Rapid detection of Ceftazidine/Avibactam sensitivity in carbapenem-resistant Enterobacteriaceae

doi:10.21203/rs.3.rs-3144091/v1

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Rapid detection of Ceftazidine/Avibactam sensitivity in carbapenem-resistant Enterobacteriaceae

https://doi.org/10.21203/rs.3.rs-3144091/v1

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Currently, ceftazidime-avibactam (CZA) is the primary antibiotic for treating carbapenem-resistant Enterobacteriaceae (CRE) infections in KPC and OXA-48 families. However, due to rising CZA resistance, a fast, simple, and effective technique is needed to detect CRE sensitivity to CZA. The rapidCZA test proposed in this study only takes 4 hours to determine the CRE sensitivity to CZA. In the presence of sufficient CZA (low concentration: 8 mg/mL ceftazidime and 4 mg/mL avibactam, high concentration: 16 mg/mL ceftazidime and 4 mg/mL avibactam), glucose metabolism of CZA-resistant bacteria changes bromocresol purple from purple to yellow. After K-B testing of CZA, 83 of 241 CRE strains were resistant (< 21 mm), and 158 were susceptible to CZA (≥ 21 mm). Furthermore, eCIM testing revealed that all 83 CZA-resistant strains were NDM type, while 121 of the 158 CZA-sensitive strains were KPC type and 37 were non-enzyme producing. After the rapidCZA test, 83 CRE strains (NDM type) were resistant to CZA, and 158 CRE strains (including KPC and non-carbapenemase production) were sensitive. The sensitivity, specificity, and positive predictive values and negative predictive values of this test were all 100%. This assay is fast, accurate, and offers clinical feedback on CRE strains' CZA sensitivity in 4 hours.

CRE

RapidCZA test

CZA

KPC

NDM

Ceftazidime-avibactam (CZA), a third-generation head broad-spectrum cephalosporin, inhibits bacterial cell wall synthesis by attaching to penicillin-binding proteins. Therefore, CZA can expand antibacterial activity and is active against most AmpC, KPC, and ESBL-producing enterobacteria[1], mainly for treating complex intra-abdominal infections caused by gram-negative bacteria, hospital-acquired pneumonia, and ventilator-associated pneumonia, particularly for CRE, CRPA and other infections with limited treatment options[2].

Currently, Enterobacteriaceae bacteria have a low CZA resistance rate globally (< 2.6%)[3], whereas Pseudomonas aeruginosa's resistance rate is comparatively high, ranging from 4 to 8%[4]. In 2017, 46.8% of all CRKP strains in China were KPC type[5]. Carbapenem-resistant strains have boosted CZA resistance to 24.7%, according to certain research[6].

The main mechanisms of bacterial resistance to CZA include amino acid substitutions at key sites of β-lactamase, altered membrane permeability due to membrane protein mutation, overexpression of the efflux pump and penicillin-binding protein, and ESBLs mutant[7]. Currently, CZA resistance is detected using PCR and enzyme-type techniques (additional specific detection methods are required). However, none of these methods can detect CZA resistance caused by gene mutations. Therefore, this study intends to develop the rapid identification of CRE strain to CZA can be judged based on the experimental results after 4 h.

Bacterial strain

To conduct the test, 241 CRE strains were chosen, including 81 Escherichia coli, 132 Klebsiella pneumoniae (including the KPN142 strain[8] and other strains), and 28 Enterobacter cloacae strains. As a reference, Klebsiella pneumoniae ATCC 700603 and Escherichia coli ATCC 25922 were used as negative control strains, and two NDM-type CRE-producing strains resistant to CZA were used as positive control strains.

Antimicrobial susceptibility testing and enzyme-based assays

An E-test was conducted to review the MICs of imipenem, meropenem, and ertapenem for 241 CRE strains. Afterward, 241 CRE strains were evaluated for enzyme production by means of the eCIM assay. Hence, isolates with CZA MICs of ≤ 8/4 mg/L were categorized as susceptible, while those with MICs of ≥ 16/4 mg/L were categorized as resistant. The K-B method was used to assess the sensitivity of 241 CRE strains to CZA. And isolates with the CZA K-B method of bacteriostatic circle ≥ 21 mm were categorized as susceptible, while those with MICs of bacteriostatic circle <21 mm were categorized as resistant. And the Clinical breakpoints were established according to the 2023 Clinical and Laboratory Standards Institute (CLSI).

Reagents and solutions

This experiment utilized a commercial bacterial microbiochemical reaction tube (Glucose biochemical reaction solution) from Hangzhou Binhe Microbial Reagent Co., Ltd., China. The reagents were 10 g peptone, 3 g beef extract, 5 g NaCl, 10 g glucose, 0.01 g bromocresol violet, and 1000 mL of sterile water with a pH of 7.2 ± 0.4. Low concentrations of ceftazidime and avibactam were 8 mg/L and 4 mg/L, respectively, while high concentrations of ceftazidime and avibactam were 16 mg/L and 4 mg/L, respectively.

Test and reading

The monoclonal strains were picked to configure 0.5 MaFarland standard optical density (10⁸ CFU/mL). Negative control group 1: 10 µL of 0.45% sterile saline was added to 990 µL of the solution (Glucose biochemical reaction solution); Negative control group 2: 5 µL ceftazidime (3.2 mg/mL) and 5 µL avibactam (0.8 mg/mL) was added to 990 µL of the solution (Glucose biochemical reaction solution); positive control group: 10 µL of 0.5 MaFarland standard optical density bacterial suspension was added to 990 µL solution; low concentration CZA experimental group: 10 µL of 0.5 MaFarland standard optical density bacterial suspension, 5 µL of ceftazidime (1.6 mg/mL) and 5 µL of Avibactam (0.8 mg/mL) were added to 980 µL of the solution; High concentration CZA experimental group: 10 µL of 0.5 MaFarland standard optical density suspension, 5 µL of ceftazidime (3.2 mg/mL) and 5 µL of avibactam (0.8 mg/mL) were added to 980 µL of the solution. Typically, 100 µL was applied to 96-well plates at 37°C for 4 h. If the color shifts from purple to yellow, it is indicative of resistance, whereas if the color remains the same, it is indicative of sensitivity. All experiments were repeated thrice.

In the development step, 241 CRE strains were tested. The eCIM experiment results indicated that 83 CRE strains were of the NDM type, 121 of the KPC type, while 37 were non-carbapenemase production. By the K-B method of CZA, 83 NDM type CRE strains were found to be resistant to CZA (< 21 mm), whereas 158 CRE strains (including KPC and non-carbapenemase production) were observed to be susceptible to CZA (> 21 mm). In the RapidCZA test, 83 NDM CRE strains were positive (color changed from purple to yellow), and 158 CRE strains (including KPC and non-carbapenemase production) were negative (color was purple) (As is shown in Fig. 1). The positive and negative predictive values, sensitivity, and specificity were 100% (95% CI: 94.5–100%), 100% (95% CI: 97.1–100%), 100% (95% CI: 94.5–100%), and 100% (95% CI: 97.1–100%), respectively. (As is shown in Table 1)

Table 1

Comparison of K-B method and RapidCZA test results on 241 CRE strains
		K-B method		Sensitivity % [95% CI]	Specificity % [95% CI]	PPV % [95% CI]	NPV % [95% CI]	K
		R	S
RapidCZA test	R	83(TP)	0 (FP)	100	100	100	100	1
	S	0 (FN)	158 (TN)	94.5–100	97.1–100	94.5–100	97.1–100
R, resistant strains; S, susceptible strains; CI, Confidence Interval; TP, true positive, FP, false positive; TN, true negative; FN, false negative; PPV, positive predictive value; NPV, negative predictive value; K,Cohen’s Kappa.

The China Bacterial Resistance Monitoring Network found a fast growth in Klebsiella pneumoniae clinical isolates resistant to carbapenem antibiotics, from 3% in 2005 to 23% in 2021[3, 5]. According to the 2021 data from the National Bacterial Resistance Monitoring Network (CARSS), the average resistance rate of Klebsiella pneumoniae clinically isolated from 1429 hospitals across the country to Carbapenem antibiotics is 11.3%, with some provinces and cities even exceeding 20%[4]. The China Bacterial Resistance Monitoring Network studied 935 CRE strains from 39 hospitals in China in 2018. 51.6% (482/935), 35.7% (334/935), and 7.3% (68/935) produced KPC, NDM, and OXA-48 carbapenem enzymes, respectively, among which a few strains simultaneously produced a variety of carbapenemases, which indicates that the resistance rate of carbapenem antibiotics is increasing year by year, making research into CRE resistance particularly important[9].

CZA is the main treatment for CRE infections that produce KPC and OXA-48 because it is effective against gram-negative bacilli, specifically Enterobacteriaceae[6]. It can drastically reduce the mortality rate of infected patients. Therefore, it is essential to quickly identify the genotype of CRE and administer effective treatment to reduce the mortality rate of those infected[10]. Various methods are available for detecting the enzyme type and genotype of CRE strains, for example, the eCIM test[11], Carba NP assay[11–13], MALDI-TOF MS (detecting the enzyme type of CRE strains)[14–17], enzyme immunochromatography[18], and GeneXpert@Carba-R (detect the genotype of CRE strain)[19, 20]. However, these approaches may take a long time or require professional equipment, and they cannot detect carbapenem resistance induced by gene mutations or other processes.

CZA is thought to treat KPC-producing CRE infections clinically. However, certain strains develop Extended-spectrum β lactam and AmpC enzymes and down-regulate or delete outer membrane porin expression, making them carbapenem-resistant[9]. Additionally, KPC-31 and KPC-33 invading the D179Y 1/- H274Y mutation, and KPC-8 (V240G-H274Y), KPC-23 (V240A-H274Y), or KPC-40 (T237S-H274Y) can all make bacteria resistant to CZA[21]. Moreover, point mutation of KPC-2 (D179Y)[22] or the nonfunctional ompK35-ompK37 and the mutant ompK36 pore protein combined with the high copy number of the bla_KPC gene can also make bacteria resistant to CZA[23]. Current methods like the eCIM test, Carba NP assay, MALDI-TOF MS, enzyme immunochromatography, and GeneXpert@Carba-R cannot accurately detect such resistance mechanisms, which may lead to incorrect results and patients not receiving timely and effective treatment. To address this issue, RapidCZA test may identify CZA-resistant Enterobacteriaceae bacteria in 4 hours, with good sensitivity and specificity, low cost, and low laboratory requirements, making it suited for routine detection. Accordingly, this test can provide timely and effective feedback on the sensitivity of CRE strains to CZA to the clinic.

In this research, 241 CRE strains were tested by the RapidCZA test, including NDM type, KPC type, and non-carbapenemase production. Out of these, 83 NDM type CRE strains were found to be resistant to CZA, and the remaining 158 CRE strains were sensitive to CZA (comprising KPC type and non-carbapenemase production). This result coincides with the K-B method of CZA. After comparing the RapidCZA test and K-B method, it was seen that the sensitivity, specificity, positive predictive value, and negative predictive value of the RapidCZA test were all 100%, indicating that the RapidCZA test is a highly sensitive and accurate technique. The RapidCZA test is more suitable for laboratories as it only takes 4 hours to determine the results based on color changes, which is much shorter than traditional drug sensitivity identification.

In the RapidCZA test, CRE strains that are resistant to CZA can decompose glucose, causing the pH to drop and bromocresol to change from purple to yellow. Visual recognition of a color change can signify the presence of CZA-resistant strains; however, if these strains have only low levels of resistance, false negatives may be produced. To ensure more accurate readings, it may be necessary to have a second person present.

Other tests with similar methods have been developed, such as the Rapid Polymyxin NP[24], which was commercialized and based on the resazurin color change in response to bacterial growth in the presence of a polymyxin drug, or the rapid fosfomycin/Escherichia coli NP test[25], which detects fosfomycin resistance in E. coli isolates through an orange-to-yellow color change of red phenol. Unlike the latter methods, RapidTZP test uses 10 µL from an inoculum with a 0.5 McFarland standard, being lower than the 3 to 3.5 McFarland standard used by the Rapid Polymyxin NP and the rapid fosfomycin/Escherichia coli NP test. Because clinical samples with less bacterial inoculum may be analyzed, this lowered inoculum may speed up results[26]. In contrast to RapidCZA test, the RapidTZP test requires the individual to prepare reaction solutions and adjust the pH themselves. And RapidCZA test employs commercial reagents and does not require pH adjustment or reagent configuration, making it convenient and efficient.

Now, CZA is still usually prescribed as a single drug. A study found that CZA monotherapy is more likely to cause drug resistance than combined therapy. CZA reduces the strain's MIC value when administered with other antibiotics, indicating a synergistic effect. Therefore, other effective antibiotics or CZA in combination with other antibiotics should be considered for strains that have already developed resistance.

The RapidCZA test is a simple, highly effective, sensitive, and specific detection method. With the growing number of individuals affected by bacteria that are resistant to carbapenem, the importance of CZA is becoming increasingly clear. This experiment aims to determine the susceptibility of CRE to CZA and enhance the survival rate, thus achieving improved clinical outcomes and expediting microbiological diagnosis.

Acknowledgements

We would like to express our appreciation to Huixin Chen, Chengfang Gong, and Ting Zhang for their valuable contributions to this study, which was conducted in a teaching hospital, where some of the co-authors are from different institutions.

Authors’ contributions

All authors contributed to the study conception and design. Material preparation were performed by Ya Huang, Huixin Chen, and Fei Lv. Data collection and analysis were performed by Ya Huang, Huixin Chen, Chengfang Gong, and Congzhu Yang. Data analysis were performed by Ya Huang, Huixin Chen and Fei Lv. The first draft of the manuscript was written by Ya Huang and all authors commented on previous versions of the manuscript. Ya Huang, and Ting Zhang prepared figures 1. Quan Zheng and Ruiwen Xie prepared table 1. Fei Lv finalized the final manuscript. All authors read and approved the final manuscript.

Funding

This work was supported by Dongguan Social Science and Technology Development (General) Project (20211800900702), and Dongguan Social Science and Technology Development (General) Project (20231800904962).

Availability of data and materials

The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare that there are no conficts of interest.

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No competing interests reported.

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Rapid detection of Ceftazidine/Avibactam sensitivity in carbapenem-resistant Enterobacteriaceae

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Abstract

Figures

Introduction

Materials and Methods

Bacterial strain

Antimicrobial susceptibility testing and enzyme-based assays

Reagents and solutions

Test and reading

Results

Discussion

Conclusion

Declarations

References

Additional Declarations

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