Prevalence of Carbapenem-Resistant Hypervirulent Klebsiella pneumoniae and Hypervirulent Carbapenem-Resistant Klebsiella pneumoniae in China Determined via Mouse Lethality Tests

Introduction

Klebsiella pneumoniae is a gram-negative and rod-shaped bacterium that belongs to the Enterobacteriaceae family (Adeolu et al., 2016), and was first described by Carl Friedlander in 1882. K. pneumoniae is considered a prominent nosocomial pathogen worldwide, and is a member of the “ESKAPE” (Enterococcus faecium, Staphylococcus aureus, K. pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) pathogens (Pendleton et al., 2013). General nosocomial infections caused by K. pneumoniae include pneumonia, bacteraemia, and urinary tract infections (UTIs) (Paczosa and Mecsas, 2016). The frequent use of antimicrobials has resulted in the development of carbapenem-resistant K. pneumoniae (CRKP) strains which first emerged in 1996 (Yigit et al., 2001). CRKP strains generally contain mobile genetic elements harbouring a variety of antimicrobial resistance genes, including beta-lactamase K. pneumoniae carbapenemase gene (bla_KPC), New Delhi metallo-β-lactamase gene (bla_NDM), and oxacillinase-48 gene (bla_OXA-48) (Lee et al., 2016; Zhang et al., 2015), among which bla_KPC has been found to be shared in approximately 78.6% (44/56) (Lin et al., 2020) and 89.5% (34/38) of isolates in two studies (Lin et al., 2018). CRKP strains account for over 30.0% of K. pneumoniae strains and present great challenges in clinical practice (Effah et al., 2020). CRKP is associated with mortality rates of 34.7% (17/49) for pneumonia, 37.8% (34/90) for bacteraemia, and 7.4% (9/121) for UTI (Hauck et al., 2016). Furthermore, CRKP treatment is associated with a higher medical cost than that of carbapenem-susceptible K. pneumoniae (Huang et al., 2018). CRKP constitutes a major public health issue, especially in endemic countries (Karampatakis et al., 2016). Therefore, CRKP control is considered a priority by the World Health Organization (World Health Organization, 2017). CRKP is usually denoted as classical K. pneumoniae (cKP) regarding its virulence (Russo and Marr, 2019; Zhang et al., 2020).

Hypervirulence in K. pneumoniae represents another major concern. Hypervirulent K. pneumoniae (HvKP) was first reported to cause pyogenic liver abscess (PLA) and septic endophthalmitis in seven healthy individuals (Liu et al., 1986). HvKP, which has a considerably lower median lethal dose (LD₅₀) than that of cKP in mouse model, generally produces various virulence factors such as hypercapsules, excessive siderophores, exopolysaccharides, and fimbriae (Paczosa and Mecsas, 2016; Russo and Marr, 2019). Apart from PLA, HvKP can also cause multiple invasive infectious diseases such as endogenous endophthalmitis, necrotising fasciitis, and meningitis, and the infection can undergo metastatic spread. PLA is endemic to East Asia and associated with a morbidity rate of 15.45 per 100,000 person-years in 2011 and a mortality rate of 8.2% (Chen et al., 2016; Siu et al., 2012). It has been estimated that 60% of endogenous endophthalmitis cases are associated with PLA caused by K. pneumoniae (Wong et al., 2000). Even with intravenous and intravitreal antimicrobial treatment, 89% of endophthalmitis cases show visual acuity of light perception or worse, and over 40% of affected eyes require evisceration or enucleation (Yang et al., 2007).

Recently, a combination of hypervirulence and extreme drug resistance has been reported in K. pneumoniae, thereby exacerbating the scarcity of effective treatments and resulting in high mortality (Zhang et al., 2015; Gu et al., 2018). The prevalence of infections caused by carbapenem-resistant hypervirulent K. pneumoniae (CR-HvKP) and hypervirulent carbapenem-resistant K. pneumoniae (Hv-CRKP) presents a global concern and a great challenge in clinical practice. However, the epidemiology of CR-HvKP and Hv-CRKP has not been extensively studied. The estimated prevalence of CR-HvKP/Hv-CRKP strains ranges among 5.0–15.0% among CRKP strains in mainland China, based on molecular determination or the Galleria mellonella (greater wax moth) lethality test (Zhang et al., 2020; Zhan et al., 2017). The gold standard method for evaluating the virulence of K. pneumoniae involves the use of mouse models, rather than the G. mellonella lethality test (Russo and MacDonald, 2020). Here, we analysed 436 clinical K. pneumoniae strains using molecular techniques and mouse lethality tests to elucidate the prevalence of CR-HvKP and Hv-CRKP strains.

Materials and Methods

K. pneumoniae Strains

In this study, 436 non-duplicate and consecutive K. pneumoniae isolates were collected from seven hospitals across six provinces in China (Huashan Hospital, 180 strains; Jinshan Hospital, 28 strains; Taizhou Municipal Hospital, 84 strains; The First Affiliated Hospital of Guangxi Medical University, 20 strains; Kunming Yan’an Hospital, 34 strains; Sixth Hospital of Shanxi Medical University, 60 strains; Shandong Provincial Hospital Affiliated to Shandong University, 30 strains) from January 2017 to February 2018. The 436 isolates were collected from diverse sources: 255 isolates (58.5%) were obtained from sputum samples, 98 isolates (22.5%) from urine samples, 29 isolates (6.7%) from blood samples, and 54 isolates (12.4%) were obtained from other sources. All the isolates were cultured on sheep blood agar plates and kept at -80°C prior to use. Identification of K. pneumoniae was performed using a matrix-assisted laser desorption/ionization time-of-flight mass spectrometry system (Bruker Daltonics Inc., Fremont, CA, USA) using the standard strains P. aeruginosa ATCC 27853, K. pneumoniae ATCC 700603, and E. coli ATCC 25922 as controls.

K. pneumoniae NTUH-K2044 (Accession number: AP006725.1) obtained from the Department of Internal Medicine, National Taiwan University Hospital, Taipei, Taiwan, is a typical hypervirulent K. pneumoniae serotype K1 strain (Fang et al., 2004). K. pneumoniae HS11286 (Accession number: CP003200.1) isolated from the Department of Laboratory Medicine, Huashan Hospital, Fudan University, Shanghai, China, is a K. pneumoniae serotype K47 strain containing bla_KPC and low virulence (Liu et al., 2012).

All the K. pneumoniae strains were investigated as the flow chart in Figure 1.

FIGURE 1

Figure 1 Experimental methodology of the study. KP, Klebsiella pneumoniae; HvKP, hypervirulent KP; bla_KPC, beta-lactamase K. pneumoniae carbapenemase gene.

Multilocus Sequence Typing

DNA was extracted from the 436 K. pneumoniae strains using the QIAamp DNA mini kit (QIAGEN, Düsseldorf, Germany) according to the manufacturer’s protocol. Seven housekeeping genes (gapA, infB, mdh, pgi, phoE, rpoB, and tonB) were amplified via polymerase chain reaction (PCR) (Diancourt et al., 2005) and then sequenced using an ABI 3730XL DNA Analyser (Applied Biosystems, San Ramon, CA, USA), and then compared with sequences available on the K. pneumoniae MLST database (http://www.pasteur.fr/recherche/genopole/PF8/mlst/Kpneumoniae.html). The primers used are shown in Table S1.

Determination of Serotypes, Antimicrobial-Resistance, and Virulence Genes

The capsule type was determined via PCR amplification and sequencing of the wzi loci (Brisse et al., 2013), followed by comparison with sequences on the database of Institute Pasteur (https://bigsdb.pasteur.fr/klebsiella/klebsiella.html).

The antimicrobial resistance gene (bla_KPC) and virulence genes (wzy-K1, allS, entB, irp2, iroN, iucA, fimH, mrkD, p-rmpA2, c-rmpA, p-rmpA, peg-344, and wzi) (Compain et al., 2014; Gu et al., 2018; Russo et al., 2018) were analysed via PCR amplification, using an Applied Biosystems Veriti PCR system (Applied Biosystems). The primers used are shown in Table S1.

Determination of Putative HvKP, cKP, Hv-bla_KPC(+)-KP, and bla_KPC(+)-HvKP Strains

On the basis of molecular characteristics, HvKP and cKP were putatively defined as a reference (Hu et al., 2021) (Figure 1). Hv-bla_KPC(+)-KP was defined as bla_KPC-positive cKP which acquired key virulence genes that conferred hypervirulence. bla_KPC(+)-HvKP (K1, K2, K5, K10, K20, K25, K27, and K57) (Chen et al., 2016; Siu et al., 2012) was defined as HvKP that acquired a bla_KPC gene.

String Test

Overnight cultured K. pneumoniae colonies on sheep blood agar plates were stretched outward using an inoculation loop as described previously (Shon et al., 2013). The string test was considered positive when a viscous string produced was over 5 mm in length. Strain NTUH-K2044 was used as a positive control and HS11286 was used as a negative control.

Capsule Staining

Capsule staining of K. pneumoniae strains was performed according to the manufacturer’s instructions (catalog number: BA-4039; BASO, Zhuhai, China). NTUH-K2044 was used as a positive control and HS11286 was used as a negative control.

Periodic Acid-Schiff Staining

Periodic acid-Schiff staining was performed according to the manufacturer’s protocol (catalog number: BA4080A; BASO, Zhuhai, China). Strains NTUH-K2044 and HS11286 were used as positive and negative controls, respectively.

Fitness Analysis

A growth curve was generated to evaluate the fitness of K. pneumoniae strains (Liu et al., 2016). These strains were cultured overnight in Luria-Bertani broth, diluted to an optical density at 600 nm (OD₆₀₀) of 0.001, and cultured at 37°C under aerobic conditions (BioTek Synergy H1, Winooski, VT, USA). The OD₆₀₀ values were measured every 30 min and plotted as a curve. Strains NTUH-K2044 and HS11286 were used as positive and negative controls, respectively.

Quantitative PCR Analysis

Quantitative PCR analysis of galF mRNA together with 16S rRNA was performed using an Applied Biosystems 7500 system (Applied Biosystems, San Ramon, CA, USA). The primers used are shown in Table S1. Strains NTUH-K2044 and HS11286 were used as positive and negative controls, respectively. The analyses were performed according to the manufacturer’s protocol (catalog number: FS-Q1002; FOREVER STAR, Beijing, China).

Mouse Lethality Test

Mouse experiments were approved by the Institutional Animal Care and Use Committee of the School of Pharmacy, Fudan University (Shanghai, China) (ethical approval document NO. 201603-TY-MQ-01). Pathogen-free female BALB/c mice (age, 6 weeks), four per group, were intraperitoneally inoculated with 100 μL of K. pneumoniae strains at the mid-logarithmic growth phase (Mizuta et al., 1983). Before inoculation, K. pneumoniae strains were washed twice with normal saline and centrifuged at 10,621 × g for 4 min. A 0.6 McFarland standard equivalent to 2.0 × 10⁸ colony forming units (CFU)/mL was prepared. The final inoculation was 10²–10⁷ CFU/mL. The mice were observed for 14 d after inoculation. LD₅₀ was determined according to a previous study (Reed and Muench, 1938). Strains NTUH-K2044 and HS11286 were used as positive and negative controls. K. pneumoniae strains with LD₅₀ ≤ 10 times of that of NTUH-K2044 were regarded as hypervirulent; those with LD₅₀ > 10 times of that of NTUH-K2044 were denoted as hypovirulent.

Statistical Analysis

GraphPad Prism 8 software (GraphPad Software Inc., Sand Diego, CA, USA) was used to perform Chi-square test, one-way ANOVA, and Kruskal-Wallis test between groups; p < 0.05 was considered significant.

Results

Distribution of Key Virulence Genes

A varying distribution of virulence genes was observed, ranging from 0.0% (c-rmpA) to 100.0% (entB) (Figure 2). The 13 virulence genes could be classified into 4 categories based on rates of distribution: ≤ 10.0% (allS, wzy-K1, and c-rmpA), approximately 11.0–30.0% (peg-344, p-rmpA, p-rmpA2, iucA, and iroN), approximately 50.0–80.0% (irp2), and approximately 81.0–100.0% (fimH, mrkD, entB, and wzi).

FIGURE 2

Figure 2 Distribution of 13 key virulence genes among the Klebsiella pneumoniae isolates.

Distribution of Predicted HvKP, bla_KPC(+)-KP, bla_KPC(+)-HvKP, and Hv-bla_KPC(+)-KP

In total, 127 (29.1%), 186 (42.7%), 9 (2.1%), and 26 (6.0%) strains were putatively denoted as HvKP, bla_KPC(+)-KP, bla_KPC(+)-HvKP, and Hv-bla_KPC(+)-KP strains, respectively.

Distribution of Key Virulence Genes in Putative bla_KPC(+)-HvKP and Hv-bla_KPC(+)-KP

The distribution of 13 key virulence genes among the putative bla_KPC(+)-HvKP and Hv-bla_KPC(+)-KP strains is shown in Figure 3. The 13 virulence genes were classified into four categories based on the rates of distribution: ≤ 10.0% (allS, wzy-K1, and c-rmpA), approximately 31.0–50.0% (p-rmpA and iroN), approximately 51.0–80.0% (peg-344 and p-rmpA2), and approximately 81.0–100.0% (fimH, mrkD, iucA, irp2, entB, and wzi).

FIGURE 3

Figure 3 Distribution of key virulence genes among putative bla_KPC(+)-HvKP and Hv-bla_KPC(+)-KP strains. bla_KPC, beta-lactamase K. pneumoniae carbapenemase gene; bla_KPC(+)-HvKP, bla_KPC(+) hypervirulent K. pneumoniae; Hv-bla_KPC(+)-KP, hypervirulent bla_KPC(+) K. pneumoniae.

Morphological Characteristics

In total, 34 putative bla_KPC(+)-HvKP and Hv-bla_KPC(+)-KP strains, except for bla_KPC(+)-HvKP strain JS184, demonstrated negative string test results. No hypercapsule was found among the 35 putative bla_KPC(+)-HvKP and Hv-bla_KPC(+)-KP strains (Figure 4). No exopolysaccharides were found to be produced by the putative bla_KPC(+)-HvKP and Hv-bla_KPC(+)-KP strains, except for JS184 (Figure 5).

FIGURE 4

Figure 4 Capsule staining of 35 putative bla_KPC(+)-HvKP and Hv-bla_KPC(+)-KP strains. (A) Klebsiella pneumoniae NTUH-K2044, (B) K. pneumoniae HS11286, (C) JS184, and (D) TZ20. K. pneumoniae strains are purple and rod-shaped, and their transparent surroundings are hypercapsules (×1000). bla_KPC, beta-lactamase K. pneumoniae carbapenemase gene; bla_KPC(+)-HvKP, bla_KPC(+) hypervirulent K. pneumoniae; Hv-bla_KPC(+)-KP, hypervirulent bla_KPC(+) K. pneumoniae.

FIGURE 5

Figure 5 Periodic acid-Schiff staining of 35 putative bla_KPC(+)-HvKP and Hv-bla_KPC(+)-KP strains. (A) Klebsiella pneumoniae NTUH-K2044, (B) K. pneumoniae HS11286, (C) JS184, and (D) TZ20. K. pneumoniae strains were purple/red and rod-shaped; the red fluffy masses were exopolysaccharides. bla_KPC, beta-lactamase K. pneumoniae carbapenemase gene; bla_KPC(+)-HvKP, bla_KPC(+) hypervirulent K. pneumoniae; Hv-bla_KPC(+)-KP, hypervirulent bla_KPC(+) K. pneumoniae.

Fitness Analysis

Among the 9 putative bla_KPC(+)-HvKP and 26 Hv-bla_KPC(+)-KP strains, 8 strains were chosen to represent each serotype: JS184 (K2, ST65), JS185 (K2, ST977), JS210 (K47, ST11), TZ16 (K64, ST11), TZ19 (K20, ST81), TZ20 (K20, ST81), TZ58 (K57, ST not defined), and JSH17 (K24, ST15). One-way ANOVA analysis indicated F = 0.9081 and p = 0.5178, which demonstrated similar growth and no fitness cost for the 8 strains (Figure 6).

FIGURE 6

Figure 6 Growth curves of 10 putative bla_KPC(+)-HvKP and Hv-bla_KPC(+)-KP strains. bla_KPC, beta-lactamase K. pneumoniae carbapenemase gene; bla_KPC(+)-HvKP, bla_KPC(+) hypervirulent K. pneumoniae; Hv-bla_KPC(+)-KP, hypervirulent bla_KPC(+) K. pneumoniae; OD₆₀₀, optical density at 600 nm.

Expression of galF

Figure 7 shows the relative expression of galF in the putative strains compared to that in the control strains NTUH-K2044 and HS11286. JS184, TZ20, JSH17, and HS11286 showed high galF expression.

FIGURE 7

Figure 7 Expression of galF among 35 putative bla_KPC(+)-HvKP and Hv-bla_KPC(+)-KP strains. Klebsiella pneumoniae NTUH-K2044 was used as the standard to which others were compared. Kruskal-Wallis test was used for comparison. bla_KPC, beta-lactamase K. pneumoniae carbapenemase gene; bla_KPC(+)-HvKP, bla_KPC(+) hypervirulent K. pneumoniae; Hv-bla_KPC(+)-KP, hypervirulent bla_KPC(+) K. pneumoniae; ns, not significant; ****, p < 0.0001; *, p < 0.05.

Mouse Lethality Tests

The survival curve for mice inoculated (10⁶ CFU) with the two bla_KPC(+)-HvKP strains, JS184 and TZ20, is shown in Figure 8. Log-rank (Mantel-Cox) test yielded values of χ² = 11.4286, p = 0.0096 for the four groups (JS184, TZ20, HS11286, and NTUH-K2044); χ² = 1.5521, p = 0.4602 for three groups (JS184, TZ20, and NTUH-K2044). Therefore, the virulence of JS184 and TZ20 was similar to that of NTUH-K2044, and was higher than that of HS11286. The LD₅₀ values were 10⁶ CFU for NTUH-K2044, 10³ CFU for JS184, < 10⁶ CFU for TZ20, > 10⁷ CFU for HS11286 and the other 33 putative bla_KPC(+)-HvKP/Hv-bla_KPC(+)-KP strains.

FIGURE 8

Figure 8 Survival curves of mice inoculated with two bla_KPC(+)-HvKP strains. bla_KPC(+)-HvKP, beta-lactamase K. pneumoniae carbapenemase gene-positive hypervirulent Klebsiella pneumoniae.

Traits of Confirmed bla_KPC(+)-HvKP Strains

The two confirmed bla_KPC(+)-HvKP strains, JS184 and TZ20, showed differences in ST, irp2 expression, and serotype (Table 1).

TABLE 1

Table 1 Traits of confirmed bla_KPC(+)-HvKP strains.

Discussion

A combination of carbapenem resistance and hypervirulence in õõõõõK. pneumoniae strains has been recently reported (Gu et al., 2018). However, the epidemiology of CR-HvKP and Hv-CRKP has not been extensively studied. To our knowledge, this is the first epidemiological surveillance study on bla_KPC(+)-HvKP and Hv-bla_KPC(+)-KP strains in China using a mouse lethality test to evaluate their prevalence.

In total, 13 key virulence genes in K. pneumoniae were investigated in this study; Figure 2 shows a remarkable divergence in their distribution. The extremely high detection rates of fimH, mrkD, entB, and wzi indicate ubiquitous production of type 1 fimbriae, type 2 fimbriae, enterobactins, and capsules. Another siderophore gene irp2 was present in a large proportion of the strains (76.1%), and is typically carried by ICEKp1 in the chromosome; The remaining 2 siderophore genes iucA and iroN showed detection rates of approximately 20.0–30.0%, and are usually harboured by pK2044 and pLVPK-like virulence plasmids (Struve et al., 2015). The virulence genes peg-344, p-rmpA, and p-rmpA2 are also present in pK2044- and pLVPK-like virulence plasmids (Russo and Marr, 2019), and therefore yielded similar detection rates to those of iucA and iroN (p > 0.05). The c-rmpA gene is present in the chromosome and often found in PLA specimens (Hsu et al., 2011). No such specimens were included in this study, which may explain the detection rate of 0.0%. The low detection rate of wzy-K1 (6.4%) suggests the rarity of K1 K. pneumoniae in clinical practice.

Various molecular factors were evaluated to screen for CR-HvKP and Hv-CRKP. Zhang et al. previously evaluated iucA, iroN, rmpA, and rmpA2 to check for the presence of virulence plasmids, and performed the G. mellonella lethality test to identify CR-HvKP, which yielded a rate of 5.2% (55/1052) for CR-HvKP (Zhang et al., 2020). We previously defined HvKP as: positive wzy-K1, ≥3 positive siderophore genes (entB, irp2, iroN, and iucA), or ≥1 positive capsule-regulating genes (p-rmpA2, c-rmpA/A2, and p-rmpA), and estimated a rate of 5.6% (29/521) for Hv-bla_KPC(+)-KP (Shon et al., 2013), which was also applied in this study. Harada et al. defined HvKP as strains carrying virulence genes, rmpA, rmpA2, iroBCDN, iucABCD, or iutA (Harada et al., 2019). Russo et al. confirmed that the G. mellonella lethality experiment cannot accurately differentiate HvKP from cKP (Russo and MacDonald, 2020). Thus, the mouse lethality test may represent the only approach to determine the exact prevalence of CR-HvKP/Hv-CRKP. In this study, only two bla_KPC(+)-HvKP strains, but no Hv-bla_KPC(+)-KP strains, were eventually confirmed using a mouse lethality test, showing a rate of 0.5% (2/436) for CR-HvKP which was far lower than that reported in other studies (Zhang et al., 2020; Hu et al., 2021). Owing to the predominance of KPC-induced carbapenem resistance (Lin et al., 2018), the actual prevalence of CR-HvKP should be approximately 0.5–1.0% in mainland China in 2017. The considerable difference in prevalence rates determined between this study and other reports highlights the need to elucidate why such biomarkers are not reliable and the difference between mouse and G. mellonella lethality tests. Zhang et al., reported that only one K. pneumoniae strain has been confirmed as CR-HvKP among three strains that harbour rmpA based on a mouse lethality test (Zhang et al., 2015). The fact that rmpA genes are non-functional in cKP may be attributed to different genetic backgrounds, although rmpA-related genes, such as kvrA, kvrB, and rcsB (Palacios et al., 2018; Walker et al., 2019), were found to be widely distributed in both cKP and HvKP (data not shown).

Although Hv-CRKP and CR-HvKP strains are currently emerging worldwide (Gu et al., 2018; Karlsson et al., 2019), our study revealed that the emergence of CR-HvKP is a relatively greater concern owing to its prevalence. In this study, two confirmed bla_KPC(+)-HvKP strains were found, including JS184 (K2) and TZ20 (K20), which showed no fitness cost, no hypercapsule production, and high expression of galF which is responsible for the synthesis of capsule precursor (Peng et al., 2018; Walker et al., 2019). However, JS184 showed a positive string test and exopolysaccharide production in contrast to TZ20. The reason for this is not known. In addition, JS184 and TZ20 also showed different ST and irp2 expression.

This study had a few limitations. First, only typical siderophore genes were referred to, but not their expression. Second, capsule staining is not sufficient to differentiate capsules of various thicknesses, which may impact virulence.

Taken together, our findings indicate that CR-HvKP may emerge more often than Hv-CRKP; the former accounted for less than 1.0% of the strains evaluated via mouse lethality tests among clinical K. pneumoniae strains in mainland China in 2017.

Data Availability Statement

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/Supplementary Material.

Ethics Statement

The animal study was reviewed and approved by the Institutional Animal Care and Use Committee of the School of Pharmacy, Fudan University (Shanghai, China).

Author Contributions

DH, WC, and QZ contributed to conception of the study. ML, LY, ZY, YW, YH, GL, and XJ collected and identified the strains. DH, WC, QZ, PF, DT, and WW performed PCR and MLST analyses, string tests, capsular staining, periodic acid-Schiff staining, and fitness tests. PR and QM performed mouse lethality tests. DH, WC, and QZ wrote the manuscript which was revised by XJ and LY. All authors read and approved the final manuscript.

Funding

This study was supported by research grants from the National Natural Science Foundation of China (grants 81871692, 82172315, 82172315 and 81572031), Shanghai Municipal Key Clinical Specialty (Laboratory Medicine, No. shslczdzk03303), and the Shanghai Municipal Science and Technology Commission (grant number 19JC1413002).

Conflict of Interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Publisher’s Note

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.

Acknowledgments

We thank Professor Jin-Town Wang from the Department of Internal Medicine, National Taiwan University Hospital, for providing the strain NTUH-K2044.

Supplementary Material

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fcimb.2022.882210/full#supplementary-material

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