Gas Chromatography-Ion Mobility Spectrometry Reveals Acetoin as a Biomarker for Carbapenemase-Producing

Introduction

Recently, the extensive use of carbapenem antibiotics in clinical practice has led to a concerning rise in carbapenem resistance among Klebsiella pneumoniae (K. pneumoniae). Since the 1990s, the prevalence of carbapenem-resistant K. pneumoniae (CRKP) has progressively increased globally, resulting in high morbidity and mortality rates associated with these infections [1]. Furthermore, the emergence of antimicrobial-resistant K. pneumoniae carbapenemase (KPC)-producing bacteria has become a pressing global issue [2–5].

Carbapenemase production is the main mechanism of resistance to carbapenem antibiotics in CRKP strains, with K. pneumoniae carbapenemase (KPC-type), New Delhi metallo-beta-lactamase (NDM-type) carbapenemase, and oxacillinase-48 (OXA-48-type) carbapenemase being the most prevalent types [6,7]. In China, the main genetic determinant of CRKP is K. pneumoniae carbapenemase-2 (KPC-2), accounting for approximately 70% of cases [8]. Carbapenemases are classified according to the Ambler molecular classification into classes A, B, and D. Class A and D carbapenemases are serine-like enzymes, and class B are metalloenzymes [9]. Notably, antimicrobial drugs exhibited varying in vitro antimicrobial activities against different carbapenemase-producing strains [10]. Metalloenzyme-producing CRKP strains often show sensitivity to aztreonam. Conversely, the broad-spectrum antibiotic ceftazidime-avibactam (CAZ-AVI) possesses robust antimicrobial properties against carbapenemase (serine-like enzymes)-producing CRKP strains but lacks efficacy against metalloenzyme-producing CRKP counterparts. Therefore, the accurate and prompt identification and classification of carbapenemases in CRKP are crucial for the appropriate clinical administration of anti-infective medication.

Currently, the laboratory tests for carbapenemases rely on the modified carbapenem inactivation method (mCIM) and the Carbapenemase Non-Phenotypic (Carba NP) test, as recommended by the Clinical and Laboratory Standards Institute (CLSI) [11, 12]. However, these techniques require routine cultivation of pure colonies, with certain methods also necessitating overnight culture for a minimum of 1-2 days [13]. This delay hinders prompt clinical diagnosis and treatment. Despite developing various carbapenemase detection techniques [14–16], their implementation has been limited due to intricate procedures or high costs.

Recently, researchers have explored the potential of volatile organic compounds (VOCs) for strain identification [17–19], and applying volatile metabolites to antibiotic drug susceptibility testing has gained increasing attention [20-23]. For instance, gas chromatography-mass spectrometry (GC-MS) detection of 3-methyl-1-butanol enabled early identification of carbapenemase-positive CRKP strains [22], while non-targeted GC-MS analysis of VOC changes shows promise in the early CRKP strains identification [20]. Moreover, Smart et al. [23] employed thermal desorption-GC-MS to identify nine compounds that distinguished between cephalexin-sensitive and cephalexin-resistant isolates.

Gas chromatography-ion mobility spectrometry (GC-IMS) has emerged as a cutting-edge detection method in recent years. This innovative technique leverages the principles of chromatographic separation and ionization reactions to detect VOCs. The process begins with the pre-separation through a chromatographic column and is followed by their introduction into an ionization reaction zone by a carrier gas (nitrogen or air). Under the influence of an ion source, the carrier gas molecules and sample molecules undergo a series of ionization and ion-molecule reactions, resulting in the charging of sample molecules and their transformation into molecular ions. These ions are then driven by an electric field into a drift region through periodically opened ion gates, where they are separated and detected based on their varying migration rates due to continuous collisions with counterflowing neutral drift gas molecules [24]. Moreover, GC-IMS integrates the superior separation capabilities of gas chromatography (GC) with the enhanced sensitivity of ion mobility spectrometry (IMS). This innovative method eliminates the necessity for solid-phase microextraction by allowing the analysis of headspace components from solid or liquid samples through direct headspace injection. With a limit of detection reaching the parts per billion by volume (ppbv) level, GC-IMS facilitates both qualitative and quantitative analysis of individual compounds or labelers [25,26].

3-hydroxy-2-butanone (acetoin) emerges as a pivotal physiological metabolite synthesized by a diverse array of microorganisms thriving in glucose-enriched environments or other fermentable carbon sources. The catabolic conversion of this metabolite is primarily mediated by the acetoin dehydrogenase enzyme system (AoDH ES). Detecting acetoin-forming capacity, frequently achieved through the Voges-Proskauer reaction, serves as a valuable tool for classifying microorganisms. In bacteria grown in glucose-containing media, the release of 3-hydroxy-2-butanone (acetoin) is primarily regulated by the coordinated action of two enzymes: α-acetolactate synthase and α-acetolactate decarboxylase. 3-hydroxy-2-butanone (acetoin) plays a crucial role in these microorganisms, with its physiological significance encompassing acidification avoidance, nicotinamide adenine dinucleotide (NAD)/NADH (reduced form of NAD) ratio regulation, and carbon storage facilitation, ultimately maintaining the metabolic homeostasis and adaptability of microbial communities [27,28]. Notably, previous studies have demonstrated that K. pneumoniae releases 3-hydroxy-2-butanone (acetoin) during blood cultures [21,29] and in trypticase soy broth (TSB) [30].

Given the preceding justification and the escalating detection rate of CRKP in blood cultures [31,32], this study builds upon previous findings [21]. The previous research demonstrated the potential of GC-IMS for non-targeted analysis in simulated blood cultures, effectively identifying CRKP strains [21]. Therefore, this study aimed to utilize GC-IMS technology to detect a specific VOC biomarker, 3-hydroxy-2-butanone (acetoin), in antimicrobial-resistant KPC-producing bacteria, facilitating early identification of carbapenemase-producing CRKP strains.

Material and Methods

ETHICS STATEMENT:

In this study, K. pneumoniae isolation was conducted following hospital laboratory protocol without identifiable patient data linked to the samples. Consequently, the Medical Research Ethics Committee of the Second Affiliated Hospital of Nanchang University exempted the study from ethics approval.

SOURCES OF THE STRAINS AND CARBAPENEMASE DETECTION:

The standard strains of K. pneumoniae, including ATCC BAA-700603 (carbapenemase-negative), ATCC BAA-1706 (carbapenemase-negative), ATCC BAA-1705 (bla_KPC-positive), ATCC BAA-2146 (bla_NDM-positive), and ATCC BAA-2524 (bla_OXA-48-positive), were obtained from the American Type Culture Collection (ATCC, USA).

Additionally, 69 clinical strains were isolated from the Second Affiliated Hospital of Nanchang University and are currently maintained in the research team’s strain bank [21,22]. Among these, 25 strains were carbapenem-susceptible K. pneumoniae (CSKP) strains, while the remaining 44 strains (CRKP) exhibited resistance to carbapenem. All K. pneumoniae strains (standard and clinical strains) underwent re-testing for antimicrobial susceptibility, interpreted according to the CLSI 2022 [12] guidelines. The mCIM and modified ethylenediaminetetraacetic acid (EDTA)-carbapenem inactivation method (eCIM) [12] were performed again to validate the carbapenemase type, confirmed by polymerase chain reaction (PCR). Moreover, all clinical isolates were evaluated for the presence of carbapenemase-related genes utilizing macrogenomic next-generation sequencing (mNGS) technology, provided by Qiantang Life Science Technology Co. Ltd. (Suzhou, China). Subsequently, all K. pneumoniae strains were stored at −80°C for further analysis.

BACTERIAL CULTURE AND SAMPLE PREPARATION:

Figure 1 shows the growth curve of K. pneumoniae (ATCC BAA-700603) in blood culture bottle medium (BacT/ALERT® SA; Ref. 259789; Biomérieux, Nürtingen, Germany), with an initial concentration of 107 colony-forming units per milliliter (CFU/mL) bacteria at 37°C and agitation of 200 revolutions per minute (rpm) in a 6 mL volume. The growth curve indicates that K. pneumoniae grew fastest after 3 h of incubation and reached the end of the exponential growth phase at around 5 h.

All experimental strains were inoculated on Columbia blood agar plates and incubated overnight at 37°C. Bacterial suspensions were prepared and added to test tubes containing 6 mL of blood culture bottle medium (initially 107 CFU/mL), capped, and incubated at 37°C with agitation at 200 rpm. Blank culture media served as control. To evaluate the effect of IPM on 3-hydroxy-2-butanone (acetoin) release from K. pneumoniae, a final concentration of 0.25 mg/mL of IPM (Solarbio, China) was added after 3 h (T0) of incubation. The final concentration of IPM in carbapenemase-negative CRKP strains was 16 μg/mL. To assess the impact of carbapenemase inhibitors on 3-hydroxy-2-butanone (acetoin) emission, IPM and avibactam sodium (1 mg/L) [33] or DPA (100 μg/mL) [34] were added after 3 h (T0). The standard strains and clinical strains were processed identically. Experiments were repeated six times for standard strains and in triplicate for clinical strains.

Following the inoculation of standard strains, with or without IPM, into a blood culture bottle medium, 500 μL samples were collected for GC-IMS analysis at various incubation periods: 3h (T0), 4h (T1), 5h (T2), 6h (T3), and 7h (T4). GC-IMS analysis of the standard strain with carbapenemase inhibitors and clinical strains was performed at the T2 time point.

THE MEASUREMENT OF 3-HYDROXY-2-BUTANONE (ACETOIN) BY GC-IMS:

The FlavourSpec^® GC-IMS equipment (G.A.S., Shandong HaiNeng Scientific Instrument Co., Ltd., Shandong, China) was employed to detect 3-hydroxy-2-butanone (acetoin). The GC-IMS parameters were set as follows: incubation time: 3 min; incubation temperature: 60°C; rotating speed: 500 rpm; injector temperature: 85°C; headspace gas volume: 1 mL; drift tube temperature (T1): 45°C; chromatographic column temperature (T2): 80°C; chromatographic column: MXT-WAX column (high-polar column, 15 m×0.53 mm, 0.1 μm, RESTEK, Bellefonte, PA, USA); carrier and drift gases: nitrogen (N₂) of 99.99% purity (Jiangzhu Industrial Co., Ltd., Nanchang, China); ionization source: tritium source; average radiation energy: 5.68 keV; drift tube length: 98 mm; ionization mode: positive ionization; drift tube flow: 150 mL/min; chromatographic column flow: 2 mL/min (0–3 min), 10 mL/min (3–10 min); total analysis time: 10 min.

The fingerprint of VOCs emitted by K. pneumoniae (ATCC BAA-700603) after 5 h of incubation in the medium of blood culture bottles (BacT/ALERT® SA) is depicted in Figure 2, and the details of VOCs have been described in previous studies [21]. The VOC fingerprints generated by all the understudied K. pneumoniae strains, following a 5-h incubation period in a blood culture flask substrate, aligned with the pattern displayed in Figure 2 (without IPM addition).

Using C4-C9 ketones (2-butanone, 2-pentanone, 2-hexanone, 2-heptanone, 2-octanone, 2-nonanone), from Shandong HaiNeng Scientific Instrument Co., Ltd. (Shandong, China), as reference standards for data calibration, 3-hydroxy-2-butanone (acetoin) was identified based on the retention index (RI) and drift time (RIP relative) in the GC-IMS library (NIST library and IMS library) [21, 35]. GC-IMS detection of 3-hydroxy-2-butanone (acetoin) is characterized as follows: Chemical Abstract Service Registry Number (CAS#): C513860; Formula: C4H8O2; Molecular weight (MW): 88.1; RI: 1304.2; Retention time (Rt): 226.606 s; and Drift time (Dt): 1.33401 a.u. (arbitrary units).

STATISTICAL ANALYSIS:

The VOC fingerprint emitted by K. pneumoniae and the pseudo-3D plots of 3-hydroxy-2-butanone (acetoin) were generated by VOCal software (version 0.1.3; G.A.S., Shandong HaiNeng Scientific Instrument Co., Ltd., Shandong, China). The relative peak volume values (integrating peak intensities within specific regions after comparing calibrations) of 3-hydroxy-2-butanone (acetoin) were extracted from the VOCal software and exported to Microsoft Excel (Excel for MacOS, 2020) for further analysis. All statistical analyses were performed using R (version 4.2.3; The R Foundation, Vienna, Austria). As the data did not follow a normal distribution, the Mann-Whitney U test was used for comparisons between groups. A P-value less than 0.05 was considered statistically significant (P<0.05). Data visualizations were created using the online tool Chiplot (https://www.chiplot.online) [36].

Results

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The content of 3-hydroxy-2-butanone (acetoin) released by all standard strains increases progressively over time (T0–T4) without IPM addition (Figure 3A). However, after a 3-h incubation period (T0), IPM addition to the culture medium resulted in a consistent level of 3-hydroxy-2-butanone (acetoin) release by K. pneumoniae ATCC BAA-1706 (carbapenemase-negative), with no further changes in content observed from T0 to T4. In contrast, carbapenemase-positive standard strains displayed a temporal variation in 3-hydroxy-2-butanone (acetoin) release, similar to the same trend without IPM addition (Figure 3B). The content of 3-hydroxy-2-butanone (acetoin) exhibited a pronounced escalation between T1 and T2, aligning with the logarithmic phase of the growth curve (Figure 1). Therefore, subsequent analysis focused mainly on the T2 time point.

With IPM addition, the pseudo-3D plots illustrating the change of 3-hydroxy-2-butanone (acetoin) in standard strains are shown in Figure 4A (T2). Notably, IPM addition resulted in a significant decrease in 3-hydroxy-2-butanone (acetoin) contents emitted by K. pneumoniae ATCC BAA-1706 compared to its release without IPM. Interestingly, no statistically significant difference was observed in the relative peak volume (semi-quantified) of 3-hydroxy-2-butanone (acetoin) emitted by K. pneumoniae ATCC BAA-1706 at T2 with IPM addition, compared to the blank medium (P>0.05) (Figure 4B).

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The effect of carbapenemase inhibitors on the content of 3-hydroxy-2-butanone (acetoin) emitted by K. pneumoniae was further investigated. Figure 5A shows the pseudo-3D plots illustrating the changes in 3-hydroxy-2-butanone (acetoin) content following the introduction of carbapenemase inhibitors in various standard strains. The combination of IPM and avibactam sodium significantly decreased 3-hydroxy-2-butanone (acetoin) content in K. pneumoniae ATCC BAA-1705 (blaKPC-positive), compared to IPM alone or IPM with DPA (both P<0.05; Figure 5B). Similarly, the combination of IPM and DPA significantly decreased the 3-hydroxy-2-butanone (acetoin) content in K. pneumoniae ATCC BAA-2146 (blaNDM-positive), compared to IPM alone or IPM with avibactam sodium (both P<0.05; Figure 5B). However, compared with the addition of IPM alone, the remaining standard strains showed no notable changes in 3-hydroxy-2-butanone (acetoin) contents after introducing carbapenemase inhibitors (despite P<0.05, but no obvious visual changes observed in the pseudo-3D plots).

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A validation study utilizing clinical strains was conducted to further elucidate the role of 3-hydroxy-2-butanone (acetoin) in identifying carbapenemase-producing K. pneumoniae. As described in Table 1, among 44 CRKP isolates, the mNGS examination discovered five carbapenemase-negative CRKP strains that were devoid of carbapenemase genes. Subsequently, based on the results of mCIM, eCIM, PCR and mNGS, these CRKP isolates were further classified based on carbapenemase type: KPC-positive CRKP strains (n=20), NDM-positive CRKP strains (n=15), imipenem-hydrolyzing β-lactamase (IMP)-positive CRKP strains (n=4), and carbapenemase-negative CRKP strains (n=5).

The addition of IPM resulted in higher 3-hydroxy-2-butanone (acetoin) in the KPC-positive, NDM-positive, and IMP-positive CRKP groups compared to the CSKP group (n=25) (all P<0.05; Figure 6A, 6B). However, the carbapenemase-negative CRKP group did not show a statistically significant difference in 3-hydroxy-2-butanone (acetoin) content when compared to the CSKP group (P>0.05; Figure 6).

The addition of carbapenemase inhibitors resulted in pseudo-3D plots similar to those of K. pneumoniae ATCC BAA-1705 for the KPC-positive group and K. pneumoniae ATCC BAA-2146 for the NDM-positive and IPM-positive groups (Figure 7A). Statistical analysis revealed a significant decrease in the relative peak volume of 3-hydroxy-2-butanone (acetoin) in the KPC-positive group with avibactam sodium addition and in the NDM-positive and IMP-positive groups with the DPA addition.

Finally, Table 1 summarizes the carbapenemase types and genes of the clinical CRKP isolates, the minimum inhibitory concentration (MIC) of IPM, and the GC-IMS detection results of 3-hydroxy-2-butanone (acetoin) released by each CRKP strain under three different treatments. It is noteworthy that the trends of 3-hydroxy-2-butanone (acetoin) content under different treatments for each CRKP strain were consistent with the results above.

Discussion

LIMITATIONS:

This study has certain limitations. First, the impact of blood addition on 3-hydroxy-2-butanone (acetoin) release remains uncertain, consistent with the previous studies [30] indicating that blood addition has no influence on the VOC content produced by K. pneumoniae in blood-free media. This phenomenon warrants further research to understand its impact on VOC contents. Second, the exclusive use of clinical strains from a single medical center limits the generalizability of our method, highlighting the need for larger sample sizes and diverse carbapenemases to reinforce our research outcomes. Third, the GC-IMS assay technique, while valuable, requires specialized instrumentation and expertise and incurs higher costs, making it less accessible in the medical field. Fourth, detecting carbapenemase-positive strains based on the matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) method is recommended by the CLSI guidelines [12]. Regrettably, this study did not employ this method because: 1) Although MALDI-TOF MS technology can rapidly detect the types of carbapenemases, its disadvantage lies in the fact that there are currently only a limited number of studies focusing on specific carbapenemases, and there is no unified and feasible methodology [47]. 2) Although MALDI-TOF MS technology has established a relatively comprehensive database for microbial identification, the existing databases may still be incomplete or have accuracy issues when detecting specific strains or resistance patterns. This may lead to misdiagnosis or missed detection of carbapenemase-producing strains. Instead, this research proposed GC-IMS as a new strategy for detecting carbapenemase-positive CRKP strains by identifying changes in 3-hydroxy-2-butanone (acetoin). This method complements the MALDI-TOF MS-based method and aims to improve the speed and accuracy of detecting carbapenemase-positive CRKP strains. We believe that combining both methods can effectively combat the spread of drug-resistant strains, ensuring patient health and safety. Lastly, further evaluation is needed to confirm the specificity of 3-hydroxy-2-butanone (acetoin) for K. pneumoniae; however, utilizing a fingerprint, as depicted in Figure 2, will aid in differentiating K. pneumoniae from other bacterial species.

Conclusions

In conclusion, GC-IMS was successfully utilized to identify carbapenemase-producing K. pneumoniae through changes in 3-hydroxy-2-butanone (acetoin) content after IPM addition. Simultaneously, the inclusion of carbapenemase inhibitors enabled the differentiation between class A and B carbapenemases. However, further research is warranted to confirm these findings.