Abstract
Objective
This study aimed to characterize hypervirulent Acinetobacter baumannii (Hv-AB) strains and elucidate their clinical and microbiological profiles in a hospital setting.
Methods
We conducted a retrospective analysis of 49 A. baumannii strains isolated from a Chinese hospital between July 2019 and June 2020. Strains were classified as Hv-AB or non-Hv-AB using Galleria mellonella infection models and survival curve analysis. Phenotypic assessments included antimicrobial susceptibility testing, serum resistance assays, biofilm formation quantification, and string tests.
Results
Among the 49 strains analyzed, 17 (34.7%) demonstrated hypervirulence (Hv-AB). These hypervirulent strains belonged to 9 distinct multilocus sequence typing(MLST) sequence types: ST191, ST195, ST208, ST381, ST540, ST1145, ST1474, ST1893, and ST2207. Phylogenetic analysis indicated that ST208 served as the founder sequence type (ST). Compared with ST208, ST191, ST195, ST540, ST1145, and ST1893 differed in only one housekeeping gene, classifying them as single-locus variants (SLVs); whereas ST381 and ST2207 differed in two loci, classifying them as double-locus variants (DLVs). Intensive Care Unit (ICU) is the main source for detecting high-virulence strains of A.baumannii. Hv-AB showed higher resistance rates to commonly used antibiotics, particularly sulfamethoxazole-trimethoprim, compared to non-Hv-AB. Patients with higher red blood cell counts or hemoglobin levels may be more susceptible to Hv-AB infection. No high viscosity of Hv-AB was found. Serum resistance and biofilm formation were not unique characteristics of Hv-AB.
Conclusions
This study identifies the emergence of highly virulent Hv-AB strains in clinical settings and underscores their genetic homology. These findings contribute to a deeper understanding of Hv-AB.
Keywords: Acinetobacter baumannii, Hv-AB, Virulence, Multilocus sequence typing
Background
Acinetobacter baumannii is a Gram-negative, opportunistic pathogen that has emerged as a significant threat in healthcare settings worldwide. Known for its remarkable ability to survive in harsh environments and develop resistance to multiple antibiotics, A.baumannii is a leading cause of hospital-acquired infections, including pneumonia, bloodstream infections, and wound infections, particularly in critically ill patients [1]. The emergence of multidrug-resistant (MDR) and extensively drug-resistant (XDR) strains has significantly limited therapeutic options, leading to higher mortality rates, particularly among immunocompromised patients [2].
In recent years, specific strains of A. baumannii, characterized by heightened virulence, have garnered considerable attention due to their increased prevalence and resistance profiles, particularly in high-risk environments such as intensive care units(ICUs) [3–5]. These strains exhibit unique virulence traits, such as increased biofilm formation, enhanced immune evasion mechanisms, and the production of virulence factors like phospholipases, proteases, and siderophores [6]. Furthermore, the convergence of hypervirulence and antibiotic resistance in some strains have raised concerns about their potential to cause untreatable infections [7].
The emergence of hypervirulent A.baumannii (Hv-AB) poses a significant challenge to both clinical and public health settings. A comprehensive understanding of its clinical and microbiological characteristics is crucial to address the growing threat it represents. This study aims to identify hypervirulent A.baumannii strains and analyze their clinical and microbiological features, thereby providing a scientific foundation for the prevention and control of Hv-AB infections.
Methods
Sample collection and susceptibility identification
Between July 2019 and June 2020, 131 A.baumannii isolates were initially obtained from clinical specimens at the First Affiliated Hospital of Nanchang University (Jiangxi Province, China). After applying exclusion criteria to remove duplicate isolates from identical anatomical sites in the same patient (n = 63) and strains with incomplete clinical records (n = 19), 49 unique strains were retained for analysis. Species identification was performed using the VITEK-2 Compact system (BioMérieux, Marseille, France) according to the manufacturer’s specifications. Virulence stratification was achieved through Galleria mellonella (G. mellonella) infection models with 72-hour survival monitoring, categorizing strains into hypervirulent A.baumannii (Hv-AB) and non-hypervirulent counterparts (non-Hv-AB). Reference strains AB5075 (Hv-AB control, provided by Zhejiang University) and ATCC 19,606 were included as experimental controls. All isolates were cryopreserved at − 80℃ in brain-heart infusion broth containing 20% glycerol. Clinical data encompassing demographic characteristics, hematological parameters (including erythrocyte counts and hemoglobin levels), comorbidities, and invasive procedures were retrospectively extracted from electronic medical records using standardized data collection protocols.
The antibiotic susceptibility of A. baumannii strains was also tested using the VITEK-2 automated platform (bioMérieux, Marcy l’ Etoile, France). Antimicrobial susceptibility testing was performed by the microdilution method and interpreted according to the guidelines provided by the Clinical and Laboratory Standards Institute (CLSI) in 2023 [8]. The evaluated antimicrobial agents included ampicillin/sulbactam, ceftriaxone, cefotaxime, cefepime, levofloxacin, trimethoprim-sulfamethoxazole, amikacin, ceftazidime, ciprofloxacin, gentamicin, meropenem, and tobramycin.
G. mellonella infection models
G. mellonella larvae, about 2–3 cm in length and 300 mg in weight, purchased from Tianjin Huiyu De Biotechnology Company, were used to determine the virulence level of the strain. As described in the literature [9], 3 ml of bacteria cultured in the logarithmic growth phase were centrifuged and resuspended in PBS to prepare a bacterial suspension with a concentration of 1.0 × 108 CFU/ml. Next, 10 µl of the bacterial suspension was injected into the abdominal cavity of G. mellonella larvae using a 25 µl Hamilton syringe, with 10 larvae injected per group. The larvae were observed for 96 h, and the number of deaths was recorded every 12 h. Three parallel experiments were performed in total. Finally, survival curves were plotted to show the mortality of G. mellonella larvae. Three control groups were included in all experiments: (1) A. baumannii ATCC 19,606 (moderate virulence reference strain), (2) A. baumannii AB5075 (high virulence reference strain), and (3) phosphate-buffered saline (PBS, sterile negative control). Reference strains were cultured under identical conditions as test strains (37 °C in LB broth to OD600 0.6 ± 0.05), with inoculum concentrations verified by both McFarland standards and plate counting. Control injections were performed daily alongside experimental groups to account for potential inter-day variability. According to the methods of references [3], the strain meeting the following three criteria at the same time was determined as the high virulence A.baumannii, while the strain not meeting all the above criteria was identified as the non-high virulence A.baumannii. The criteria are as follows: the survival rate of G. mellonella larvae infected is less than or equal to 20% at 96 h; the survival rate of G. mellonella larvae infected is significantly lower than that of A.baumannii ATCC 19,606 strain; the survival rate of G. mellonella larvae infected is lower than that of A.baumannii AB5075 strain or there is no statistical difference. This led to the identification of hypervirulent and non-hypervirulent strains of A.baumannii.
Multilocus sequence typing and strain homology analysis
The method we used is similar to that in the study of Huang et al. [10]. Seven housekeeping genes (gltA, gyrB, gdhB, recA, cpn60, gpi, and rpoD) were amplified by PCR, and the amplification products were sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The sequencing results were then compared with the MLST database of A.baumannii in PubMed (http://pubmlst.org/abaumannii/) to obtain the ST typing of A.baumannii. PCR primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd. The total PCR reaction system was 50 µl: 17 µl sterile ultrapure water, 6 µl DNA template, 25 µl Tap Mix, 1 µl upstream primer, and 1 µl downstream primer. The homology of strains was analyzed by goeBURST of Phyloviz software.
Serum resistance assay
Briefly, serum was isolated from healthy individuals, packaged, and stored at -80 °C. A.baumannii isolates were grown to mid-logarithmic phase in Luria-Bertani broth, and the bacterial suspension was diluted to a concentration of 1 × 106cfu/mL. Subsequently, 25ul of the bacterial suspension was added to Eppendorf tubes containing 75ul of normal human serum (NHS) and heat-inactivated serum (HIS), respectively. The mixtures were vortexed and incubated at 37 °C without shaking for 2 h. After incubation, colony counts were performed by serial dilution and incubation on Mueller-Hinton (MH) agar at 37 °C for 18 h to determine the viable bacterial count for each sample.The serum bactericidal effect was calculated with the following formula: Serum resistance=CFUs-NHS/CFUs-HIS. All experiments were repeated three times, and the results were expressed as survival rates. The experimental procedures described in the reference were adjusted appropriately [11].
String test
As described in the literature [12], the string test was performed on a single fresh colony grown overnight on a plate at 37 °C.A bacterial inoculating loop was used to gently lift the colony of A.baumannii from the plate and was repeatedly stretched for more than two times. A positive mucoid string test was defined as the length of the mucoid string of A.baumannii lifted was not less than 5 mm; otherwise, it was negative.
Biofilm formation assay
The biofilm was measured using crystal violet method, and the experimental operation and interpretation criteria referred to the literature [13, 14]. The optical density value (A) was read at the wavelength of 540 nm, and the A value of the negative control group was measured 18 times. The mean value of the negative control group plus three times the standard deviation (x ± 3s) was used as the negative control value Ac. If the value was greater than Ac, it was judged as positive biofilm formation, and the specific classification was as follows: strongly positive (4×Ac< A); positive (2×Ac< A ≤ 4×Ac); weakly positive (Ac< A ≤ 2×Ac); (4) negative (A ≤ Ac value).
Statistical methods
SPSS 19.0 software and Graphpad Prism 7.0 software were used for statistical analysis. Variables of measurement data conforming to normal distribution were expressed as X ± S, and the differences between groups were compared using t-test. Variables of measurement data not conforming to normal distribution were expressed as median and interquartile range [M(P25-P75)], and the differences between groups were compared using rank sum test. The survival curve was plotted using Kaplan-Meier method, and the P value was calculated using log-rank test. All statistical analyses were considered significant when P < 0.05.
Results
G. mellonella infection models and virulence distribution of strains
The 49 strains under investigation were designated as AB15, AB28, AB31, AB34, AB40, AB57, AB62, AB83, AB133, AB135, AB136, AB137, AB140, AB142, AB145, AB147, AB154, AB155, AB156, AB158, AB159, AB161, AB162, AB163, AB164, AB166, AB167, AB168, AB169, AB170, AB180, AB202, AB204, AB207, AB208, AB209, AB210, AB212, AB215, AB216, AB217, AB225, AB229, AB231, AB232, AB234, AB241, AB42, and AB245. Upon infection with A.baumannii, G. mellonella larvae exhibited decreased activity, with their bodies gradually turning black until death. Notably, a small number of G. mellonella larvae with robust resistance survived the infection, and the majority of these resilient larvae regained their vitality and gradually resumed normal activities within 48 h post-infection. The G. mellonella mortality rate reached 80% after 96 h of infection with the highly virulent reference strain AB5075, while the mortality rate with the standard strain ATCC19606 was only 10% after 96 h. Specifically, 100% mortality was observed with strains AB28, AB34, AB40, AB62, AB83, AB133, AB136, and AB140 at 96 h post-infection; 90% mortality with AB57, AB142, and AB147; 80% mortality with AB135, AB145, AB154, AB207, AB212, and AB242; 70% mortality with AB15, AB31, AB137, AB209, and AB241; 60% mortality with AB161, AB210, and AB231; 50% mortality with AB180, AB216, and AB245; 40% mortality with AB163, AB169, AB215, AB225, and AB234; 30% mortality with AB156, AB167, and AB208; 20% mortality with AB158, AB170, AB202, AB204, AB217, and AB229; and 10% mortality with AB155, AB159, AB162, AB164, AB166, and AB168. Notably, no mortality was recorded 96 h after injection with PBS (Fig. 1). Utilizing survival analysis in conjunction with the previously established criteria, we conclusively pinpointed 17 strains of A.baumannii exhibiting high virulence, specifically AB28, AB34, AB40, AB57, AB62, AB83, AB133, AB135, AB136, AB140, AB142, AB145, AB147, AB154, AB207, AB212, and AB242. Conversely, AB15, AB31, AB137, AB155, AB156, AB158, AB159, AB161, AB162, AB163, AB164, AB166, AB167, AB168, AB169, AB170, AB180, AB202, AB204, AB208, AB209, AB210, AB215, AB216, AB217, AB225, AB229, AB231, AB232, AB234, AB241, and AB245 were classified as non-Hv-AB.
Fig. 1.
Survival curve of G. mellonella infection. The number on the right side of each subfigure indicates the strain number of A.baumannii
Multilocus sequence typing and strain homology analysis
The ST typing of 49 strains of A.baumannii was identified, among which a total of 18 ST types were found in non-Hv-AB, and 9 ST types were found in Hv-AB (Fig. 2). Among the non-Hv-AB strains, 25% (8/32) belonged to ST540, 18.8% (6/32) belonged to ST208 and ST1474, and the remaining 37.5% (12/32) belonged to other ST types. In contrast, among Hv-AB, 29.4% (5/17) belonged to ST1474, and the remaining 70.6% (12/17) belonged to other ST types.A total of 9 ST types were identified among the 17 hypervirulent strains, including ST191 (11.8%,2/17), ST195 (5.9%,1/17), ST208 (11.8%,2/17), ST381 (5.9%,1/17), ST540 (11.8%,2/17), ST1145 (5.9%,1/17), ST1474 (29.4%,5/17), ST1893 (5.9%,1/17), and ST2207 (11.8%,2/17).
Fig. 2.
Distribution of Hv-AB and non-Hv-AB MLST Typing. ST: multilocus sequence typing; Hv-AB: hypervirulent A.baumannii;non-Hv-AB: non-hypervirulent A.baumannii
Using the goeBURST algorithm in Phyloviz software, we conducted an MLST-based homology analysis of Hv-AB [15, 16]. The results indicated that ST208 served as the founder ST. Compared with ST208, ST191, ST195, ST540, ST1145, and ST1893 differed in only one housekeeping gene, classifying them as single-locus variants (SLVs). On the other hand, ST381 and ST2207 differed in two loci, classifying them as double-locus variants (DLVs). Based on the above analysis, we can speculate that highly virulent A.baumannii strains exhibit a certain degree of genetic relatedness (Fig. 3).
Fig. 3.
MLST-based homology analysis of Hv-AB.The goeBURST Full MST (A) and The Neighbor-Joining algorithm (B)
Clinical characteristics and antimicrobial susceptibility of Hv-AB strains
Among the 49 strains included in this study, 17 were hypervirulent strains, and 32 were non-hypervirulent strains. The distribution of sample types and hypervirulent strains is shown in Table 1, while the department and gender distribution of hypervirulent strains are presented in Fig. 4. ICU is the main source of detecting hypervirulent strains of A.baumannii.
Table 1.
Distribution of hypervirulent A. baumannii strains across different sample types
| Sample Type | Total Isolates | Hypervirulent Strains | Percentage of Hypervirulent Strains (%) |
|---|---|---|---|
| Sputum | 41 | 14 | 34.146 |
| Blood | 4 | 2 | 50.000 |
| Wound Secretions | 2 | 0 | 0.000 |
| Urine | 1 | 1 | 100.000 |
| Catheter Tip | 1 | 0 | 0.000 |
Fig. 4.
distribution of Hv-AB strains by department (A) and gender(B)
In addition, we analyzed the differences in clinical features between Hv-AB and non-Hv-AB. Chi-square analysis showed that there were no significant differences in general information, underlying diseases, and invasive operations between the Hv-AB group and the non-Hv-AB group. However, in the blood test before infection, the Hv-AB group had higher levels of red blood cells and hemoglobin than the non-Hv-AB group (P < 0.05) (Table 2).
Table 2.
Comparison of clinical infection characteristics between hv-AB and non-hv-AB patients
| Item | Hv-AB(n=17) | non-Hv-AB(n=32) | P |
|---|---|---|---|
| Age(year) | 50.6±19.4 | 59.4±18.1 | 0.124 |
| Male(%) | 12（70.6） | 22（68.8） | 0.894 |
| Hospital time(day) | 24.1±19.1 | 34.3±23.0 | 0.127 |
| Stay in ICU (%) | 13（76.5） | 25（78.1） | 0.895 |
| Underlying disease | |||
| Hypertension(%) | 3（17.6） | 13（40.6） | 0.103 |
| Diabetes mellitus(%) | 1（5.9） | 6（18.8） | 0.22 |
| Coronary heart disease(%) | 0（0） | 3（9.4） | 0.197 |
| Cerebral hemorrhage(%) | 5（29.4） | 13（40.6） | 0.438 |
| Chronic pulmonary disease(%) | 3（17.6） | 1（3.1） | 0.077 |
| Malignant tumor(%) | 4（23.5） | 5（15.6） | 0.496 |
| Fracture(%) | 2（11.8） | 8（25） | 0.274 |
| Invasive operation | |||
| Central venous catheterization(%) | 13（76.5） | 25（78.1） | 0.895 |
| Ureter(%) | 15（88.2） | 29（90.6） | 0.793 |
| Thoracoabdominal drainage(%) | 4（23.5） | 6（18.8） | 0.693 |
| Nasogastric tube(%) | 14（82.4） | 28（87.5） | 0.624 |
| Trachea intubation or incision(%) | 10（58.8） | 25（78.1） | 0.155 |
| Blood test before infection | |||
| White blood cells(*10^9/L) | 11.8±7.3 | 11.9±6.0 | 0.959 |
| Red blood cells(*10^12/L) | 3.7±0.5 | 3.1±0.6 | 0.004 |
| Platelet(*10^9/L) | 277.1±152.0 | 249.1±135.7 | 0.529 |
| Hemoglobin(g/L) | 110.3±19.1 | 92.8±21.8 | 0.008 |
| Percentage of neutrophils(%) | 79.7±9 | 82.4±11.8 | 0.375 |
| Albumin(g/L) | 34.7±6.9 | 32.6±5.8 | 0.295 |
| Alanine aminotransferase(U/L) | 35.0（20.0~125.0） | 41.0（16.0~41.0） | 0.501 |
| Creatinine(μmoI/L) | 50.8（38.6~69.1） | 68.0（45.0~107.5） | 0.303 |
Hv-AB hypervirulent A.baumannii, non-Hv-AB non-hypervirulent A.baumannii
A.baumannii has a high level of drug resistance, with the antibiotic resistance rate of HV-AB ranging from 93.8% to 100%, while that of non-hv-AB is 75% to 96.9%. The results of chi-square analysis showed that the resistance rate of Hv-AB group to sulfamethoxazole-trimethoprim was higher than that of non-Hv-AB (P < 0.05) (Table 3).
Table 3.
Comparison of antibiotic resistance between Hv-AB group and non-Hv-AB group
| Antibiotic | Hv-AB(n=17) | non-Hv-AB(n=32) | X2 | P |
|---|---|---|---|---|
| Ampicillin/sulbactam | 17/17,100% | 30/32,93.8% | 1.085 | 0.298 |
| Ceftriaxone | 16/16,100% | 31/32,96.9% | 0.5 | 0.48 |
| Cefotaxime | 17/17,100% | 31/32,96.9% | 0.531 | 0.466 |
| Cefepime | 17/17,100% | 30/32,93.8% | 1.085 | 0.298 |
| Levofloxacin | 17/17,100% | 29/32,90.6% | 1.663 | 0.197 |
| Trimethoprim-sulfamethoxazole | 17/17,100% | 24/32,75% | 4.976 | 0.026 |
| Amikacin | 16/17,94.1% | 27/32,84.4% | 0.981 | 0.322 |
| Ceftazidime | 17/17,100% | 30/32,93.8% | 1.085 | 0.298 |
| Ciprofloxacin | 17/17, 100% | 29/32,90.6% | 1.663 | 0.197 |
| Gentamicin | 17/17, 100% | 29/32,90.6% | 1.663 | 0.197 |
| Meropenem | 17/17, 100% | 30/32,93.8% | 1.085 | 0.298 |
| Tobramycin | 15/16,93.8% | 27/32,84.4% | 0.857 | 0.36 |
Hv-AB hypervirulent A.baumannii, non-Hv-AB non-hypervirulent A.baumannii
Serum resistance assay and string test
We showed the results of serum resistance of A.baumannii, but there was no statistical difference in serum resistance between Hv-AB and non-Hv-AB(p = 0.18) ( Fig. 5C). Our experimental results showed that no String test-positive strains were found in both Hv-AB group and non-Hv-AB group.
Fig. 5.
Colonies on MH agar plate in serum resistance experiments (A-1, CFUs-NHS; A-2,CFUs-HIS). Negative result of a String test (B). Comparison of serum resistance (C) and biofilm formation (D) between Hv-AB group and non-Hv-AB group. Hv-AB: hypervirulent A.baumannii;non-Hv-AB: non-hypervirulent A.baumannii
Biofilm formation assay
A.baumannii demonstrated a remarkable biofilm-forming capability, with 48 out of 49 strains (98.0% positivity rate) exhibiting biofilm production. All 17 strains in the hypervirulent group (100% positivity) formed biofilms, categorized as 1 weakly positive, 10 positive, and 6 strongly positive. In contrast, 31 strains from the non-hypervirulent group showed biofilm formation (96.9% positivity), comprising 2 weakly positive, 17 positive, and 12 strongly positive isolates. Statistical analysis using the χ² test revealed no significant difference in biofilm-forming capacity between hypervirulent and non-hypervirulent A.baumannii strains (p = 0.46) ( Fig. 5D).
Discussion
A.baumannii is notorious for its widespread resistance to commonly used antibiotics. Traditionally, it was considered a low-grade pathogen, but subsequent research has revealed that its pathogenicity was underestimated [17, 18]. Jun Li et al. found 7 Hv-AB strains among 109 carbapenem-resistant A.baumannii isolated from blood samples from Xiangya Hospital from January 2017 to May 2019, with a positive rate of 6.4% [3]. Their research results showed that the average mortality rates of patients infected with these 7 strains at 7 and 21 days were 42.9% and 71.4%, respectively [3]. However, in this study, we identified 17 Hv-ABs out of 49 A.baumannii strains, reflecting the increasing trend of HV-ABs in clinical settings, which warrants the vigilance of clinicians and hospital infection prevention and control personnel.
To investigate the molecular epidemiology of Hv-AB, we adopted the method of Multilocus sequence typing, which is simple to operate and has good repeatability. It can be used for comparison among different laboratories worldwide and has been widely used in the molecular epidemiological investigation of bacteria and fungi [19]. Our research has revealed that Hv-AB strains exhibit a high diversity of sequence types (STs), yet they still display a certain degree of genetic relatedness. Further analysis indicates that ST208 serves as the ancestral ST type, with other ST types differing by one or two gene loci from it. ST208 belongs to the international clonal complex CC92, which is reported to be prevalent in many regions of China and has become one of the dominant sequence types [20]. Moreover, most ST208 strains carry the OXA-23 gene, producing carbapenemase, which mediates resistance to carbapenem antibiotics [21, 22].
The study highlighted the ICU as a significant source for detecting Hv-AB strains, indicating a heightened risk environment for acquisition and transmission. Hv-AB exhibited notably higher resistance rates to sulfamethoxazole-trimethoprim and other commonly used antibiotics compared to non-Hv-AB strains, posing challenges for antibiotic stewardship and treatment protocols. Interestingly, the study suggested a potential correlation between higher red blood cell counts or hemoglobin levels and susceptibility to Hv-AB infection. This finding implies that host physiological parameters may influence the clinical outcomes of Hv-AB infections, underscoring the need for further investigation into host-pathogen interactions. Contrary to previous assumptions that Hv-AB strains are typically associated with high-viscosity phenotypes, serum resistance, or enhanced biofilm formation, our results revealed no significant differences in these traits between Hv-AB and non-Hv-AB strains [23–25]. This challenges existing hypotheses about the unique virulence factors associated with hypervirulent pathogens and highlights the complexity of A.baumannii pathogenesis. The emergence of Hv-AB strains in clinical settings underscores the urgent need for enhanced surveillance, infection control measures, and targeted therapeutic strategies.
This study is limited by its retrospective design and the relatively small sample size from a single hospital in China. Future studies should involve larger, multicenter cohorts to validate these findings across diverse geographical regions and healthcare settings. Additionally, further exploration into the mechanisms underlying Hv-AB pathogenicity and virulence factor expression is essential for comprehensive control and prevention strategies.
Conclusion
This study confirms the prevalence of hypervirulent A.baumannii strains in clinical settings, revealing a degree of homology among these strains that suggests they belong to a strain type originating from the ST208 genotype. This research provides a foundation for further understanding the evolution and transmission patterns of A.baumannii, which is crucial for the prevention and control of this pathogen.
Acknowledgements
Not applicable.
Abbreviations
- AB
Acinetobacter baumannii
- Hv-AB
hypervirulent Acinetobacter baumannii
- non-Hv-AB
non-hypervirulent Acinetobacter baumannii
- MLST
Multilocus sequence typing
- ICU
Intensive Care Unit
- CLSI
Clinical and Laboratory Standards Institute
- PBS
phosphate buffered saline
- NHS
normal human serum
- HIS
heat-inactivated serum
- MH
Mueller-Hinton
- SLV
single-locus variant
- DLV
double-locus variant
Authors’ contributions
Jianglong Shi conducted the research experiment and completed the manuscript. Ren Liu and Jiehui Qiu participated in the implementation of some experiments. Chunping Wei, Dejin Pan, and Zhiyong He were primarily responsible for data collection, statistics, and analysis. Yang Li, Tianxin Xiang, and Na Cheng conceived the research plan and reviewed the manuscript. All authors reviewed the manuscript.
Funding
Financial support was provided by Jiangxi Provincial Health Commission science and technology plan project (202210370) and Science and Science and Technology Research Project of the Education Department of Jiangxi Province (GJJ2200217).
Data availability
The medical data used in this study are available upon reasonable request to the corresponding author.
Declarations
Ethics approval and consent to participate
The study was approved by ethical committees of the first affiliated hospital of Nanchang University (no. (2024)CDYFYYLK(02–020)).
Consent for publication
Not applicable.
Competing interests
The authors declare no competing interests.
Footnotes
Publisher’s Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Data Availability Statement
The medical data used in this study are available upon reasonable request to the corresponding author.