Rapid Identification and Methicillin Resistance Test to Staphylococcus aureus in Cerebrospinal by MALDI-TOF MS

doi:10.21203/rs.3.rs-5289888/v1

Background: Bacterial meningitis is a disease with high mortality and morbidity, and it primarily manifests as symptoms involving the central nervous system (CNS). Hence, it would be of great importance to make an early diagnosis and initiate empirical antimicrobial treatment in time for this disease.

Methods: In this study, we investigated the feasibility of rapid pathogen identification and drug resistance analysis through the combination of centrifugation-based enrichment of bacteria and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). Specifically, the cerebrospinal samples were treated by differential centrifugation to collect precipitates after a short-term rapid culture. Then, the precipitates were identified by MALDI-TOF MS. Subsequently, the bacterial solution (106 CFU/mL) was mixed with an equal volume of cation-adjusted Mueller-Hinton broth (CAMHB) supplemented with cefoxitin (4 µg/mL). After the culture of the mixture, the precipitates obtained by centrifugation were analyzed by MALDI-TOF MS.

Results: The efficiency of centrifugation-based enrichment of Staphylococcus aureus (S. aureus) was 87.9% at 103 CFU/mL and increased to 90.4% at 102 CFU/mL. This identification efficiency reached 100% after an 8-hour culture. The optimal testing time for bacterial resistance identification was achieved by the culture within 3 hours. The validity, sensitivity, and specificity were all 100% at this time point. The results of the rapid identification method were identical to those of the broth microdilution method.

Conclusion: Through this protocol, the identification and drug resistance analysis of S. aureus in the cerebrospinal fluid (CSF) can be completed within 11 hours. These findings are expected to provide a new method for the rapid diagnosis and treatment of patients with bacterial meningitis.

cerebrospinal fluid

MALDI-TOF MS

antibiotic resistance

pathogen identification

MRSA

Bacterial meningitis is a worldwide health problem characterized by rapid onset, high mortality and morbidity, and high outbreak and epidemic potentials^1–3. This disease is caused by bacterial invasion of the meninges and primarily manifests as symptoms involving the central nervous system (CNS), with Staphylococcus aureus (S. aureus) being one of the causative organisms⁴. S. aureus colonizes about 20–80% of the human population and provides a reservoir for subsequent transmission and infection⁵. The proliferation of multidrug-resistant strains imposes constraints on the selection of antibiotic treatment options, thereby posing challenges to clinical intervention^3,6,7. Identifying bacterial meningitis pathogens and analyzing drug resistance constitute the key to achieving effective clinical treatment.

Meningitis cannot be diagnosed based on clinical features alone, and the cerebrospinal fluid (CSF) analysis is essential for its diagnosis. The microscopic examination and microbial culture of CSF samples constitute the gold standard for the diagnosis of this disease. It is essential to make an early diagnosis and initiate empirical antimicrobial treatment in time for this disease^8–10. However, conventional bacterial cultures and drug susceptibility tests typically require 3–5 days, leading to delays in treatment. Existing techniques such as molecular diagnostic technology can be employed to rapidly identify pathogens and detect drug resistance. However, such potential factors as inhibitors and cross-contamination may result in erroneous microorganism identification¹¹. Therefore, there is an urgent demand for simpler and more cost-effective detection methods.

Currently, matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS), which features simple procedures, fast detection, and low costs, is a common method for the clinical detection of bacteria^12–14. In recent years, MALDI-TOF MS has also been developed for the detection of drug resistance in bacteria. Rhoads et al. reported that the MALDI-TOF mass spectral peak could be used to predict methicillin resistance in staphylococci¹⁵. Idelevich et al. developed a direct targeted microdrop growth assay (DOT-MGA) based on MALDI-TOF MS to rapidly detect bacterial resistance within 5 hours¹⁶. Nix ID et al. detected methicillin resistance in S. aureus from blood culture bottles and solid media¹⁷. In urinary tract infections, the DOT-MGA method can also be utilized to detect drug-resistant bacteria¹⁸. However, the detection of drug-resistant bacteria in the CSF has not been reported.

In this study, mass spectrometry based on centrifugal enrichment was used to rapidly identify pathogens and detect drug resistance, in an attempt to minimize turnaround time and provide a foundation for precise clinical treatment.

Bacterial strains

A total of 40 S. aureus isolates were included in this study, including 20 MRSA and 20 MSSA strains. All isolates were collected from the routine diagnostics at Wanbei Coal-Electricity Group General Hospital (Anhui, China). To avoid isolate duplicates, only 1 isolate per patient was included. The mecA gene was detected in all MRSA isolates. The minimum inhibitory concentration (MIC) of 40 samples was obtained by the micro broth dilution method.

Bacterial stock preparation

The bacteria were inoculated on a blood culture plate and incubated for 12 hours. Then, a single colony was placed into sterile water and shaken well. Subsequently, the absorbance of the bacterial solution at 600 nm was measured by ultraviolet-visible spectroscopy (UV-VIS) and adjusted to 0.7 to 0.8 as the bacterial stock solution. The bacterial concentration was determined by the plate counting method.

MIC determination

The MIC of cefoxitin was determined for S. aureus using the broth microdilution reference method according to the Clinical & Laboratory Standards Institute (CLSI) and International Organization for Standardization (ISO) guidelines¹⁹. The bacterial colonies on the blood agar plate were transferred into sterile water and mixed. Then, the OD₆₀₀ of the bacterial suspension was measured using UV-VIS and adjusted to a range of 0.7 to 0.8. Subsequently, the bacterial suspension was diluted with the cation-adjusted Mueller-Hinton broth (CAMHB) to achieve a final concentration of 10⁶ CFU/mL. Next, the serial dilution was inoculated onto triplicate nutrition agar plates, and the colonies were enumerated after an overnight culture. The bacterial suspension (100 µL) was diluted with cefoxitin at a ratio of 1:2 in a 96-well plate to achieve a final concentration of 5×10⁵ CFU/mL, and the final concentration of cefoxitin was increased in a doubling manner, ranging from 0.5 µg/mL to 256 µg/mL. The results were read after the culture for 18 ± 2 hours at 37℃. The MIC50, MIC90, and MIC ranges were calculated for further analysis.

Centrifugation-based enrichment efficiency

The bacterial stock solution was diluted 10 times with sterile water, resulting in a concentration of 10⁶ CFU/mL. Then, the bacterial suspension was diluted 10 times with sterile water until reaching a final concentration of 10² CFU/mL. The diluted bacterial suspension was centrifuged at 12,000 rpm for 5 minutes, and the supernatant and precipitate were streaked onto nutrient agar plates and incubated for 12 hours. The accurate bacterial concentration was determined by the plate counting method. The bacterial suspension was diluted to a concentration of 10 CFU/mL, and the above steps were repeated in triplicate to observe the efficiency of centrifugation-based enrichment.

Rapid identification of pathogens in the CSF

The bacterial stock solution was diluted to 10⁴ CFU/mL with sterile water, and the bacterial suspension (1µL) was added to the Luria-Bertani (LB) broth (9µL, 19µL, and 39µL) in varying volumes. The CSF was utilized as a growth control. Then, the mixture was incubated at 37℃ under shaking at 900 rpm for 8 hours, and the OD₆₀₀ was measured every hour. When the OD₆₀₀ was > 0.8, the culture was stopped and the mixture was adjusted to 0.7–0.8 with sterile water. When the OD₆₀₀ was 0.7–0.8, the mixture was centrifuged at 12,000 rpm for 5 min, followed by supernatant removal and washing with 100 µL sterile water three times as well as resuspension in 5 µL of 70% formic acid and 5 µL of acetonitrile (ACN). Subsequently, the analysis was performed based on MALDI-TOF MS. The optimal volume of the LB broth was determined by comparing the OD₆₀₀ values of three groups with varying volumes. Finally, 40 strains of S. aureus were rapidly identified.

Bacterial resistance analysis of the CSF by MALDI-TOF MS

The bacterial stock solution was diluted to a concentration of 5×10⁵ CFU/mL with CAMHB. Subsequently, different volumes of the bacterial suspension (10µL, 20µL, 40µL, 80µL, and 160µL) were incubated at 37℃ under shaking at 900 rpm, and the OD₆₀₀ was measured every hour (1 to 4 hours). After the measurement, the bacterial suspension was centrifuged at 12,000 rpm for 5 minutes, followed by supernatant removal and washing with sterile water three times, as well as resuspension in 5µL of 70% formic acid and 5 µL of ACN. Then, the analysis was performed based on MALDI-TOF MS. The optimal volume of CHAMB was determined by comparing the OD₆₀₀ values of five groups with varying volumes.

After a short-term culture for bacterial identification, 10µL of bacterial suspension was mixed with 990µL of CHAMB solution. Based on the above experimental results, the optimal volume for the bacterial culture was determined to be 40µL. Cefoxitin was dissolved in the CHAMB solution at a concentration of 8µg/mL, and then 40µL of Cefoxitin solution was added to the diluted bacterial suspension (40µL) in CAMHB. Eventually, an expected final bacterial inoculation volume of approximately 5×10⁵ CFU/mL was obtained. The final concentration of cefoxitin reached 4µg/mL. For this experiment, one MRSA strain and one MSSA strain were selected. Cefoxitin was added to the bacterial suspension as the experimental group, while CHAMB without the addition of antibiotics served as the control group. The bacteria were incubated at 37°C under shaking at 900 rpm for 8 hours, with OD₆₀₀ values measured every hour in this period. At the end of the culture, the sample was centrifuged at 12,000 rpm for 5 minutes, followed by supernatant removal and washing with sterile water three times. The analysis was performed based on MALDI-TOF MS. The remaining strains were measured by the above method.

MALDI-TOF MS identification

The bacterial protein solution (1µL) treated with 70% formic acid and ACN was applied onto the MALDI-TOF MS target plate, followed by the addition of theα-cyano-4-hydroxycinnamic acid (CHCA) matrix (1 µL, 10 mg/mL in ACN/H2O [vol/vol = 1/1] containing 2% trifluoroacetic acid [TFA]) and subsequent drying for MALDI-TOF MS analysis. If the species identification score for the growth control without cefoxitin was ≥ 1.7, the test was considered valid; a score of < 1.7 indicated an invalid test. For samples with cefoxitin, successful species identification (score ≥ 1.7) was interpreted as a non-susceptible result for the given isolate, whereas failed species identification (score<1.7) was classified as a susceptible isolate. A median result for three spots was calculated and used for further analysis.

Statistical analysis

SPSS 26.0 was utilized to conduct a statistical credit analysis. The continuous calibration χ2 test ratio was employed to demonstrate the effectiveness rate. The results were expressed as mean ± standard deviation (SD) or percentage. The consistency of the DOT-MGA and microbroth dilution method was evaluated using the Kappa test. A Kappa value of 1 indicated identical results; a Kappa value between 1 and 0.75 indicated good consistency; a Kappa value between 0.75 and 0.4 indicated general consistency; a Kappa value below 0.4 indicated poor consistency. The significance level (α) for testing was set at 0.05.

Enrichment efficiency of the bacterial solution

The enrichment efficiency of the bacterial solution was determined by centrifugation. As illustrated in Figs. 1A-D, in bacterial solutions of varying concentrations, the enrichment efficiency was measured at 87.9 ± 1.4% and 90.4 ± 1.8%, respectively, indicating the successful extraction of the bacteria solution. The remaining data are shown in Figure S1.

Rapid identification of pathogens through a microbroth culture in the CSF

The identification of bacteria was achieved through a microbroth culture, as illustrated in Fig. 2. The bacterial solution was diluted to about 10⁵ CFU/mL with sterile water, and 1 µL of the bacterial suspension was added to the LB broth and CSF with different volumes (9µL, 19µL, and 39µL). The changes in the OD₆₀₀ value are shown in Table S1. In the CSF without the LB broth, there was no significant change in the OD₆₀₀ value after an 8-hour culture. The OD₆₀₀ value was 1.12 ± 0.10, 0.89 ± 0.18, and 0.66 ± 0.14 when the samples were cultured with 9 µL, 19 µL, and 39 µL of the LB broth for 7 hours, respectively. The LB broth of 9 µL exhibited the highest absorbance ever recorded. The specific OD₆₀₀ values of 40 strains of S. aureus cultured in 9µL of LB broth are depicted in Table 1. When the OD₆₀₀ value was > 0.8, the culture could be stopped for bacterial identification.

Table 1

OD₆₀₀ value changes during pathogen identification.
Strain	4 h	5 h	6 h	7 h	8 h
R-1	0.04	0.15	0.46	1.17	-
R-2	0.02	0.06	0.13	0.91	-
R-3	0.02	0.05	0.09	0.77	1.16
R-4	0.03	0.13	0.38	1.12	-
R-5	0.01	0.02	0.04	0.32	0.79
R-6	0.02	0.02	0.13	0.86	-
R-7	0.03	0.14	0.27	0.69	1.88
R-8	0.02	0.02	0.17	0.53	1.42
R-9	0.01	0.01	0.08	0.89	-
R-10	0.02	0.05	0.09	0.82	-
R-11	0.01	0.01	0.07	0.69	1.14
R-12	0.03	0.07	0.13	0.86	-
R-13	0.04	0.11	0.24	1.10	-
R-14	0.01	0.01	0.04	0.26	0.91
R-15	0.01	0.01	0.03	0.35	1.28
R-16	0.02	0.06	0.12	0.84	-
R-17	0.01	0.04	0.08	0.58	1.79
R-18	0.02	0.02	0.11	0.29	0.86
R-19	0.01	0.01	0.03	0.14	0.51
R-20	0.01	0.02	0.07	0.33	0.92
S-1	0.04	0.17	0.36	1.75	-
S-2	0.02	0.06	0.11	0.78	1.51
S-3	0.01	0.04	0.09	0.70	1.18
S-4	0.02	0.03	0.14	0.85	-
S-5	0.01	0.06	0.12	0.69	0.97
S-6	0.04	0.19	0.38	1.36	-
S-7	0.02	0.02	0.06	0.54	1.83
S-8	0.03	0.14	0.37	1.52	-
S-9	0.01	0.04	0.09	0.38	0.96
S-10	0.01	0.03	0.08	0.21	1.02
S-11	0.04	0.16	0.42	1.77	-
S-12	0.02	0.03	0.08	0.36	1.13
S-13	0.02	0.04	0.09	0.58	0.91
S-14	0.03	0.11	0.26	1.17	-
S-15	0.02	0.09	0.18	0.67	1.64
S-16	0.01	0.04	0.08	0.40	0.88
S-17	0.02	0.08	0.16	0.34	1.27
S-18	0.04	0.19	0.44	1.77	-
S-19	0.01	0.05	0.12	0.42	0.93
S-20	0.02	0.03	0.08	0.44	1.22
The dash (-) indicates that the OD value of the liquid medium was > 0.8 and incubation stopped.

Characterization of bacterial isolates using standard methods

As shown in Table S2, for MRSA isolates, the MIC50, MIC90, and MIC of cefoxitin ranged from 16, 128, and 8 to > 256 mg/mL, respectively. For MSSA isolates, the MIC50, MIC90, and MIC of cefoxitin ranged from 2, 4, and 2 to 4 µg/mL, respectively. The MIC of the QC strain S. aureus (ATCC 25923) was within the recommended range throughout the study. The mecA gene was detected in all clinical MRSA isolates.

Rapid antimicrobial susceptibility testing by MALDI-TOF MS

The percentage of bacterial identification achieved through the culture based on various volumes is presented in Table S3. When cultured for 2 hours, the identification scores for bacteria cultured using CHAMB with a volume of 40 µL were nearly indistinguishable from those obtained with a volume of 80 µL and 160 µL but superior to those obtained with a volume of 10 µL and 20 µL. This trend persisted after a 3-hour culture. Without affecting the results of the experiment, the bacterial solutions with smaller volumes were selected in this study. Thus, 40 µL was selected as the optimal culture volume for the CAMHB bacterial suspension.

As shown in Fig. 3, MRSA exhibited a slow growth rate in the CAMHB broth with cefoxitin (4µg/mL), and a significant difference in OD₆₀₀ was observed between the control group and the drug group after a 7-hour culture (P < 0.036). Conversely, MSSA failed to proliferate in the CAMHB containing cefoxitin (4µg/mL). There was a significant difference in OD₆₀₀ values between the control group and the drug group after a 2-hour culture (P < 0.001). The OD₆₀₀ values are presented in Table S4. Based on previous experiments (Table S3), most of the bacteria can be identified after a 3-hour culture. Therefore, to achieve accurate identification, the detection time lengths of 2, 3, and 4 hours were selected for the drug resistance experiment.

As shown in Table S5, MRSA grew faster in the CHAMB without cefoxitin, with the OD₆₀₀ values reaching 0.08 ± 0.03, 0.28 ± 0.07, and 0.76 ± 0.10 after a 2-hour, 3-hour, and 4-hour culture, respectively. MRSA exhibited a slower growth rate in CAMHB (4µg/mL cefoxitin), with the OD₆₀₀ values reaching 0.07 ± 0.03, 0.24 ± 0.09, and 0.41 ± 0.18, respectively, at the same time points. As shown in Table S6, the OD₆₀₀ of MSSA was 0.08 ± 0.03, 0.32 ± 0.07, and 0.72 ± 0.12 when cultured in CAMHB for 2, 3, and 4 hours, respectively; the OD₆₀₀ of MSSA was 0.02 ± 0.01, 0.02 ± 0.01, and 0.02 ± 0.01 when cultured in CAMHB (4 µ g/mL cefoxitin) for 2, 3, and 4 hours, respectively.

The identification of 40 strains of S. aureus in CAMHB (4µg/mL cefoxitin) was performed using MALDI-TOF MS after the bacterial culture was completed, as presented in Tables 2–3. Based on different enrichment methods, the bacteria were categorized into the non-centrifuge group and the centrifuge group. For the non-centrifuge group, 20 strains of MRSA were not successfully identified after a 2-hour culture. After a 3-hour culture, 13 strains of MRSA were not identified successfully. Besides, 3 strains of MRSA were not successfully identified after a 4-hour culture. In the centrifuge group, 12 strains of MRSA were not identified successfully after a 2-hour culture. All strains were successfully identified after a 3-hour and 4-hour culture. In contrast, the strains of MSSA were not identified successfully after a 2-hour, 3-hour, and 4-hour culture in both groups. The MALDI-TOF MS spectra of MSSA and MRSA are shown in Fig. 4. As presented in Table 4, the validity, sensitivity, specificity, positive predictive value, and negative predictive value of MRSA were all 100%, which was identified by centrifugation-based enrichment after a 3-hour culture. The results remained consistent with the aforementioned observations after a 4-hour culture.

Table 2

MRSA identification scores of different protocols in CHAMB (4 µg/mL cefoxitin).
Number	MIC (µg/mL)	Enrichment by non centrifugation			Enrichment by centrifugation
Number	MIC (µg/mL)	2 h	3 h	4 h	2 h	3 h	4 h
R-1	8	-	-	-	2.22	2.46	2.35
R-2	32	-	2.25	2.44	-	2.36	2.35
R-3	16	-	-	2.34	-	2.46	2.36
R-4	8	-	-	-	2.04	2.53	2.45
R-5	128	-	2.35	2.45	-	2.51	2.29
R-6	64	-	2.10	2.25	-	2.28	2.30
R-7	64	-	2.18	2.44	-	2.26	2.35
R-8	32	-	2.28	2.35	2.46	2.45	2.34
R-9	8	-	-	2.09	-	2.42	2.49
R-10	32	-	2.39	2.41	-	2.40	2.46
R-11	16	-	2.42	2.36	2.30	2.26	2.42
R-12	16	-	2.43	2.41	2.42	2.32	2.23
R-13	32	-	2.45	2.30	2.42	2.38	2.44
R-14	16	-	-	2.39	-	2.33	2.35
R-15	128	-	2.24	2.28	-	2.32	2.40
R-16	> 256	-	2.25	2.36	-	2.20	2.33
R-17	8	-	-	-	-	2.36	2.41
R-18	64	-	2.44	2.38	2.39	2.43	2.42
R-19	8	-	-	2.44	2.40	2.28	2.36
R-20	16	-	2.23	2.35	-	2.32	2.37
		0	13	17	8	20	20
The dash (-) indicates that the bacteria have not been conclusively identified.

Table 3

MSSA identification scores of different protocols in CHAMB (4 µg/mL cefoxitin).
Number	MIC (µg/mL)	Enrichment by non centrifugation			Enrichment by centrifugation
Number	MIC (µg/mL)	2 h	3 h	4 h	2 h	3 h	4 h
S-1	2	-	-	-	-	-	-
S-2	4	-	-	-	-	-	-
S-3	2	-	-	-	-	-	-
S-4	2	-	-	-	-	-	-
S-5	2	-	-	-	-	-	-
S-6	2	-	-	-	-	-	-
S-7	4	-	-	-	-	-	-
S-8	4	-	-	-	-	-	-
S-9	2	-	-	-	-	-	-
S-10	4	-	-	-	-	-	-
S-11	2	-	-	-	-	-	-
S-12	4	-	-	-	-	-	-
S-13	2	-	-	-	-	-	-
S-14	4	-	-	-	-	-	-
S-15	4	-	-	-	-	-	-
S-16	4	-	-	-	-	-	-
S-17	4	-	-	-	-	-	-
S-18	2	-	-	-	-	-	-
S-19	2	-	-	-	-	-	-
S-20	4	-	-	-	-	-	-
		0	0	0	0	0	0
The dash (-) indicates that the bacteria have not been conclusively identified.

Table 4

The resistance of *S. aureus* to methicillin was identified by MALDI-TOF MS in the cerebrospinal fluid.
	Enrichment by non centrifugation			Enrichment by centrifugation
	2 h	3 h	4 h	2 h	3 h	4 h
Validity (%)	0	82.5	92.5	70	100	100
Sensitivity (%)	0	65	85	40	100	100
Specificity (%)		100	100	100	100	100
Positive predictive value (%)	0	100	100	100	100	100
Negative predictive value (%)	0	74.1	86.9	80	100	100

Coherence analysis

The results of centrifugation-based enrichment combined with mass spectrometry and microbroth culture of 40 strains of S. aureus after a 3-hour culture were analyzed by the kappa test (Table S7). It was validated that the results of both methods were identical (kappa = 1).

As a severe infectious disease involving the central nervous system (CNS), bacterial meningitis commonly affects adults and is particularly prevalent in children, with a high mortality rate and accompanied by an increased incidence of sequelae^20,21. Infection with S. aureus is one of the causes of bacterial meningitis, presenting a particular challenge in its management and treatment²². A timely and effective antibiotic treatment is considered essential for improving the cure rate of this disease. However, the rapid development of antibiotic resistance, particularly with MRSA, poses a significant challenge to the cure of this disease^23–25. The rapid identification of MRSA is considered beneficial to the early treatment of patients, thus reducing the risk of death^16,26. However, potential contamination conditions may impact bacterial identification.

In this study, the enrichment efficiency of bacteria was evaluated. The results demonstrated that the efficiency of the commonly used centrifugation method can reach over 85%, highlighting the simplicity and reliability of this method. In terms of bacterial identification, it was found that a rapid broth culture of 7 or 8 hours was sufficient to meet the requirements for mass spectrometry detection, significantly reducing the time required by conventional methods. For the identification of antibiotic resistance, the differentiation between MRSA and MSSA was made based on their distinct sensitivity to antibiotics. It was observed that MSSA could not grow in the broth culture media containing cefoxitin, and none of the 20 sensitive strains were successfully identified. For MRSA, 12 strains of S. aureus were not successfully identified after a 2-hour culture, but the successful identification of these strains was achieved after a 3-hour and 4-hour culture. Therefore, 3 hours can be considered the optimal culture time for MRSA identification. In comparison, the optimal culture time of the DOT-MGA method used by Idelevich et al. to identify MRSA was determined to be 5 hours¹⁶. In this study, the detection of the OD₆₀₀ value of bacterial suspensions can be achieved through ultraviolet-visible spectroscopy (UV-VIS). In CHAMB with cefoxitin, the changes in the OD₆₀₀ value appeared after a 2-hour culture, and the lower absorbance at this stage was correlated with a lower identification rate. A significant change in the OD₆₀₀ value was observed after a 3-hour culture, marking this time length become the optimal culture time for MRSA identification. As a result, the OD₆₀₀ value can serve as an auxiliary method to ascertain the necessity for the enrichment medium and mass spectrometry.

This report offers a mass spectrometry-based method that utilized centrifugal enrichment was employed for the rapid identification of pathogens and their resistance, and the identification and resistance analysis of S. aureus in the cerebrospinal fluid (CSF) was completed within 11 hours. Hence, this method can be applied to various pathogens for the rapid verification of their resistance, aiding in early clinical treatment. In this study, the CSF was used to simulate the environment by adding bacteria, thus achieving clinical application. Nevertheless, the results should be validated in a study based on a larger sample size. Moreover, it is necessary to standardize and optimize testing conditions and evaluation criteria in further research. It is expected that this method will guide clinicians in the timely and correct application of antibiotics after the validation of various resistant bacteria.

CNS	central nervous system
MALDI-TOF MS	matrix-assisted laser desorption ionization time-of-flight mass spectrometry
CAMHB	cation-adjusted Mueller-Hinton broth
S. aureus	Staphylococcus aureus
CSF	cerebrospinal fluid
DOT-MGA	direct targeted microdrop growth assay
MIC	minimum inhibitory concentration
UV-VIS	ultraviolet-visible spectroscopy
LB	Luria-Bertani
ACN	acetonitrile
MRSA	Methicillin-resistant Staphylococcus aureus
MSSA	Methicillin-sensitive Staphylococcus aureus
CHCA	α-cyano-4-hydroxycinnamic acid
TFA	trifluoroacetic acid

Clinical trial number

Not applicable

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests

Funding

This work was supported by Suzhou Municipal Health Commission Project (SZWJ2023a056).

Author Contribution

All authors made substantial contributions to the conception and design, acquisition of data, or analysis and interpretation of data; took part in drafting the article or revising it critically for important intellectual content; agreed to submit it to the current journal; gave final approval for the version to be published; agreed to be accountable for all aspects of the work.

Acknowledgements

Not applicable.

Data availability statement

Data is provided within the manuscript or supplementary information files. The MALDI-TOF MS datasets for the current study are available from the corresponding author on reasonable request.

van de Beek D, Brouwer M, Hasbun R, Koedel U, Whitney CG, Wijdicks E. Community-acquired bacterial meningitis. NAT REV DIS PRIMERS. 2016 2016-11-03;2(1).
Hasbun R. Progress and Challenges in Bacterial Meningitis: A Review. JAMA-J AM MED ASSOC. 2022 2022-12-06;328(21):2147-54.
Aguilar J, Urday-Cornejo V, Donabedian S, Perri M, Tibbetts R, Zervos M. Staphylococcus aureus Meningitis. MEDICINE. 2010;89(2):117-25.
Wall EC, Chan JM, Gil E, Heyderman RS. Acute bacterial meningitis. CURR OPIN NEUROL. 2021;34(3):386-95.
Zhang F, Ledue O, Jun M, et al. Protection against Staphylococcus aureus Colonization and Infection by B- and T-Cell-Mediated Mechanisms. MBIO. 2018 2018-01-01;9(5).
van Duin D, Paterson DL. Multidrug-Resistant Bacteria in the Community: An Update. INFECT DIS CLIN N AM. 2020 2020-01-01;34(4):709-22.
Stadler M, Dersch P. Tackling Threats and Future Problems of Multidrug-Resistant Bacteria. *398*398. Switzerland: Springer International Publishing AG, 2016:3-33.
Alamarat Z, Hasbun R. Management of Acute Bacterial Meningitis in Children. INFECT DRUG RESIST. 2020 2020-01-01; 13:4077-89.
Smets I, Verelst S, Meyfroidt G, et al. Community-acquired bacterial meningitis in adults: emergency department management protocol. ACTA NEUROL BELG. 2020 2020-01-01;120(5):1033-43.
Garcia S, Echevarri J, Arana-Arri E, et al. Outpatient management of children at low risk for bacterial meningitis. Emergency medicine journal: EMJ. 2018 2018-01-01;35(6):361-6.
Cai HY, Caswell JL, Prescott JF. Nonculture molecular techniques for diagnosis of bacterial disease in animals: a diagnostic laboratory perspective. VET PATHOL. 2014 2014-03-01;51(2):341-50.
Tsuchida S, Umemura H, Nakayama T. Current Status of Matrix-Assisted Laser Desorption/Ionization-Time-of-Flight Mass Spectrometry (MALDI-TOF MS) in Clinical Diagnostic Microbiology. Molecules (Basel, Switzerland). 2020 2020-01-01;25(20):4775.
Uzuriaga M, Leiva J, Guillén-Grima F, Rua M, Yuste JR. Clinical Impact of Rapid Bacterial Microbiological Identification with the MALDI-TOF MS. ANTIBIOTICS-BASEL. 2023 2023-01-01;12(12):1660.
Birhanu AG. Mass spectrometry-based proteomics as an emerging tool in clinical laboratories. CLIN PROTEOM. 2023 2023-08-26;20(1):32.
Rhoads DD, Wang H, Karichu J, Richter SS. The Presence of a Single MALDI-TOF Mass Spectral Peak Predicts Methicillin Resistance in Staphylococci. DIAGN MICR INFEC DIS. 2016 2016-01-01;86(3):257-61.
Nix ID, Idelevich EA, Storck LM, et al. Detection of Methicillin Resistance in Staphylococcus aureus From Agar Cultures and Directly From Positive Blood Cultures Using MALDI-TOF Mass Spectrometry-Based Direct-on-Target Microdroplet Growth Assay. FRONT MICROBIOL. 2020 2020-01-20;11:232.
Nix ID, Idelevich EA, Storck LM, et al. Detection of Methicillin Resistance in Staphylococcus aureus From Agar Cultures and Directly from Positive Blood Cultures Using MALDI-TOF Mass Spectrometry-Based Direct-on-Target Microdroplet Growth Assay. FRONT MICROBIOL. 2020 2020-02-14;11.
Liu Z, Tang H, Xu H, et al. Rapid Identification and Drug Sensitivity Test to Urinary Tract Infection Pathogens by DOT-MGA. INFECT DRUG RESIST. 2022 2022-01-01; 15:1391-7.
M100-S11, Performance standards for antimicrobial susceptibility testing. Clinical microbiology newsletter. 2001 2001-01-01;23(6):49.
Deliran SS, Brouwer MC, van de Beek D. Subarachnoid Hemorrhage in Bacterial Meningitis Patients. Cerebrovascular diseases (Basel, Switzerland). 2022 2022-01-01;51(1):118-24.
Lepage P, Dan B. Infantile and childhood bacterial meningitis. Handb Clin Neurol. 2013 2013-01-20; 112:1115-25.
Antonello RM, Riccardi N. How we deal with Staphylococcus aureus (MSSA, MRSA) central nervous system infections. Front Biosci (Schol Ed). 2022 2022-01-12;14(1):1.
Turner NA, Sharma-Kuinkel BK, Maskarinec SA, et al. Methicillin-resistant Staphylococcus aureus: an overview of basic and clinical research. NAT REV MICROBIOL. 2019 2019-04-01;17(4):203-18.
Deurenberg RH, Stobberingh EE. The evolution of Staphylococcus aureus. Infection, genetics and evolution. 2008 2008-01-01;8(6):747-63.
Tran NN, Morrisette T, Jorgensen SCJ, Orench Benvenutti JM, Kebriaei R. Current therapies and challenges for the treatment of Staphylococcus aureus biofilm‐related infections. PHARMACOTHERAPY. 2023 2023-01-01;43(8):816-32.
Rensner JJ, Lueth P, Bellaire BH, Sahin O, Lee YJ. Rapid detection of antimicrobial resistance in methicillin-resistant Staphylococcus aureus using MALDI-TOF mass spectrometry. FRONT CELL INFECT MI. 2023 2023-01-01; 13:1281155.

No competing interests reported.

supplementmaterial.docx

Rapid Identification and Methicillin Resistance Test to Staphylococcus aureus in Cerebrospinal by MALDI-TOF MS

Status:

Version 1

Abstract

Figures

Background

Materials and Methods

Bacterial strains

Bacterial stock preparation

MIC determination

Centrifugation-based enrichment efficiency

Rapid identification of pathogens in the CSF

Bacterial resistance analysis of the CSF by MALDI-TOF MS

MALDI-TOF MS identification

Statistical analysis

Results

Enrichment efficiency of the bacterial solution

Rapid identification of pathogens through a microbroth culture in the CSF

Characterization of bacterial isolates using standard methods

Rapid antimicrobial susceptibility testing by MALDI-TOF MS

Coherence analysis

Discussion

Conclusion

Abbreviations

Declarations

Clinical trial number

Funding

Author Contribution

Acknowledgements

Data availability statement

References

Additional Declarations

Supplementary Files

Status:

Version 1