A Rapid Tigecycline and Polymyxin B Susceptibility Test for Klebsiella pneumoniae Using Direct-on-Target Micro-droplet Growth Assay

doi:10.21203/rs.3.rs-1507631/v1

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A Rapid Tigecycline and Polymyxin B Susceptibility Test for Klebsiella pneumoniae Using Direct-on-Target Micro-droplet Growth Assay

https://doi.org/10.21203/rs.3.rs-1507631/v1

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Background

Tigecycline (TGC) and polymyxin B (PMB)are important antibiotics for the treatment of multidrug-resistant bacterial infections. However, with the widespread applications, the resistant strains have been isolated. To optimize the use of TGC and PMB, there is an urgent need to develop a rapid detection method for the antibiotics. In recent years, a novel Direct-on-Target Micro-droplet Growth Assay (DOT-MGA) has been developed that can detect antibiotic resistance within hours. The study aims to verify the feasibility and accuracy of DOT-MGA in rapidly identification of TGC and PMB-susceptible and non-susceptible bacteria.

Methods

A total of 67 strains of Klebsiella pneumoniae were collected for DOT-MGA. According to FDA/EUCAST breakpoint concentrations, 6 µL droplets with or without TGC or PMB were added in triplicate into the wells of the MALDI plate with four incubation time points (3 h, 4 h, 6 h, and 8 h). The results were compared with standard databases using Bruker Biotyper software and classified as susceptible and non-susceptible based on the scores.

Results

Compared with the micro-broth dilution method, the DOT-MGA achieved the best detection performance after 4 h of incubation. The results showed that the growth efficiency of TGC and PMB were both 100%, and the classification agreement rates were 98.15% and 96.15%, respectively.

Conclusion

With the 4h incubation time, DOT-MGA can accurately identify the resistance of TGC and PMB, which has good consistency compared with the micro-dilution method. DOT-MGA can provide rapid and reliable drug susceptibility diagnosis, which is of great significance for the clinical anti-infective treatment.

MALDI-TOF

DOT-MGA

antibiotic susceptibility test rapid assay

tigecycline

polymyxin

Multidrug-resistant Klebsiella pneumoniae (MDR-KP) is a common nosocomial pathogen associated with high mortality and morbidity. The globally increasing drug resistance limits the therapeutics of MDR-KP, which becomes a major public health problem [1]. Due to their broad antibacterial spectrum, strong antibacterial activity, and relatively low drug resistance rate, tigecycline (TGC) and polymyxin B (PMB) have become effective therapeutics for the treatment of multidrug-resistant bacterial infections, particularly the most effective drugs in clinical treatment of MDR-KP [2, 3]. However, with the widespread application of TGC and PMB in anti-infective therapy, resistant strains have been isolated in recent years [4, 5]. CHINET data show that in 2021, the TGC resistance rate of K. pneumoniae was increased to 4.2%, the intermediate rate was 7%. The PMB resistance rate of in K. pneumoniae also reached 3.9%. The increasing resistance rates have attracted attention among clinicians. To optimize the use of TGC and PMB, there is an urgent need to develop new rapid phenotypic assays to enhance the monitoring of TGC and PMB susceptibility.

Common phenotypic assays employed in clinical microbiology laboratories include: E-tests, disk diffusion, and the VITEK 2 automated system. These traditional antibiotic susceptibility assays typically take up to 24 hours, potentially delaying effective treatment [6, 7]. Many molecular methods have excellent sensitivity and are capable to provide same-day results, but these assays are usually expensive, only provide information on the existence of resistance markers, with no phenotypic data, thus lack clinical practicality [8].

Matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) technology is an emerging detection method in modern clinical laboratory diagnosis, which is widely used in the identification of microorganisms.

At present, it has been proposed to apply this technology to the rapid detection of microbial resistance, which has developed direct-on-target droplet growth assay (DOT-MGA). DOT-MGA greatly shortens the time for bacterial identification and phenotypic detection, which not only brings great convenience to clinical microbiological diagnosis, but also significantly improves the accuracy and shortens the examination time [9, 10].

This study aims to detect the resistance of TGC and PMB by DOT-MGA using MALDI-TOF MS, and to achieve rapid detection of the drug-resistant phenotype of K. pneumoniae within hours for same-day adjustment of antibacterial treatment, which is of great significance for the anti-infective treatment of clinical multidrug-resistant bacteria. The assay can also potentially be applied to phenotypic detection of pathogenic microorganisms other than Enterobacteriaceae, and analysis their resistance to multiple antibiotics.

Bacterial strains

A total of 67 clinical isolates of Klebsiella pneumoniae were collected, including tigecycline (TGC)-susceptible or non-susceptible strains, and polymyxin B(PMB)-susceptible or non-susceptible strains. The strains included in the study were all identified by MALDI-TOF MS (BRUKER) to determine the bacterial species and analyzed by Mérieux VITEK 2 Compact automatic microbiological analysis system to determine the minimum inhibitory concentrations (MIC).

Determination of minimum inhibitory concentration (MIC) by micro-broth dilution method

The MIC of TGC was determined by the micro-broth dilution method. For the specific operation process, please refer to the CLSI M100 document. According to CLSI clinical breakpoints, TGC susceptibility was interpreted (MIC ≥ 8 mg/L is resistant, MIC = 4 mg/L is intermediate, MIC ≤ 2 mg/L is sensitive), ATCC25922 was used as the reference strain. According to ECUAST clinical breakpoints, polymyxin susceptibility was interpreted (MIC ≥ 4mg/L is resistant, MIC ≤ 2 mg/L is sensitive), ATCC27853 and ATCC25922 were used as reference strains.

DOT-MGA

For each well of of 96-well plate, 100 µL bacterial solution was added (the preparation method was the same as the micro-broth dilution method), followed by adding 100 µL MH broth (with or without 2 µg/mL TGC/PMB). Each sample were tested in triplicate. The wells without antibiotics were used as controls.

After inoculating the above-mentioned 96-well plate, 6 µL mixed droplets were taken and transferred onto the MALDI-TOF MS in triplicate: 3 test spots with tigecycline and 3 growth control spots without antibiotics. The inoculated target plate was placed in a plastic shipping box (Bruker Daltonik), and 4 mL of deionized water was added to the bottom of the box to serve as a wet box. After incubation at 35 ± 1°C in an incubator, the broth was removed from the target plate using lint-free paper.

After the remaining bacterial solution on the target plate was completely dried, 1 µL of mass spectrometry sample matrix solution (IVD Matrix HCCA-portioned, Bruker Daltonik) was added to cover the target. MALDI-TOF mass spectra were manually collected and evaluated by standard MALDI Biotyper 3 software (Bruker Daltonik).

In the pilot study, thirteen susceptible strains and thirteen resistant strains were randomly selected, and incubated for 3h, 4h, 6h, or 8h. The optimal incubation time was determined according to the detection performance. For the rest of the study, all strains were subjected to DOT-MGA using the optimal incubation time.

The test was considered valid when growth control identification score was ≥ 1.7. For samples containing tigecycline or polymyxin B, a successful identification (score ≥ 1.7) was interpreted as non-susceptible (NS, intermediate or resistant); while a failed identification (score < 1.7) was defined as sensitive (S).

Data analysis

The final results of DOT-MGA were compared with those of the micro-broth dilution method. The efficiency, sensitivity, specificity, negative predictive value, positive predictive value, and classification agreement were calculated, respectively.

Previous studies have shown that DOT-MGA can achieve the best detection performance with 6 µL droplet volume [9, 11]. In our previous experiments, we compared 4 µL, 6 µL, and 8 µL droplet volume. We found that the growth of bacteria was impeded if the droplet volume was not sufficient. While if the droplet volume exceeded 6 µL, samples may overflow the target wells, causing cross-contamination. With a droplet volume of 6 µL, the samples could be retained in the wells to obtain the best growth condition. Therefore, 6 µL droplet volume was used to explore the best incubation volume for DOT-MGA in this study.

To explore the best incubation time, we randomly selected 13 tigecycline-susceptible strains and 13 tigecycline-non-susceptible strains, and incubated them for 3 h, 4 h, 6 h, and 8 h, respectively. The results of this pilot study showed that after culturing for 3 h, the effective rate of bacterial growth was 88.46%, and the classification agreement rate was 73.91%. Under this condition, the growth condition was not good and the bacteria could not be detected correctly. After 4 h incubation, the effective rate of bacterial growth was 100%, the classification agreement rate was 96.15%. The sensitivity, specificity, positive predictive value, and negative predictive value were 92.31%, 100%, 100%, and 92.86%, respectively (Table 1, Appendix Table 1, Appendix Table 2). The 4 h incubation time showed the optimal condition for bacteria growth. When the incubation time was further extended to 6h and 8h, the detection performance was not significantly improved. Therefore, 4 h was determined as the best incubation time. All the collected 54 strains were incubated with 6 µL droplets for 4 hours, and were detected by MALDI-TOF mass spectrometer. By comparing to micro-broth dilution method, the effective rate, classification agreement rate, sensitivity, specificity, positive predictive value, and negative predictive value were 100%, 98.15%, 96.30%, 100%, 100%, and 98.15%, respectively. These results were better than the detection performance of the pilot study using 4 h incubation, which suggests a good consistency in the identification of tigecycline-non-susceptible strains (Table 1, Appendix Table 3).

Table 1

Identification performance of DOT-MGA method for tigecycline-insensitive strains
Incubation time	Effective rate	Classification agreement rate	Sensitivity	Specificity	Positive predictive value	Negative predictive value
3h (13S,13NS)	88.46%	73.91%	45.45%	100%	100%	66.67%
4h (13S,13NS)	100%	96.15%	92.31%	100%	100%	92.86%
6h (13S,13NS)	100%	96.15%	100%	92.31%	92.86%	100%
8h (13S,13NS)	96.15%	96%	100%	92.31%	92.31%	100%
4h (27S,27NS)	100%	98.15%	96.30%	100%	100%	98.15%
NS(I/R): tigecycline- intermediate or resistant strains, S: tigecycline-susceptible strains

A total of 13 polymyxin B-non-susceptible strains and 13 polymyxin B-susceptible strains were collected for DOT-MGA experiment. Our results showed that the growth efficiency of growth control wells was 96.15% under the incubation time of 3 h. Under the incubation time of 4 h, 6 h, and 8 h, the growth efficiency was 100%, the strains could be effectively identified under these incubation times. After incubation for 4h, DOT-MGA showed the best identification performance, under which the classification agreement rate, sensitivity, specificity, positive predictive value, and negative predictive value were 96.15%, 92.31%, 100%, 100%, and 92.86%, respectively. Further prolonged the incubation time to 6 h and 8 h did not significantly improve the performance (Table 2, Appendix Table 1, Appendix Table 4). In conclusion, the DOT-MGA can accurately identify polymyxin-susceptible and non-susceptible strains in only 4 h incubation time, which has good consistency compared with the micro-broth dilution method.

Table 2

Identification performance of DOT-MGA for polymyxin B-non-susceptible strains
Incubation time	Effective rate	Classification agreement rate	Sensitivity	Specificity	Positive predictive value	Negative predictive value
3h (13S,13NS)	96.15%	88%	75%	100%	100%	81.25%
4h (13S,13NS)	100%	96.15%	92.31%	100%	100%	92.86%
6h (13S,13NS)	100%	96.15%	92.31%	100%	100%	92.86%
8h (13S,13NS)	100%	96.15%	92.31%	100%	100%	92.86%
NS(I/R): polymyxin B- intermediate or resistant strains, S: polymyxin B-susceptible strains

At present, the micro-broth dilution method is the gold standard method for interpreting the clinical microbial resistance phenotype. The commonly used VITEK 2 Compact automatic microbial analysis system is based on this method to detect the MIC value of drugs, but the results are usually not available within the same day, which may delay effective treatment. Therefore, a rapid testing method for antimicrobial susceptibility of microorganisms remains an urgent clinical problem.

The DOT-MGA is also based on the principle of the micro-broth dilution method. Relying on the high detection performance of MALDI-TOF MS, DOT-MGA shortens the incubation time compared to the conventional broth dilution method, thus achieves rapid detection. Compared with the naked eye observation, DOT-MGA can not only exclude subjective interference, but also improve its accuracy and sensitivity. Due to these advantages, the DOT-MGA has received extensive attention [12–15]. Previously Idelevich, EA et al. co-incubated meropenem with K. pneumoniae and Enterobacteriaceae, followed by using DOT-MGA for rapid detection of the carbapenemase non-susceptibility and the classification of bacterial carbapenemase within 4 hours [16]. In subsequent study, they applied this method to cefuroxime, cefotaxime, ceftazidime/avibactam, ertapenem, ciprofloxacin, levofloxacin, gentamicin, and other antibiotics. Their results validated the detection performance of DOT-MGA and demonstrated DOT-MGA is suitable for rapid and simultaneous detection of the susceptibility of clinical Enterobacteriaceae isolates against multiple antibiotics [17]. However, the number of some antibiotic-nun-susceptible strains was limited in the previous studies. For example, tigecycline- and polymyxin-non-susceptible strains were not investigated, and the evaluation of these two antibiotics was lacking. In this study, 27 tigecycline-non-susceptible strains and 13 polymyxin-non-susceptible K. pneumoniae strains were collected to verify the phenotypic detection performance of DOT-MGA. Our results showed that after co-incubating with tigecycline or polymyxin for 4 h, most Enterobacteriaceae could be detected by MALDI-TOF MS with high consistency when compared to micro-broth dilution method.

DOT-MGA could be applied to many different microbial and antibiotic assays, but each assay needs optimization of the antibiotic-organism incubation conditions and incubation times to obtain the best detection performance. In this study, we found that some growth controls could not be detected with 3 h incubation time, which may due to the insufficient incubation time, low bacterial content, and poor spectrum. In the samples incubated with antibiotics, although the growth control wells exceeded the detection threshold, there were occasionally a few non-susceptible strains still failed to meet the detection requirements, which was mainly due to the growth status of different strains. Slow-growing bacteria take longer to compete with antibiotics, but excessive incubation time would lead to bacterial overgrowth and may produce other miscellaneous bacteria, thus reduces the detection accuracy. Appropriate incubation time should allow sufficient bacterial growth for successful detection, and accurate classification of susceptibility to antibiotics.

In this study, we evaluated tigecycline and polymyxin and found that DOT-MGA could accurately distinguish susceptible and non-susceptible strains to tigecycline and polymyxin after 4 h incubation. Although other antibiotics have not been evaluated in this study, the detection of other antibiotics, especially carbapenems, has been previously reported. For example, Idelevich, EA et al. verified that multiple antibiotics including carbapenems can achieve the best detection performance after 4 h incubation [17]. Because strains resistant to tigecycline and polymyxin are difficult to collect, this study mainly verified K. pneumonia. The study by Horseman TS et al. proved the high accuracy of DOT-MGA in drug susceptibility tests of four common pathogens, Staphylococcus aureus, Enterococcus, Escherichia coli and K. pneumoniae [12]. Therefore, we speculate that DOT-MGA can be widely used in the identification of different bacterial species and the susceptibility of different antibiotics. After optimizing the best detection conditions for different antibiotic-bacteria combinations, a disposable target plate containing a fixed amount of antibiotics can be developed, by which the detection of antibiotic susceptibility can be performed by simply adding a certain volume of bacterial solution and incubating for several hours. This technology would greatly shorten the time of drug susceptibility test, provide new ideas for the rapid detection of microorganisms, and is of great significance to the anti-infective treatment of clinical multidrug-resistant bacteria.

In conclusion, DOT-MGA can correctly identify tigecycline and polymyxin B sensitive and non-sensitive bacteria after only 4 hours of incubation, which is in high consistency with the results of the micro-broth dilution method. The method has high potential application value in the rapid detection of drug susceptibility, but further research is still needed to standardize and optimize the test conditions and evaluation criteria.

Ethical Approval and Consent to Participate

This study involved the use of excess or discarded sputum, feces, and blood samples from anonymous patients in our hospital. The samples were collected as part of standard treatment with consent to participate of the patients. This research will not affect the health and privacy of the patient. All procedures performed in this study involving human participants were in compliance with the ethical standards of the Medical Ethics Committee of Anhui Medical University (Reference number: LISC20210802).

Consent for publication

Not applicable.

Availability of data and materials

All data generated or analysed during this study are included in this published article and its supplementary information files.

Competing interests

The authors declare that they have no competing interests.

Authors’ contributions

Zhaoxin Xia, Wensu Yang, Yi Zhu, Huaming Xu, Hao Tang and Jilu Shen are participated in the study design and study management. Zhaoxin Xia, Wensu Yang, Guoping Lu, Zhen Liu, Xinyi Hu, Chunhui Lin and Tong Jiang participated in the study of antimicrobial susceptibility experiments and Direct-on-Target Micro-droplet Growth Assay. Zhaoxin Xia, Wensu Yang and Jilu Shen participated in data interpretation and writing of the manuscript. All authors participated in data collection and reviewed the manuscript. All authors have agreed on the publication of this manuscript, and agreed to be responsible for all their research work.

Funding

This study is supported by 2020 Anhui Provincial University Cooperative Research and Public Health Collaborative Innovation Project of Anhui Provincial Department of Education (Grant No. GXXT-2020-016), 2021 Anhui Provincial Health and Health Commission Key Scientific Research Project (Grant No. AHWJ2021a011), 2021 Anhui Provincial Medical and Health Key Specialty Construction Project (Approval number: serial number 95), 2021 Anhui Provincial Key Project of Natural Science Research in Colleges and Universities (Approval number: KJ2021ZD0032).

Acknowledgments

Not applicable.

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No competing interests reported.

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A Rapid Tigecycline and Polymyxin B Susceptibility Test for Klebsiella pneumoniae Using Direct-on-Target Micro-droplet Growth Assay

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Version 1

Abstract

Background

Methods

Results

Conclusion

Introduction

Materials And Methods

Bacterial strains

Determination of minimum inhibitory concentration (MIC) by micro-broth dilution method

DOT-MGA

Data analysis

Results

Discussion

Conclusions

Declarations

References

Additional Declarations

Supplementary Files

Status:

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