Clinical and molecular characteristics of carbapenem-resistant Hypervi

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Clinical and molecular characteristics of carbapenem-resistant high-virulence Klebsiella pneumoniae in a tertiary hospital in Shanghai
Zhou Cong, 1 Wu Qiang, 1 He Leqi, 1 Zhang Hui, 1 Xu Maosuo, 1 Bao Yuyuan, 2 Jin Zhi, 3 Fang Shen 11 Department of Clinical Laboratory Medicine, Shanghai Fifth People’s Hospital, Fudan University, Shanghai, People’s Republic of China; 2 Shanghai Jiaotong Department of Laboratory Medicine, Shanghai Children’s Hospital, Shanghai, People’s Republic of China; 3 Department of Neurology, Shanghai Fifth People’s Hospital, Fudan University Corresponding author: Fang Shen, Department of Clinical Laboratory Medicine, Shanghai Fifth People’s Hospital, Fudan University, No. 128 Ruili Road, Minhang District, Shanghai, Postcode 200240 of ChinaTel +86 18021073261 Email [email protected] Background: The fusion of carbapenem resistance and hypervirulence in Klebsiella pneumoniae has led to major public health challenges. In recent years, there have been more and more reports on carbapenem-resistant high-virulence Klebsiella pneumoniae (CR-hvKP) isolates. Materials and methods: A retrospective analysis of the clinical data evaluation of patients infected with CR-hvKP from January 2019 to December 2020 in a tertiary hospital. Calculate the Klebsiella pneumoniae, Klebsiella pneumoniae (hmKP), carbapenem-resistant Klebsiella pneumoniae (CR-hmKP) and carbapenem-resistant high-virulence pneumonia collected within 2 years The number of isolates of Leberella (CR-hvKP). PCR detection of resistance genes, virulence-related genes, capsular serotype genes and multilocus sequence typing (MLST) of CR-hvKP isolates. Results: A total of 1081 non-repetitive Klebsiella pneumoniae strains were isolated during the study. , Including 392 strains of Klebsiella pneumoniae (36.3%), 39 strains of CR-hmKP (3.6%) and 16 strains of CR-hvKP (1.5%). Approximately 31.2% (5/16) of CR-hvKP will be isolated in 2019, and approximately 68.8% (11/16) of CR-hvKP will be isolated in 2020. Among the 16 CR-hvKP strains, 13 strains are ST11 and serotype K64, 1 strain is ST11 and K47 serotypes, 1 strain is ST23 and K1 serotypes, and 1 strain is ST86 and K2 serotypes. The virulence-related genes entB, fimH, rmpA2, iutA, and iucA are present in all 16 CR-hvKP isolates, followed by mrkD (n=14), rmpA (n=13), aerobactin (n=2) , AllS (n=1). The 16 CR-hvKP isolates all carry the carbapenemase gene blaKPC-2 and the extended-spectrum β-lactamase gene blaSHV. The results of ERIC-PCR DNA fingerprinting showed that 16 CR-hvKP strains were highly polymorphic, and the bands of each strain were significantly different, showing a sporadic state. Conclusion: Although CR-hvKP is distributed sporadically, it is increasing year by year. year. Therefore, clinical attention should be aroused, and necessary measures should be taken to avoid the cloning and spread of superbug CR-hvKP. Keywords: Klebsiella pneumoniae, carbapenem resistance, high virulence, high mucus, epidemiology
Klebsiella pneumoniae is an opportunistic pathogen that can cause a variety of infections, including pneumonia, urinary tract infections, bacteremia, and meningitis. 1 In the past thirty years, unlike the classic Klebsiella pneumoniae (cKP), a new highly virulent Klebsiella pneumoniae (hvKP) hypermucosal mucus has become a clinically important pathogen, which can be found in Highly aggressive infections such as liver abscesses are caused in healthy and immunocompromised individuals. 2 It is worth noting that these infections are usually accompanied by destructive disseminated infections, including endophthalmitis and meningitis. 3 The production of high mucosal mucosal phenotype hvKP is usually due to the increased production of capsular polysaccharides and the presence of specific virulence genes, such as rmpA and rmpA2.4. The high mucus phenotype is usually determined by the “string test”. The Klebsiella pneumoniae colonies grown overnight on blood agar plates are stretched with a loop. When a viscous rope with a length of >5mm is formed, the “rope test” is positive. 5 A recent study showed that peg-344, iroB, iucA, rmpA rmpA2 and rmpA2 are biomarkers that can accurately identify hvkp. 6 In this study, the highly virulent Klebsiella pneumoniae was defined as having a highly mucus viscous phenotype (positive string test result) and carrying Klebsiella pneumoniae virulence plasmid related sites (rmpA2, iutA, iucA) In the 1980s, Taiwan’s case reports first described community-acquired liver abscesses caused by hvKP, accompanied by severe end-organ damage, such as meningitis and endophthalmitis. 7,8 hvKP has sporadic transmission in many countries in Asia, Europe and America. Although several cases of hvKP have been reported in Europe and the Americas, the prevalence of hvKP mainly occurred in Asian countries, especially China. 9
In general, hvKP is more sensitive to antibiotics, while carbapenem-resistant Klebsiella pneumonia (CRKP) is less toxic. However, with the spread of drug resistance and virulence plasmids, CR-hvKP was first described by Zhang et al. in 2015, and there are more and more domestic reports. 10 Since CR-hvKP can cause serious and difficult-to-treat infections, if a pandemic clone appears, it may become the next “superbug”. To date, most infections caused by CR-hvKP have occurred in sporadic cases, and small-scale outbreaks are rare. 11,12
At present, the detection rate of CR-hvKP is low, and there are few related studies. The molecular epidemiology of CR-hvKP is different in different regions, so it is necessary to study the clinical distribution and molecular epidemiological characteristics of CR-hvKP in this region. This study comprehensively analyzed the resistance genes, virulence-related genes and MLST of CR-hvKP. We tried to investigate the prevalence and molecular epidemiology of CR-hvKP in a tertiary hospital in Shanghai, eastern China. This study is of great significance for understanding the molecular epidemiology of CR-hvKP in Shanghai.
The non-repetitive Klebsiella pneumoniae isolates from Shanghai Fifth People’s Hospital affiliated to Fudan University from January 2019 to December 2020 were retrospectively collected, and the percentages of hmKP, CRKP, CR-hmkp and CR-hvKP were calculated. All isolates were identified by the VITEK-2 compact automatic microbial analyzer (Biomerieux, Marcy L’Etoile, France). Maldi-Tof mass spectrometry (Bruker Daltonics, Billerica, MA, USA) was used to recheck the identification of bacterial strains. The high mucus phenotype is determined by the “string test”. When imipenem or meropenem is resistant, carbapenem resistance is determined through a drug susceptibility test. Highly virulent Klebsiella pneumoniae is defined as having a high mucus phenotype (positive string test result) and carrying Klebsiella pneumoniae virulence plasmid related sites (rmpA2, iutA, iucA)6.
A single Klebsiella pneumoniae colony was inoculated on a 5% sheep blood agar plate. After incubating overnight at 37°C, gently pull up the colony with an inoculating loop and repeat 3 times. If a viscous line is formed three times and the length is greater than 5mm, the “line test” is considered positive, and the strain has a high mucus phenotype.
In the VITEK-2 compact automatic microbial analyzer (Biomerieux, Marcy L’Etoile, France), the antimicrobial susceptibility to several commonly used antibiotics was detected by broth micro-dilution. The results are interpreted according to the guidance document developed by the Clinical and Laboratory Standards Institute (CLSI, 2019). E. coli ATCC 25922 and Klebsiella pneumoniae ATCC 700603 were used as controls for antimicrobial susceptibility testing.
Genomic DNA of all Klebsiella pneumoniae isolates were extracted by TIANamp Bacteria Genomic DNA Kit (Tiangen Biotech Co. Ltd., Beijing, China). Extended-spectrum β-lactamase genes (blaCTX-M, blaSHV and blaTEM), carbapenemase genes (blaKPC, blaNDM, blaVIM, blaIMP and blaOXA-48) and 9 representative virulence-related genes, including pLVPK Plasmid-like loci (allS, fimH, mrkD, entB, iutA, rmpA, rmpA2, iucA, and aerobactin) were amplified by PCR as previously described. 13,14 Capsular serotype-specific genes (K1, K2, K5, K20, K54, and K57) were amplified by PCR as described above. 14 If negative, amplify and sequence the wzi locus to determine the capsular serotype-specific genes. 15 The primers used in this study are listed in Table S1. The positive PCR products were sequenced by the NextSeq 500 sequencing platform (Illumina, San Diego, CA, USA). Compare nucleotide sequences by running BLAST on the NCBI website (http://blast.ncbi.nlm.nih.gov/Blast.cgi).
Multi-site sequence typing (MLST) was performed as described in the Pasteur Institute MLST website (https://bigsdb.pasteur.fr/klebsiella/klebsiella.html). The seven housekeeping genes gapA, infB, mdh, pgi, phoE, rpoB and tonB were amplified by PCR and sequenced. The sequence type (ST) is determined by comparing the sequencing results with the MLST database.
The homology of Klebsiella pneumoniae was analyzed. Klebsiella pneumoniae genomic DNA was extracted as a template, and the ERIC primers are shown in Table S1. PCR amplifies genomic DNA and constructs a fingerprint of genomic DNA. 16 PCR products were detected by 2% agarose gel electrophoresis. DNA fingerprinting results were identified using QuantityOne software band recognition, and genetic analysis was performed using the unweighted paired group method (UPGMA) of arithmetic mean. The isolates with similarity> 75% are considered to be the same genotype, and those with similarity <75% are considered to be different genotypes.
Use statistical software package SPSS for Windows 22.0 to analyze the data. The data are described as mean ± standard deviation (SD). Categorical variables were evaluated by chi-square test or Fisher’s exact test. All statistical tests are 2-tailed, and a P value of <0.05 is considered statistically significant.
Shanghai Fifth People’s Hospital affiliated to Fudan University collected 1081 Klebsiella pneumoniae isolates from January 1, 2019 to December 31, 2020, and excluded duplicate isolates from the same patient. Among them, 392 strains (36.3%) were hmKP, 341 strains (31.5%) were CRKP, 39 strains (3.6%) were CR-hmKP, and 16 strains (1.5%) were CR-hvKP. It is worth noting that 33.3% (13/39) of CR-hmKP and 31.2% (5/16) of CR-hvKP are from 2019, 66.7% (26/39) of CR-hmKP and 68.8% (11/ 16) The CR-hvKP was separated from 2020. From sputum (17 strains), urine (12 strains), drainage fluid (4 strains), blood (2 strains), pus (2 strains), bile (1 isolation) and pleural effusion (1 isolation), respectively. Sixteen types of CR-hvKP were recovered from sputum (9 isolates), urine (5 isolates), blood (1 isolate) and pleural effusion (1 isolate).
Through strain identification, drug sensitivity test, string test and virulence-related gene detection, 16 CR-hvKP strains were screened. The clinical characteristics of the 16 patients infected with CR-hvKP isolates are summarized in Table 1. 13 of the 16 patients (81.3%) were men, and all patients were older than 62 years (mean age: 83.1±10.5 years). They came from 8 wards, and more than half came from the central ICU (9 cases). Basic diseases include cerebrovascular disease (75%, 12/16), hypertension (50%, 8/16), chronic obstructive pulmonary disease (50%, 8/16), etc. Invasive surgery includes mechanical ventilation (62.5%, 10/16), urinary catheter (37.5%, 6/16), gastric tube (18.8%, 3/16), surgery (12.5%, 2/16) and intravenous catheter (6.3%, 1/16). Nine of the 16 patients died, and 7 patients improved and were discharged.
The 39 CR-hmKP isolates were divided into two groups according to the length of the sticky string. Among them, 20 CR-hmKP isolates with viscous string length ≤ 25 mm were divided into one group, and 19 CR-hmKP isolates with viscous string length> 25 mm were divided into another group. PCR method detects the positive rate of virulence-related genes rmpA, rmpA2, iutA and iucA. The positive rates of CR-hmKP virulence-related genes in the two groups are shown in Table 2. There was no statistical difference in the positive rate of CR-hmKP virulence-related genes between the two groups.
Table 3 lists the detailed antimicrobial resistance profiles of the 16 drugs. 16 CR-hvKP isolates showed multi-drug resistance. All isolates were treated with ampicillin, ampicillin/sulbactam, cefoperazone/sulbactam, piperacillin/tazobactam, cefazolin, cefuroxime, ceftazidime, ceftriaxone, cefepime, Cefoxitin, imipenem, and meropenem are resistant. Trimethoprim-sulfamethoxazole had the lowest resistance rate (43.8%), followed by amikacin (62.5%), gentamicin (68.8%) and ciprofloxacin (87.5%).
The distribution of virulence-related genes, antimicrobial resistance genes, capsular serotype genes and MLST of 16 CR-hvKP isolates is shown in Figure 1. The results of agarose gel electrophoresis of some virulence-related genes, antimicrobial resistance genes and capsular serotype genes are shown in Figure 1. Figure 2. MLST analysis shows a total of 3 STs, ST11 is the most dominant ST (87.5%, 14/16), followed by ST23 (6.25%, 1/16) and ST86 (6.25%, 1/16). According to the results of wzi typing, 4 different capsular serotypes were identified (Figure 1). Among the 16 carbapenem-resistant hvKP isolates, K64 is the most common serotype (n=13), followed by K1 (n=1), K2 (n=1) and K47 (n=1). In addition, the capsular serotype K1 strain is ST23, the capsular serotype K2 strain is ST86, and the remaining 13 strains of K64 and 1 strain of K47 are all ST11. The positive rates of 9 virulence genes in 16 CR-hvKP isolates are shown in Figure 1. , The virulence-related genes entB, fimH, rmpA2, iutA, and iucA are present in 16 CR-hvKP strains, followed by mrkD (n = 14), rmpA (n = 13), aerobacterin (n = 2) , AllS (n=1). The 16 CR-hvKP isolates all carry the carbapenemase gene blaKPC-2 and the extended-spectrum β-lactamase gene blaSHV. 16 CR-hvKP isolates did not carry carbapenem genes blaNDM, blaVIM, blaIMP, blaOXA-48 and extended-spectrum β-lactamase genes blaTEM, blaCTX-M-2 group, and blaCTX-M-8 group. Among the 16 CR-hvKP strains, 5 strains carried the extended-spectrum β-lactamase gene blaCTX-M-1 group, and 6 strains carried the extended-spectrum β-lactamase gene blaCTX-M-9 group.
Figure 1 The virulence-related genes, antimicrobial resistance genes, capsular serotype genes and MLST of 16 CR-hvKP isolates.
Figure 2 Agarose gel electrophoresis of some virulence-related genes, antimicrobial resistance genes and capsular serotype genes.
Note: M, DNA marker; 1, blaKPC (893bp); 2, entB (400bp); 3, rmpA2 (609bp); 4, rmpA (429bp); 5, iucA (239bp); 6, iutA (880bp); 7 , Aerobacterin (556bp); 8, K1 (1283bp); 9, K2 (641bp); 10, all S (508bp); 11, mrkD (340bp); 12, fimH (609bp).
ERIC-PCR was used to analyze the homology of 16 CR-hvKP isolates. After PCR amplification and agarose gel electrophoresis, there are 3-9 DNA fragments. The fingerprinting results showed that 16 CR-hvKP isolates were highly polymorphic, and there were obvious differences among the isolates (Figure 3).
In recent years, there have been more and more reports on CR-hvKP isolates. The appearance of CR-hvKP isolates poses a major threat to public health because they can cause serious, difficult-to-treat infections in healthy people. In this study, the prevalence and molecular epidemiological characteristics of CR-hvKP in a tertiary hospital in Shanghai from 2019 to 2020 were studied to assess whether there is a risk of CR-hvKP outbreak and its development trend in this area. At the same time, this study can provide a more comprehensive evaluation of clinical infectivity, which is of great significance for preventing the further spread of such isolates.
This study retrospectively analyzed the clinical distribution and trend of CR-hvKP from 2019 to 2020. From 2019 to 2020, CR-hvKP isolates showed an increasing trend. Approximately 31.2% (5/16) of CR-hvKP was isolated in 2019, and 68.8% (11/16) of CR-hvKP was isolated in 2020, which is consistent with the upward trend of CR-hvKP reported in the literature. Since Zhang et al. first described CR-hvKP in 2015,10 more and more CR-hvKP literature has been reported, 17-20 mainly in the Asia-Pacific region, especially in China. CR-hvKP is a super bacterium with super virulence and multi-drug resistance. It is harmful to people’s health and has a high mortality rate. Therefore, attention should be paid and measures should be taken to prevent its spread.
The antibiotic resistance analysis of 16 CR-hvKP isolates showed a high rate of antibiotic resistance. All isolates were treated with ampicillin, ampicillin/sulbactam, cefoperazone/sulbactam, piperacillin/tazobactam, cefazolin, cefuroxime, ceftazidime, ceftriaxone, cefepime, Cefoxitin, imipenem, and meropenem are resistant. Trimethoprim-sulfamethoxazole had the lowest resistance rate (43.8%), followed by amikacin (62.5%), gentamicin (68.8%) and ciprofloxacin (87.5%). The resistance rate of CR-hmkp studied by Lingling Zhan and others is similar to this study [12]. Patients infected with CR-hvKP have many basic diseases, low immunity, and weak independent sterilization ability. Therefore, timely treatment based on the results of the antimicrobial sensitivity test is very important. If necessary, the infected site can be found and treated by drainage, debridement and other methods.
The 39 CR-hmKP isolates were divided into two groups according to the length of the sticky string. Among them, 20 CR-hmKP isolates with viscous string length ≤ 25 mm were divided into one group, and 19 CR-hmKP isolates with viscous string length> 25 mm were divided into another group. Comparing the positive rates of CR-hmKP virulence-related genes between the two groups, there was no statistically significant difference in the positive rates of virulence genes between the two groups. Research by Lin Ze et al. showed that the positive rate of virulence genes of Klebsiella pneumoniae was significantly higher than that of classic Klebsiella pneumoniae. 21 However, whether the positive rate of virulence genes is positively correlated with the length of the sticky chain remains unclear. Other studies have shown that the classic Klebsiella pneumoniae may also be a highly virulent Klebsiella pneumoniae, with a higher positive rate of virulence genes. 22 This study found that the virulence gene positive rate of CR-hmKP is not positively correlated with the length of mucus. String (or does not increase with the length of the sticky string).
The ERIC PCR fingerprints of this study are polymorphic, and there is no clinical crossover between patients, so 16 patients with CR-hvKP infection are sporadic cases. In the past, most infections caused by CR-hvKP have been reported as isolated or sporadic cases, 23,24 and small-scale outbreaks of CR-hvKP are rare in the literature. 11,25 ST11 is the most common ST11 in CRKP and CR-hvKP isolates in China. 26,27 Although ST11 CR-hvKP accounted for 87.5% (14/16) of the 16 CR-hvKP isolates in this study, it cannot be assumed that the 14 ST11 CR-hvKP strains are from the same clone, so ERIC PCR fingerprinting is required. Homology analysis.
In this study, all 16 patients infected with CR-hvKP underwent invasive surgery. According to reports, the fatal outbreak of ventilator-associated pneumonia caused by CR-hvKP11 indicates that invasive procedures may increase the risk of CR-hvKP infection. At the same time, 16 patients infected with CR-hvKP have underlying diseases, of which cerebrovascular diseases are the most common. A previous study showed that cerebrovascular disease is a significant independent risk factor for CR-hvKP infection. 28 The reason for this phenomenon may be the weakened immunity of patients with cerebrovascular disease, the pathogenic bacteria cannot be excluded independently, and only their bactericidal effect is relied on. Antibiotics will lead to a combination of multi-drug resistance and hypervirulence in the long run. Among the 16 patients, 9 died, and the mortality rate was 56.3% (9/16). The mortality rate is higher than 10,12 in previous studies, and lower than 11,21 reported in previous studies. The average age of 16 patients was 83.1±10.5 years, indicating that the elderly are more susceptible to CR-hvKP. Previous studies have shown that young people are more susceptible to infection. The virulence of Klebsiella pneumoniae. 29 However, other studies have shown that the elderly are susceptible to the highly virulent Klebsiella pneumoniae24,28. This study is consistent with this.
Among the 16 CR-hvKP strains, except for one ST23 CR-hvKP and one ST86 CR-hvKP, the other 14 strains are all ST11 CR-hvKP. The capsular serotype corresponding to ST23 CR-hvKP is K1, and the corresponding capsular serotype of ST86 CR-HVKP is K2, similar to previous studies. 30-32 Patients infected with ST23 (K1) CR-hvKP or ST86 (K2) CR-hvKP died, and the mortality rate (100%) was significantly higher than that of patients infected with ST11 CR-hvKP (50%). As shown in Figure 1, the positive rate of ST23 (K1) or ST86 (K2) strains of virulence-related genes is higher than that of ST11 (K64) strains. The mortality may be related to the positive rate of virulence-related genes. In this study, 16 strains of CR- hvKP all carry the carbapenemase gene blaKPC-2 and the extended-spectrum β-lactamase gene blaSHV. blaKPC-2 is the most common carbapenemase gene in CR-hvKP in China. 33 In the study of Zhao et al., 25blaSHV is the extended-spectrum β-lactamase gene with the highest positive rate. The virulence genes entB, fimH, rmpA2, iutA, and iucA are present in all 16 CR-hvKP isolates, followed by mrkD (n=14), rmpA (n=13), anaerobicin (n=2), allS (n = 1), which is similar to the previous study. 34 Some studies have shown that rmpA and rmpA2 (modulators of mucus phenotype genes) can promote the secretion of capsular polysaccharides, leading to hypermucoid phenotypes and increased virulence. 35 Aerobacterins are encoded by the iucABCD gene, and their homologous receptors are encoded by the iutA gene, so they have a higher level of virulence in the G. mellonella infection assay. allS is a marker of K1-ST23, not in pLVPK, pLVPK is a virulence plasmid from K2 super virulence type. allS is an HTH type transcription activator. These virulence genes are known to contribute to virulence and are responsible for colonization, invasion and pathogenicity. 36
This study describes the prevalence and molecular epidemiology of CR-hvKP in Shanghai, China. Although the infection caused by CR-hvKP is sporadic, it is increasing year by year. The results support previous research and show that ST11 CR-hvKP is the most popular CR-hvKP in China. ST23 and ST86 CR-hvKP showed higher virulence than ST11 CR-hvKP, although they are both highly virulent Klebsiella pneumoniae. As the percentage of highly virulent Klebsiella pneumoniae increases, the resistance rate of Klebsiella pneumoniae may decrease, which will lead to blind optimism in clinical practice. Therefore, it is necessary to study the virulence and drug resistance of Klebsiella pneumoniae.
This study was approved by the Medical Ethics Committee of Shanghai Fifth People’s Hospital (No. 104, 2020). Clinical samples are part of routine hospital laboratory procedures.
Thank you to all the staff of the Central Laboratory of Shanghai Fifth People’s Hospital for providing technical guidance for this study.
This work was supported by the Natural Science Foundation of Minhang District, Shanghai (approval number: 2020MHZ039).
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