of CRE.
The CRE strains used in this study showed extensive drug resistance, consistent with China's surveillance data [26,30]. All isolates exhibited resistance to carbapenems and cephalosporins. There were apparent interspecies variations in susceptibility; for example, Escherichia coli was less susceptible to aztreonam than other species.
Consistent with changing domestic trends [31,32], our research revealed that 93.13% of CRE strains produced carbapenemases, among which KPC was predominant in K. pneumoniae isolates, and NDM-5 was predominant in Escherichia coli isolates; NDM-1 was the most common type in Enterobacter cloacae. Interestingly, studies have shown that the resistance mechanisms of CRE clinical strains isolated from children or adults, or from different geographical distributions differ. In particular, KPC-2-producers are widespread in adult patients and can be spread horizontally through plasmids, thus causing potential outbreaks. Notably, CRE strains that co-produce NDM and KPC carry many resistance genes, making them highly resistant to the most commonly used antibiotics [33].
Seventy-one K. pneumoniae strains carrying the bla KPC-2 gene were classified into five STs, including ST11, ST15, ST48, ST25, and ST86. MLST showed subtype diversity and more heterogeneous clonal backgrounds of CRKP isolates related to nosocomial pneumonia. All ST48 isolates were detected in the neurosurgery ward, and may have resulted from the nosocomial transmission. Moreover, the proportion of bla NDM (22.2%, 16/72) in ST11 K. pneumoniae significantly increased in 2018-2019. This finding may indicate local epidemics, suggesting that more attention should be paid to the spread of bla NDM in the ST11 strain.
K47 was the most common K. pneumoniae serotype in our study. Similarly, in a study by Zhou et al., K64 was the most common serotype among 16 carbapenem-resistant hvKP isolates [34]. This single-center study showed that K64 had a higher virulence gene carrier rate than K47. The hypermucoviscous phenotype of hypervirulent K. pneumoniae (hvKP) is typically due to the increased production of capsular polysaccharides and specific virulence genes, such as rmpA and rmpA2. The emergence of and rapid increase in hvKP strains in recent years, especially carbapenemase-producing hvKP-related infections in immunocompromised patients, are worrisome and pose a severe threat to patients [25,[35][36][37].
Our in vitro competition study revealed that K. pneumoniae-bla KPC overgrew K. pneumoniae-bla NDM . Meanwhile, the proportion of NDM-1-positive K. pneumoniae isolates with biofilm formation capacity was higher than KPC-2-positive K. pneumoniae isolates. However, K. pneumoniae-bla KPC exhibited greater defense against bactericidal activity than K. pneumoniae-bla NDM . The virulence evaluation criterion was the hypermucoviscous phenotype, biofilm formation ability, and serum resistance [38]. In this study, virulence factors were not detected in the NDM-1-positive K. pneumoniae group, which is consistent with the drawing experiment and the K111 capsule serotypes. Fuursted et al. reported that K. pneumoniae carrying NDM-1 have an intrinsic virulence potential [39]. Interestingly, Montanari claims that bacteria lose certain virulence genes to obtain resistance genes for optimal adaptability [40]. Therefore, virulence influences the decrease in biofilm formation and growth rate. Indeed, there are several cases of carbapenem-resistant hvKP carrying bla NDM-1 in China [41,42].
Through our research, we came to the following conclusions. ST11 K. pneumoniae carrying bla NDM has strong adaptability and can locally replace K. pneumoniae carrying bla KPC to become an epidemic strain. Infection control and preventive measures should focus on high-risk ST11-K. pneumoniae strains.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/antibiotics11101373/s1, Figure S1: Capsule production measured by uronic acid extraction; Table S1: Data Analysis of Growth Curves; Table S2: The competition index of the three groups of strains; Table S3: t-test analysis of competition index of strains at different time points.

Institutional Review Board Statement:
The study was conducted in accordance with the Declaration of Helsinki, and approved by the Institutional Review Board (or Ethics Committee) of Research Ethics Committee of Shanxi Provincial People's Hospital.
Informed Consent Statement: Informed consent was not required because the medical records and patient information were anonymously reviewed and collected in this observational study.