Comparative evaluation of five β-Lactamase inhibitors in combination with β-Lactams against multidrug-resistant Mycobacterium tuberculosis in vitro

Abstract

Objective

Evaluating the activity of six β-lactams in combination with different β-lactamase inhibitors to identify the most potent combination against Mycobacterium tuberculosis(MTB) in vitro.

Methods

A total of 105 MDR-TB strains from different regions of Henan province were included in this study.Drug susceptibility of sixβ-lactams alone or in combination with β-lactamase inhibitors was examined by broth dilution method against 105 clinical isolates.Mutations of blaC, ldt_mt1,dacB2and ldt_mt2 were analyzed by PCR and DNA sequencing.

Results

Out of the β-lactams used herein, tebipenem was the most effective against MDR-TB and had an MIC₉₀ value of 16 µg/ml.Clavulanic acid, tazobactam, and sulbactam, demonstrated the best synergy with tebipenem, resultingin an 32-fold reduction in theMIC values for 12, 5, and 20 strains, respectively. Simultaneously, these three types ofβ-lactamase inhibitors had the least impact on imipenem.Clavulanic acid caused the maximum 8-fold reduction in the MIC value of imipenem, while tazobactam and sulbactam only resulted in the maximum 4-fold reduction in the MIC value of imipenem. Besides, after the addition ofβ-lactamase inhibitors, the MICs of most β-lactam drugs were reduced more evidently in the presence of avibactamand tazobactamcompared to other β-lactamase inhibitors. In addition, 13.33% (14/105) of isolates harbored mutations in the blaC gene, with three different nucleotide substitutions: AGT333AGG 、AAC638ACCand ATC786ATT. For the strains with a Ser111Arg andAsn213Thrsubstitution inBlaC, a better synergistic effect was observed in the meropenem-clavulanate and in the meropenem-sulbactam combinationsthan that in a synonymous single nucleotide polymorphism (SNP) group.

Conclusion

the combination of tebipenem and relebactam shows the most potent activity against MDR-TB isolates. In addition, the Ser111Arg and Asn213Thr substitution of BlaC may be associated with increased susceptibility of MDR-TB isolates to meropenem in thepresence of clavulanate and sulbactam.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12879-025-10730-y.

Introduction

In recent years, the emergence of multidrug-resistant (MDR) strains of Mycobacterium tuberculosis, namely the M. tuberculosis resistant to at least rifampicin and isoniazid, has become a major obstacle to global tuberculosis control [1, 2]. According to the estimation of the World Health Organization (WHO), there are about 465,000 patients with rifampicin-resistant tuberculosis worldwide, among whom 78% have MDR tuberculosis (MDR-TB). Only one-third of the MDR-TB patients have received appropriate treatment [3]. MDR bacteria are resistant to the two most important anti-TB drugs, rifampicin and isoniazid, which increase the risk of treatment failure in MDR patients.The extensively drug-resistant tuberculosis (XDR-TB), a type of MDR-TB resistant to fluoroquinolone drugs and at least one drug of bedaquiline and linezolid, has an even lower treatment success rate [4, 5]. The increase in the number of MDR-TB patients has made it difficult for the traditional first- and second-line anti-TB drugs to meet the needs for drug-resistant TBtreatment. Novel and effective treatment strategies are urgently needed to overcome the shortcomings of the current treatment regimens, especially for XDR-TB [6–8].

β-Lactam drugs impede the synthesis of bacterial cell wall by inhibiting the cross-linking reaction of peptidoglycans (PGs), thus exerting a potent bactericidal activity [9, 10]. M. tuberculosis is naturally resistant to most β-lactam drugs dues to its highly active β-lactamase BlaC [11, 12]. BlaC is a class A lactamase in the Ambler molecular structure classification. It can be inactivated by most commercial β-lactamase inhibitors [13].Therefore, the combined application of β-lactams and lactamase inhibitors can overcome the resistance of M. tuberculosis to β-lactams. Both in vivo and in vitro studies have shown that the combined application of β-lactams and β-lactamase inhibitors effectively kills M. tuberculosis [14–16]. In addition, some clinical trialshave evaluated carbapenem efficacy, safety, and tolerance in M. tuberculosis patients. The combined applicationof amoxicillin and clavulanic acid effectively inhibits the growth of M. tuberculosisin vitro and exhibits antibacterial activity in the early anti-tuberculosis treatment of MDR-TB. In macrophages infected by M. tuberculosis, combined administration of imipenem and clavulanic acid for 1 week reduces the bacterial load by more than 50% [17]. Meropenem exhibits good antibacterial activity in MDR patients with a risk of treatment failure [18, 19]. Meropenem and clavulanic acid have strongbactericidal activity against M. tuberculosis in MDR-TB and XDR-TB.Because oftheir potent antibacterial activity [20], imipenem and meropenem have been classified by the WHO as group C anti-TB drugs forthe treatment of patients with MDR-TBor rifampicin-resistant TB [21–23].

BlaC exhibits broad-spectrum antibacterial activity against penicillins and cephalosporins, while having weaker activity against the new carbapenems. Compared with penicillin and cephalosporin, the combination of β-lactamase inhibitors and carbapenems has greater antibacterial activity against MDR-TB strains [24].Several clinical trials provided preliminary evidence of the efficacy and safety of meropenem and clavulanic acid in the treatment of MDR and XDR patients [25].The potent antibacterial activity of meropenem and clavulanic acid suggests that other combinations of β-lactams and β-lactamase inhibitors might besuitable for the clinical treatment of MDR and XDR patients.

Avibactam and relebactam are two new carbapenem β-lactamase inhibitors. Avibactam is a structural inhibitor. Although it does not contain a β-lactam core, it inducesβ-lactamases to undergo covalent acylation. As a covalent β-lactamase inhibitor, avibactam has a inhibitory effect on β-lactamase activity [26]. Relebactam is a β-lactamase inhibitor structurally similar to avibactam, but the activity of this drug against MDR-TBhas beenless thoroughly evaluated [27]. In this study, we selected six β-lactams and five β-lactamase inhibitors, including the two new β-lactamase inhibitors avibactam and relebactam. We then used the broth dilution method to evaluate the bactericidal activities of β-lactams alone and the combination of β-lactams and β-lactamase inhibitors against MDR-TB strains in vitro. This study provides more potential β-lactam and β-lactamase inhibitor candidates for the clinical treatment of patients with MDR TB.

Materials and methods

Strains and drug susceptibility test

The 105 MDR clinicalstrains included in this study were isolated from culture-positive TB patients in six municipal TB control institutions of Henan province in Zhengzhou, Kaifeng, Nanyang, Luoyang, Anyang, and Zhoukou.These strains were preserved in the Strain Bank of the Institute for the Prevention and Control of Infectious Diseases, Henan Provincial Center for Disease Control and Prevention.All experiments were conducted in the TB reference laboratory of Henan Provincial Center for Disease Control and Prevention.This study was approved by the Medical Ethics Committee of the Henan Provincial Center for Disease Control and Prevention(Number: 2023-KY-002-02).

The positive strains cultured on acidic Lowenstein-Jensen (LJ) media were subjected to isoniazid, rifampicin, ethambutol, streptomycin, kanamycin, amikacin, capreomycin, and ofloxacinsusceptibility tests using the proportions method recommended by WHO [28].In addition, the strains were subjected to mycobacterial species identification using LJ media supplemented withp-nitrobenzoic acid (PNB) and 2-thiophene carboxylic acid hydrazide (TCH).

Different carbapenem-class β-lactam drugs and β-lactamase inhibitors

The present study used six lactam drugs and five β-lactamase inhibitors to determine the minimum inhibitory concentrations (MICs). The sixβ-lactam drugs were all carbapenems, including five injection drugs commonly used in clinical practice (imipenem, meropenem, doripenem, ertapenem, and biapenem) and a novel oral drug, tebipenem. The five β-lactamase inhibitors included three clinically commonly used inhibitors(clavulanic acid, tazobactam, and sulbactam) and two newer inhibitors (avibactam and relebactam). This study evaluatedthe synergistic effect of these β-lactamase inhibitors with the β-lactam drugs against MDR strains. Imipenem, meropenem, doripenem, ertapenem, biapenem, tebipenem, clavulanic acid, tazobactam, sulbactam, and avibactam were purchased from APExbio (USA). Relebactam was purchased from the MEK company (USA).

Determination of the mics

The MICs of the β-lactam drugs against clinical MDR strains were determined using the microplate Alamar blue assay [29]. Briefly, 100 µl of 7H9 liquid medium containing various concentrations of β-lactamdrugs andβ-lactamase inhibitors were added to the wells of 96-well plates. Theβ-lactam drugs were serially diluted from one column to the next, until the penultimate column of sample wells had a concentration of 0.5 µg/ml. Noβ-lactam drugs were added in the last well to act as blank control. Colonies cultured on LJ medium for 4 weeks were scraped off and transferred to normal saline containing 0.5% Tween-20. The turbidity of the bacterial suspension was adjusted to 1 McFarland concentration.Then this bacterial suspension was diluted 20-fold with Middlebrook 7H9 liquid medium containing 10% OADC medium, and 100 µl of the diluted bacterial suspension was added to the wells of 96-well plates containing various drug combinations. The final concentrations of the β-lactam drugs were 0.5–512 µg/ml. After 7 days of cultivation,20 µl Alamar Blue was added to microplate wells and reincubatedovernight until a colour change occurred. The MIC was defined as the lowest concentration that inhibited the growth of the bacteria.MIC₅₀ refers to the drug concentration that inhibits the growth of 50% among105Mycobacterium tuberculosis strains.In other words, at this concentration, 50% of the experimental samples no longer exhibited growth.MIC₉₀ denotes the drug concentration that inhibits the growth of 90% of the Mycobacterium tuberculosis strains within the same cohort of 105 strains. This indicated that 90% of the experimental samples didnot show growth at this concentration.

Genomic DNA extraction and sequencing

Genomic DNA was extracted from M. tuberculosis using the boiling method reported in the literature [30]. Freshly cultured colonies were scraped into 1.5-ml centrifuge tubes containing 200 µl of TE buffer, boiled at 100 °C for 10 min [31], and centrifuged at 13,000g for 5 min to remove sample residues. The DNA-containing supernatants were subjected topolymerase chain reaction (PCR) amplification and sequencing.

Three genes, blaC, dacB2, and ldt_Mt1, were subjected toPCR amplification [32]. The sequences of the amplification primers were as follows: blaC: 3′-ATGCGCAACAG.

AGGATTCGGTC-5′;blaC:5′-CTATGCAAGCACACCGGCAACG-3′;dacB2:3′-ACCAGCAACTGCTGGATTTC-5′;dacB2:5′-ACCAGCAACTGCTGGATTTC-3′;ldt_Mt15′-:ATGCGTCGAGTGGTTCGTTATC-3′;ldt_Mt1: 3′-ATGCCAAAGGTGGGGATTGC-5′. PCR amplification was performed in a 25-µl reaction system using the extracted genomic DNA as atemplate. The 25 µL PCR mixture was prepared as follows: 12.5 µl 2× PCR mixture(CWBIO Biotech Company, China), 5 µL of DNA template, and 0.2 µM of each upstream and downstream primer. The PCR conditions for amplification were 5 min at 94 °C followed by 35 cycles of 94 °C for 30 min, 58 °C for 1 min, 72 °C for 45s, and a final extension of 72 °C for 10 min. PCR products were sent to Shanghai Sangon Biotech Company for Sanger sequencing. The PCR products were sent forSanger sequencing at Sangon Biotech Co., Ltd. Shanghai, China. The sequencing results were compared with the reference sequence of the standard strain H37Rv to identify mutation sites using DNAMAN software.

Data analysis

The synergistic effect is declared when the ratio of MIC without beta-lactamase inhibitor to MIC with beta-lactamase inhibitor is more than 4 times.Chi-square tests were performed using SPSS 20.0 software for the comparison of count data.A p-value of 0.05 or lower is generally considered statistically significant.The synergistic effect of different β-lactamase inhibitors on the Minimum Inhibitory Concentration (MIC) values of β-lactams was analyzed using GraphPad Prism 9.0 software.

Results

Determination of the optimal inhibitory concentrations of various β-lactamase inhibitors

To determine the optimal inhibitory concentrations of various β-lactamase inhibitors, we first evaluated the MICs of different β-lactams against five MDR strains when used alone and the improvement in these MICs in the presence of concentration gradients of β-lactamase inhibitors (1.25, 2.5, 5, and 10 µg/ml). As shown in Table 1, with the increase in the concentration of β-lactamase inhibitors, the MICs of biapenem, doripenem, and tebipenem decreased significantly against the MDR strains. β-lactam inhibitors also improved the MICs of meropenem, imipenem, and ertapenem against the MDR strains. In most cases, the MICs were reduced by 4-8-fold. Overall, there was little difference in the MICs of β-lactamase drugs between the two groups with β-lactamase inhibitor concentrations of 5 µg/ml and 10 µg/ml. In light of these results, a β-lactamase inhibitor concentration of5 µg/ml was used in later experiments.

Table 1.

Antibacterial activity of different combinations of six β-lactams and five β-lactamaseinhibitors against five multidrug-resistant clinical strains

	range of MIC values after combined use of β-lactamase inhibitors
	clavulanic acid(µg/ml)						tazobactam(µg/ml)						sulbactam(µg/ml)
	0	1.25	2.5	5	10		0	1.25	2.5	5	10		0	1.25	2.5	5	10
Biapemem	16–64	8–32	4–32	2–16	2–16		16–64	8–32	4–32	2–16	2–16		16–64	4–32	2–16	1–8	0.5-8
Meropenem	32–128	16–64	8–64	4–32	4–32		32–128	32–128	16–64	8–64	8–64		32–128	16–64	8–64	2–16	2–16
Imipenem	64,512	32–64	16–32	4–16	4–16		64–512	64–512	64–256	32–128	32–128		64–512	64–512	64–256	32–128	32–128
Doripenem	16–64	16–32	8–32	0.5-8	0.5-8		16–64	8–32	4–16	2–16	2–16		16–64	8–32	2–16	0.5-4	0.5-4
Ertapenem	32–128	32–64	16–32	4–32	4–32		32–128	32–128	16–64	8–64	8–64		32–128	32–128	16–64	4–64	4–32
Tebipenem	4–16	2–8	1–4	0.5-2	0.5-2		4–16	2–16	2–8	0.5-4	0.5-4		4–16	2–8	1–4	0.5-1	0.5-1

	range of MIC values after combined use of β-lactamase inhibitors
	avibactam(µg/ml)						relebactam(µg/ml)
	0	1.25	2.5	5	10		0	1.25	2.5	5	10
Biapemem	16–64	4–16	2–8	0.5-8	0.5-8		16–64	4–32	2–16	0.5-8	0.5-8
Meropenem	32–128	16–128	8–64	4–32	4–32		32–128	16–128	4–64	2–32	2–32
Imipenem	64–512	32–512	16–512	8-256	8-256		64–512	32–512	16–512	8-256	8-256
Doripenem	16–64	8–32	2–16	0.5-8	0.5-8		16–64	8–32	4–16	0.5-8	0.5-4
Ertapenem	32–128	16–128	8–64	2–32	2–32		32–128	16–128	4–64	1–32	1–32
Tebipenem	4–16	2–8	1–4	0.5-1	0.5-1		4–16	1–4	0.5-1	0.5	0.5

Sensitivity of MDR strains to β-lactam drugs when administered in combination with clavulanic acid

A total of 105 MDR strains were included in this study. We first evaluated the MICs of the β-lactam drugs against the MDR strains without any β-lactamase inhibitors.The results showed that among the six lactamase drugs used in this study, tebipenem exhibited the best antibacterial activity against MDR-TB strains(χ² = 123.70, p < 0.001), followed by doripenem and biapenem. The MIC₅₀ and MIC₉₀ values of tebipenem were 8 µg/ml and 16 µg/ml, respectively. The MIC₉₀ values of doripenem and biapenem were both 64 µg/ml. Compared with biapenem, doripenem showed better antibacterial activity. The MIC₅₀ values of doripenem and biapenem were 16 and 32 µg/ml, respectively. Imipenem had the worst antibacterial activity, with the MIC₅₀ and MIC₉₀ values of 128 µg/ml and 512 µg/ml, respectively.

We then examined the effect of theaddition of clavulanate on the MICs of lactam drugs and further analyzed the improvement of the MICs of the lactam drugs by clavulanate. As shown in Table 2, tebipenem showed the best synergistic effect with clavulanate. The MICs of tebipenem were reduced by 32- and 16-fold, respectively, against 12 (15.23%) and 22 (24.76%) strains. The MIC₅₀ and MIC₉₀ values of tebipenem were 2 µg/ml and 0.5 µg/ml, respectively. Doripenem and biapenem also showed a good synergistic effect with clavulanic acid. In contrast, clavulanic acid appeared to have the least effect on the MICs of imipenem and ertapenem against MDR-TB strains, both of which had a MIC₉₀ of 128 µg/ml. The presence of clavulanic acid reduced the MICs of imipenem by 4-fold at most. However, compared with the other 4 lactamase inhibitors, clavulanic acid had the best synergistic effect with imipenem. It reduced the MIC₅₀ and MIC₉₀ values ofimipenem by 2-fold and 4-fold, respectively.Moreover, the MICs of imipenem were reduced by 8-fold against 8 strains (7.6%) and 4-fold against 28 strains (26.67%).

Table 2.

Effect of 5 µg/ml clavulanic acid on the mics of β-lactam drugs

	MIC50			MIC90			The range of MIC decrease multiples	MIC range (µg/ml)	MICDistribution width	SDofMIC
	-	+		-	+
Biapemem	32	16		64	32		1–32	2–64	6	46.36
Meropenem	32	16		128	64		1–16	4-128	6	49.27
Imipenem	128	64		512	128		1–8	4-128	6	107.89
Doripenem	16	8		64	16		1–32	≤ 0.5–32	≥ 7	17.67
Ertapenem	64	32		128	128		1–16	4-128	6	70.91
Tebipenem	8	0.5		16	2		1–32	≤ 0.5-8	≥ 5	2.90

Sensitivity to β-lactam drugs when administered in combination with Tazobactam

Among the three β-lactamase inhibitors, tazobactam showed the worst synergistic effect (Table 3). Compared with other inhibitors, the combined application of tazobactam andβ-lactam drugs resulted in higher MIC₅₀ values. After the addition of tazobactam, tebipenem showed the most significant change in the MIC against the MDR strains of all the β-lactam drugs. This result was similar to that of clavulanic acid. Compared with tebipenem alone, the combined application of tebipenem and tazobactam reduced the MICs by more than 4-fold in 88 MDR-TB strains.The MIC₅₀ value of tebipenem against the MDR strains was decreased from 8 µg/ml to 0.5 µg/ml, indicating that tebipenem and tazobactam had a good synergistic effect.However, supplementation withtazobactam had little effect on the MICs of meropenem and imipenem against the MDR-TB strains. Specifically, the MIC values remained unchanged against 42 and 65 strains, respectively.

Table 3.

Effect of 5 µg/ml Tazobactam on the MIC of β-lactam drugs

	MIC50			MIC90			The range of MIC decrease multiples	MIC range (µg/ml)	MIC Distribution width	SD of MIC
	-	+		-	+
Biapemem	32	16		64	32		1–32	2–64	6	50.95
Meropenem	32	16		128	128		1–8	8-256	6	115.41
Imipenem	128	128		512	512		1–4	32–512	5	205.38
Doripenem	16	8		64	32		1–32	2–64	6	46.62
Ertapenem	64	64		128	128		1–8	8-256	6	100.69
Tebipenem	8	0.5		16	4		1–32	≤ 0.5–16	≥ 6	5.82

Sensitivity to β-lactam drugs when administeredin combination with sulbactam

Sulbactam showed a good synergistic effect with four of the six β-lactam drugs (biapenem, meropenem, doripenem, and tebipenem).Sulbactam reduced the MIC₅₀ values of these four drugs to below 8 µg/ml. Moreover, the MIC₅₀ or MIC₉₀ values of the four drugs in the presence of sulbactam were lower than those in the presence of clavulanic acid and tazobactam. In particular, sulbactam showed the best synergistic effect with tebipenem. Sulbactam reduced the MICs of tebipenem by 32-fold against 18 strains and 16-fold against 38 strains (Table 4). The addition of sulbactam reduced MIC₅₀ and MIC₉₀ values of tebipenem by 16-fold against the MDR strains. It reduced the MIC₅₀ of tebipenem from 8 µg/ml to 0.5 µg/ml, while it reduced the MIC₉₀ from 16 µg/ml to 1 µg/ml. In addition, sulbactam had a good synergistic effect with doripenem and biapenem. It reduced the MICs of doripenem and biapenem by more than 16-fold in 27 (25.71%) and 17 (16.19%) strains, respectively. Like the other two inhibitors, sulbactam showed the least synergistic effect with imipenem. Sulbactam reduced the MICs of imipenem by 4-fold at most.

Table 4.

Effect of 5 µg/ml sulbactam on the mics of β-lactam drugs

	MIC50			MIC90			The range of MIC decrease multiples	MIC range (µg/ml)	MIC Distribution width	SD of MIC
	-	+		-	+
Biapemem	32	8		64	16		1–32	1–64	7	20.36
Meropenem	32	8		128	32		1–6	2–64	6	36.59
Imipenem	128	64		512	512		1–4	32–512	5	206.00
Doripenem	16	4		64	16		1–32	≤ 0.5–64	≥ 8	12.38
Ertapenem	64	32		128	128		1–16	4-128	6	71.72
Tebipenem	8	0.5		16	2		1–32	≤ 0.5–16	≥ 6	2.07

Sensitivity to β-lactam drugs when applied in combination with avibactam

We then evaluated the synergistic effect of avibactam with the lactam drugs. Compared with clavulanic acid, avibactam had a superior synergistic effect with fourof the six β-lactam drugs (biapenem, meropenem, doripenem, and tebipenem). Avibactam reduced the MIC₅₀ values of the 4 drugs to below 8 µg/ml.The MIC₅₀and MIC₉₀values of the four drugs were lower in the presence of avibactam than they werein the presence of clavulanic acid. After the addition of avibactam, tebipenem showed the most significant change in MIC values against the MDR strains among the β-lactam drugs. Theseresultswere similar to those of clavulanic acid. The addition of avibactam reduced the MICs oftebipenem by16-fold and 32-fold against 36 and 20 MDR-TB strains, respectively(Table 5).The MIC₅₀ values of tebipenem were decreased from 8 µg/ml to 0.5 µg/ml against theMDR strains. These findings indicate a good synergistic effect between avibactam and tebipenem.

Table 5.

Effect of 5 µg/ml avibactam on the mics of β-lactam drugs

	MIC50			MIC90			The range of MIC decrease multiples	MIC range (µg/ml)	MIC Distribution width	SD of MIC
	-	+		-	+
Biapemem	32	4		64	8		1–32	0.5–64	8	11.05
Meropenem	32	8		128	32		1–16	4–64	5	25.29
Imipenem	128	64		512	256		1–4	8-256	6	147.33
Doripenem	16	4		64	16		1–32	≤ 0.5–32	≥ 7	11.55
Ertapenem	64	32		128	128		1–16	2-128	7	52.03
Tebipenem	8	0.5		16	1		1–32	≤ 0.5-8	≥ 5	1.51

Sensitivity to lactam drugs when applied in combination with relebactam

Finally, we examined the synergistic effect of relebactam with the six lactam drugs. With the addition of relebactam, both the MIC₅₀ and the MIC₉₀ of tebipenem were reduced by 16-fold, and the MICs of tebipenem were decreased by 32-fold and 16-fold, respectively, against 23 and 43 of the 105 strains. The above results indicate that among the six β-lactam drugs, tebipenem had the best synergistic effect with relebactam (which was similar to other inhibitors). In addition, relebactam had a good synergistic effect with doripenem and biapenem. Relebactam decreased the MICs of doripenem and biapenem by more than 8-fold against 61 (25.71%) and 54 (16.19%) strains, respectively. Similar to the other two inhibitors, relebactam showed the least synergistic effect with imipenem. Relebactam reduced the MICs of imipenem by 4-fold at most. The MIC₅₀ and MIC₉₀ of imipenem were only decreased by 1-fold in the presence of relebactam(Table 6).

Table 6.

Effect of 5 µg/ml relebactam on the mics of β-lactam drugs

	MIC50			MIC90			The range of MIC decrease multiples	MIC range (µg/ml)	Distribution width	SD of MIC
	-	+		-	+
Biapemem	32	4		64	8		1–32	0.5–32	7	5.86
Meropenem	32	8		128	32		1–16	2–64	6	16.65
Imipenem	128	64		512	256		1–4	8-256	6	135.82
Doripenem	16	4		64	16		1–32	≤ 0.5–16	≥ 6	10.30
Ertapenem	64	32		128	128		1–16	1-128	8	46.79
Tebipenem	8	0.5		16	1		1–32	≤ 0.5-2	≥ 3	1.11

Comparison of the synergistic effects of the β-lactamase inhibitors with the six lactamase drugs

We further compared the effect of the combined application of the β-lactamase inhibitors on the six lactamase drugs (Fig. 1). The synergistic effect is declared when the ratio of MIC without β-lactamase inhibitor to MIC with β-lactamase inhibitor is more than 4 times.Firstly, we compared the synergistic effects of the three traditional β-lactamase inhibitors (namely clavulanic acid, tazobactam, and sulbactam). The MICs of imipenem were decreased by more than 4-fold in 36 (34.28%) MDR strains after the addition of clavulanic acid, which was significantly higher than the decreases caused by the other two inhibitors. Moreover, clavulanic acid reduced the MICs of imipenem by more than 8-fold against 8 MDR strains, while the other two inhibitors only reduced the MICs by up to 4-fold. Therefore, clavulanic acid was considered to be the lactamase inhibitor that had the greatest effect on imipenem. Similar to imipenem, clavulanic acid had the best synergistic effect with the new β-lactam drug ertapenem. Clavulanic acid reduced the MICs of ertapenem by more than 4-fold against 55 (52.38%) strains, which was significantly more than that shown by tazobactam (30strains, 28.57%) and slightly morethan that shown by sulbactam (48 strains, 45.71%).

Fig. 1.

The synergistic effect of different β-lactamase inhibitors on the MIC values of β -lactams

X-axis: 5 types of β-lactam drugs

Y-axis: Number of bacterial strains exhibiting synergistic effects after adding 5 β-Lactamase Inhibitors

Regarding biapenem, combined application with clavulanic acid, tazobactam, and sulbactam reduced its MICs by more than 4-fold against 72 (68.57%), 65 (61.90%), and 81 (77.14%) of the 105 MDR strains, respectively. Further statistical analysis showed that the synergistic effect caused by the combined application of biapenem and sulbactam was significantly superior to that of the clavulanic acid and the tazobactam groups(χ² = 6.56, p = 0.03). Similar to biapenem, the meropenem-sulbactam combination caused a drastic MIC decrease (more than 4-fold) against more strains (62.85%, 66/105) than the other two groups of drug combinations(χ² = 35.16, p < 0.001). In addition, the meropenem–clavulanic acid produced synergistic effects at a higher ratio than the meropenem–tazobactam. Sulbactam also showed a good synergistic effect with the two newer β-lactam drugs. With the addition of sulbactam, the MICs of doripenem and tebipenem were reduced by more than 4-fold against 79 (75.23%, (χ² = 10.18, p = 0.06) )and 97 (92.83%, (χ² = 7.52, p = 0.02)) MDR strains, respectively. Thisreduction was significantly greaterthan that of the other two inhibitors.

We further compared the two newer β-lactamase inhibitors (avibactam and relebactam)with the common clinical drug clavulanic acid in terms of their effects on β-lactamase drugs. As shown in Fig. 1, the addition of clavulanic acid reduced the MICs of imipenem by more than 4-fold in 36 (34.28%) MDR strains, which was significantly greater than the effect of the addition of the two new β-lactam drugs (avibactam and relebactam, (χ² = 11.16, p = 0.004)). Moreover, clavulanic acid reduced the MICs of imipenem by more than 8-fold against 8 MDR strains, while the other two inhibitors decreased the MICs by 4-fold at most. Overall, among the six β-lactamase inhibitors, clavulanic acid was the most effective supplement to imipenem. Biapenem combined with clavulanic acid and the two novel inhibitors (avibactam and relebactam) reduced its MICs by more than 4-fold against 72 (68.57%), 82 (78.09%), and 85 (80.95%) of the 105 MDR strains, respectively. Further statistical analysis showed that the synergistic effect caused by the combined application of biapenem and the two new inhibitors was significantly superior to that caused by biapenem and the traditional drug clavulanic acid. In particular, biapenem showed the best synergistic effect with relebactam (χ² = 4.26, p = 0.04). The addition of relebactam reduced the MICs of meropenem by more than 4-fold against 76 (72.38%) MDR strains, making it significantly superior to other β-lactam inhibitors(χ² = 22.64, p < 0.001). In addition, the meropenem–avibactam showed a synergistic effect at a higher ratio than the meropenem–clavulanic acid. Like meropenem, relebactam showed a good synergistic effect with the three novel β-lactam drugs. The addition of relebactam reduced the MICs of doripenem, ertapenem, and tebipenem by more than 4-fold in 85 (80.95%, (χ² = 12.00, p = 0.001)), 65 (61.90%, (χ² = 0.37, p = 1.94)), and 100 (95.23%,(χ² = 12.83, p < 0.001)) MDR-TB strains, respectively, which were significantly greaterthan the decreases induced by the addition of avibactam or clavulanic acid. Compared with clavulanic acid, avibactam showed greater synergistic effects with the novel β-lactam drugs doripenem, ertapenem, and tebipenem. The two novel β-lactamase inhibitors, especially relebactam, showed significantly greater synergistic effects with five lactamase drugs (excluding imipenem) than clavulanic acid.

Study of the correlation between synergistic effect and BlaC gene mutation

To investigate gene mutations associated with the occurrence of the synergistic effects, we examined the blaC gene, which might be related to the synergism. In M. tuberculosis,the blaC gene encodes β-lactamase. As shown in Tables 7 and 14 strains (13.33%, 14/105) had mutations in the blaC gene. The mutations were mainly three types of substitution mutations: AGT333AGG, AAC638ACC, and ATC786ATT. The most frequent mutation was the synonymous mutation at the ATC786ATT site, which was observed in 6 strains. Moreover, the mutation occurring at this site was synonymous. The AAC638ACC mutation occurred in 5strains, which changed the Asn at position 213 to Thr. In addition, the AGT333AGG mutation occurred in 3 strains, in which the Ser at position 111 was mutated to Arg. Since the synonymous mutation of blaC occurred in these 105 MDR strains, we used the synonymous mutation ATC786ATT as the control to analyze the impact of gene mutations at two other sites of blaC on the synergism between β-lactams andβ-lactamase inhibitors. We found that compared with the synonymous control SNP, the meropenem–clavulanic acid and the meropenem–sulbactam combination showed greater synergistic effects in the presence of the Asn213Thr mutation. In addition, the synergistic effect was enhanced by 2–4 times in strains with the Ser111Arg substitution mutation compared to the stains with the synonymous mutation. No significant differences were found when examining the synergistic effect of other drug combinations.

Table 7.

Gene mutations and the correlation with synergy

gene	Nucleotide mutation	Amino acid mutation	No of strains	Effect of BlaC mutation on MIC value after the addition of meropenem
				clavulanic acid-Meropenem	sulbactam-Meropenem
blaC	AGT333AGG	Ser111Arg	3	4–8	8–16
	AAC638ACC	Asn213Thr	5	8–16	2–8
	ATC786ATT	Ile262Ile	6	1–2	1–4
dacB2	CTG659CAG	Leu220Gln	5
	449 GC deletion	Frameshift mutation	1
Idt mt1	GCC659GTC	Ala220Val	1
	GCC206GAC	Ala69Asp	1

Since the d, d-carboxypeptidase DacB2 and the transpeptidase Ldt_mtl(which catalyzes 3→3 cross-links) are also the targets of carbapenems, we examined the mutation status of these two enzymes. Sequencing ofdacB2 revealed a mutation at position 659 in five strains, which resulted in a substitution mutation of Leu to Gln. In addition, 1 strain had a GC deletion mutation at position 449. As for Ldt_mtl, two strains habored mutations, which were the substitution mutations AGC101AAC and GTC848GCC.

Discussion

β-lactams have been the most common prescription antibiotics, they include penicillin derivatives, sporins, monosporins, and carbapenems [33]. The high specificity and low toxicity of this class of drugs against bacteria make them essential for the treatment of gram-positive and gram-negative bacteria [34]. However, these compounds are rarely used in the treatment of M. tuberculosis, because the chromosome of M. tuberculosis contains the gene encoding BlaC, and the gene product confers resistance to most β-lactam drugs [35]. As a class A β-lactamase, BlaC catalyzes the opening of the β-lactam ring via the nucleophilic attack by an active site serine residue (Ser70) to produce acyl-enzyme, which is followed by the hydrolysis of the ester bond to generate an inactive ring-opened product [36]. Carbapenem drugs can be used specifically against the bacteria that have β-lactamase and are insusceptible to penicillins and cephalosporins [24]. The carbapenem drugs used clinically to treat bacterial infections include imipenem, meropenem, ertapenem, doripenem, and biapenem [37]. These drugs contain the 6-α-1R-hydroxyethyl substituent on the β-lactam ring, which sterically prevents binding to other class A β-lactamases [38]. Carbapenems have a narrower MIC spectrum and exhibit more potent activity against the clinically isolated M. tuberculosisthan aminopenicillins [25]. The differences in their antibacterial activity are related to the mechanisms of action of l, d-transpeptidases (LDTs) and Ambler class A β-lactamases. LDTs participate in the biosynthesis of 3→3 PGcross-links. Therefore, LDTs can be effectively inactivated by carbapenems but are less sensitive to aminopenicillins [39, 40]. In addition, BlaC produced by M. tuberculosis induces the hydrolysis of lactam ring, whereas carbapenems are relatively resistant to such hydrolysis [41]. Therefore, carbapenems may be the ideal compounds against MDR and XDR M. tuberculosis. Tebipenem ester is the first oral lactam drug. Compared with other β-lactams, tebipenem has a better body absorption rate.In addition, as an oral drug, tebipenem can be better toleratedthan injectable drugs.

The combinations of β-lactam andβ-lactamase inhibitor have attracted growingattention from scholars due to their efficacy against MDR M. tuberculosisin vivo and in vitro [20, 42, 43].β-lactamase inhibitors areoften used at 2.5–10 µg/ml in vitro to evaluate their enhancing effect on the anti-TB activity ofβ-lactam [44–46]. In clinical application, 125 mg or 250 mg of clavulanic acid is mainly used three times a day in combination with amoxicillin to achieve peak serum drug levels of 2.55 µg/ml and 5.9 µg/ml, respectively [47, 48]. Research indicates that the Ser111Arg substitution mutation may increase the synergistic effect of clavulanic acid with meropenem or amoxicillin [32]. Clavulanic acid shows better synergy with meropenem compared to vaborbactam. Additionally, clavulanic acid can reduce the MIC of tebipenem to 2 µg/ml [25]. In Mycobacterium tuberculosis, the combination of tebipenem and clavulanic acid results in the most significant reduction in MIC. For Mycobacterium abscessus, the combination of avibactam and relebactam exhibits the best antimicrobial activity [49].In view of these results, we selected four concentrations (1.25, 2.5, 5, and 10 µg/ml) to evaluate the MICs of β-lactams against seven clinical MDR strains when they were administered alone or in combination with β-lactamase inhibitors. As shown in Table 1, significant differenceswere observed when comparing the MICs of β-lactams administered alone to the MICs of β-lactams administered in combination with a β-lactamase inhibitor. In addition, as the concentration of β-lactamase inhibitors increased, six β-lactam drugs (biapenem, meropenem, imipenem, doripenem, avibactam, and relebactam) had significantly decreased MICs against the MDR strains. However, β-lactamase inhibitors little improved the MICs of these six drugs when theirconcentration increased from 5 µg/ml to 10 µg/ml. Therefore, the concentration of β-lactamase inhibitors was set to 5 µg/ml in the drug susceptibility tests.

In vitro experiments, various carbapenem drugs showed different bactericidal activities against MDR M. tuberculosis. Overall, the evaluation results of MIC range, MIC₅₀, and MIC₉₀ showed that among the sixβ-lactam drugs testedin this study, tebipenem had the strongestantibacterial activity against MDR-TB, followed by doripenem and biapenem. Moreover, doripenem had higher antibacterial activity than biapenem. The MIC₅₀ values of doripenem and biapenem were 16 and 32 µg/ml, respectively. The next were meropenem and ertapenem, and meropenem had an antibacterial activity superior to biapenem. Imipenem had the lowest antibacterial activity. The difference in their bactericidal activity against MDR strains might be related to the mechanisms of action of the class A Ambler β-lactamase BlaC and the LDTs.BlaC produced by M. tuberculosis causes the hydrolysis of the lactam ring, while carbapenems are relatively resistant to hydrolysis [24]. A recent biochemical study showed that tebipenem has a k_cat value of only 0.04 min^− 1 due to the low rate of tebipenem hydrolysis by BlaC. Other carbapenem drugs, such as meropenem [20], doripenem, and ertapenem [50], easily form covalent acyl intermediates with BlaC due to their very slow deacylation process. As a result, M. tuberculosis shows a stronger reactivity to tebipenem than to other carbapenems [51]. In addition, their antibacterial activity is related to l, d-transpeptidases (LDTs). LDTs are involved in the biosynthesis of peptidoglycan (PG) cross-links and can be effectively inactivated by carbapenems. In contrast, LDTs are less sensitive to aminopenicillins [40]. Carbapenems block PG synthesis by inhibiting the cross-linking reaction of diaminopimelic acid [44]. Tebipenem inhibits the formation of PGs by binding to penicillin-binding proteins on the cell wall, thus affecting the synthesis of thecell wall and resulting in bacterial lysis [52].Therefore, theaddition of carbapenems to the culture medium has a good inhibitory effect on the synthesis of thecell wall, which enhances the permeability of the bacterial envelope. Slow penetration is believed to be the major determinant ofM. tuberculosis resistance to β-lactam drugs.

The combined administration of β-lactams andβ-lactamase inhibitors provides a drug regimen for overcoming lactamase-mediated drug resistance. Early studies have shown that the antibacterial activity of β-lactamase inhibitors exhibits species-specific variations in microorganisms, which are caused by the differences in the types of β-lactamases and their three-dimensional structures in vivo [53, 54]. In this study, we found that compared with other lactamase inhibitors, relebactam and avibactam (especially relebactam) had better synergy with β-lactams against MDR M. tuberculosis. The first-generation β-lactamase inhibitors clavulanic acid and tazobactam are β-lactam derivatives. They exert their effects mainly by inactivating class A and some class C serine β-lactamases.Avibactam is a novel covalent, reversible β-lactamase inhibitor with a non-β-lactam structure. It effectively inhibits the activity of KPC-type carbapenemase [55]. Because the complex formed by avibactam and BlaC has a very low dissociation constant, it effectively inhibits the BlaC produced by M. tuberculosis [56]. Previous studies compared the MIC of avibactam with those of several commercial β-lactamase inhibitors [56, 57]. Consistent with our results, the studies found that avibactam had stronger inhibitory activity against class A β-lactamases than clavulanate and tazobactam.Avibactam and ceftazidime have been clinically used to treat infections with MDR gram-negative bacteria. In an in vitro hollow fiber system model, avibactam and ceftazidime effectively killed the rapidly growing semi-dormant M. tuberculosis in cells [51]. Relebactam is a novel β-lactamase inhibitor structurally related to avibactam.A combination drug consisting of relebactam and imipenem was approved for marketing by the Food and Drug Administration in 2019. It might become a candidate lactam drug for the treatment of MDR tuberculosis infection.A study of Mycobacterium abscessus showed that relebactam reduced the MIC₅₀ and the MIC range of meropenem and imipenem by 2-4-fold [27]. This finding was similar to our results. In addition, that study used the broth dilution method to determine the MIC values, which took 7 days to interpret. The half-life of clavulanic acid is 1.8 days [58], which is significantly lower than the half-life (6 days) of avibactam [57]. Therefore, the relative instability of clavulanic acid leads to the loss of active drug content during determining drug MIC values, thereby limiting the antibacterial synergy between clavulanic acid and β-lactam drugs against M. tuberculosis. On the other hand, in vivo experiments have shown that the effective permeability of β-lactamase inhibitors across cell membrane plays an important role in enhancing the antibacterial activity of β-lactams [59].Since clavulanic acid is more hydrophilic than avibactam and relebactam, we speculated that clavulanate might havemore difficulty penetrating the thicker lipid layer on the outer surface of M. tuberculosis, leading to the decline in its antibacterial ability. In future studies, in vivo experiments need to be conducted to examine the effect of in vivopermeability of various β-lactamase inhibitors on their anti-TB synergy with β-lactams.

Sequencing analysis of three β-lactamase resistance-related genes, blaC, ldt_Mt1and dacB2, was performed. 16, 6, and 2 of the 105 strains carried base mutations in the blaC, ldt_Mt1, and dacB2 genes, respectively. Interestingly, mutations ofthe blaC gene included two missense mutations and a synonymous mutation at Ile262 (6 strains). Settingthe six strains with blaC synonymous mutations as controls, we analyzed the correlation between the missense mutations of the blaC gene and the synergistic effect of β-lactam-lactamase inhibitor. The results showed that asparagine (Arg) at position 213 was mutated to threonine (Thr) in the blaC gene, which enhanced of the synergistic effect of clavulanic acid with meropenem and sulbactam against MDR-TB strains.Mutation of Ser to Arg at position 111 enhanced the synergistic effect of clavulanic acid with meropenem against MDR-TB strains.

The binding affinity between ligands and their receptors depends on their structural complementarity and intermolecular interactions. Structural analysis of wild-type BlaC protein revealed that both Asn 213 and Ser 111 were located near the binding motif of BlaC to β-lactams–lactamase inhibitors. In M. tuberculosis BlaC protein, this motif contains the residues R220, A244, S130, and T237.Mutation of the blaC gene has increased the resistance of M. tuberculosis to sulbactam [60]. In our study, the Arg-to-Thr mutation at position 213 of the blaC gene enhanced the synergistic effect of clavulanic acid with sulbactam against MDR-TB strains. However, there was no direct in vitro biochemical evidence that Ser 111 and Asn 213 participated in substrate binding as part of the carboxylic acid binding region. Further molecular structural analysis is needed to understand the binding sites of M. tuberculosis BlaC protein.

In summary, this study showed that tebipenem and relebactam had the highest antibacterial activity against MDR M. tuberculosis.Tazobactam, currently utilized as a β-lactamase inhibitor in clinical settings, is an injectable medication, which imposed an additional burden on treatment adherence for tuberculosis patients. In contrast, relebactam not only enhanced antimicrobial activity against MDR-TB in our study but also offers the advantage of oral administration. This mode of delivery facilitates outpatient management and significantly improved patient adherence to the therapeutic regimen.In vitro experiments showed that MDR-TB strains remained drug-resistant when imipenem was used alone or in combination with β-lactamase inhibitors. In addition, Ser at position 111 was mutated to Arg in BlaC protein, which enhanced the synergistic effect between clavulanic acid and meropenem against MDR-TB strains. The Arg-to-Thr mutation at position 213 of BlaC protein increased the resistance of the MDR-TB strains to meropenem in the presence of sulbactam. Future studiesurgently need to evaluate the potential application of tebipenem–tazobactam against MDR-TB in clinical practice.

It is necessary to acknowledge the limitations of this work. First, compared to other anti-tuberculosis drugs, the efficacy of β-lactam antibiotics in tuberculosis treatment may be relatively limited, which might necessitate their use in combination with other anti-tuberculosis medications. β-lactam antibiotics can potentially cause side effects such as allergic reactions and gastrointestinal discomfort, which may affect patient compliance.Second, this study primarily focused on observing the synergistic effects of several common β-lactamase inhibitors on beta-lactam antibiotics among MDR-TB strains in vitro. However, the study did not include in vivo experiments and clinical trials, potentially leading to the oversight of variables and interactions that may occur within a biological organism when evaluating drug synergy. Animal experiments and clinical trials can more comprehensively reveal the actual effects and potential clinical applications of drug interactions.Therefore, future researchers need to conduct additional in vivo and clinical experiments to validate our understanding.

Conclusions

In summary, varied activities of biapemem, meropenem, imipenem, doripenem, ertapenemand tebipenem alone or in combination with β-lactamase inhibitors against MTB were observed. Avibactam and relebactam have better synergistic effects on most of the lactamase drugs.In particular, tebipenem incombination with relebactam showed the most remarkable activityand has a good prospect in developing novel anti-TB regimens. Further studies are warranted to investigate the efficacyof tebipenem/relebactam in the clinical trials.

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Abbreviations

MTB

Mycobacterium tuberculosis

SNP

Synonymous single nucleotide polymorphism

MDR

Multidrug-resistant

WHO

World Health Organization

MDR-TB

MDR tuberculosis

XDR-TB

Extensively drug-resistant tuberculosis

PGs

Peptidoglycans

LJ

Lowenstein-Jensen

PNB

p-nitrobenzoic acid

MICs

Minimum inhibitory concentrations

LDTs

l, d-transpeptidases

PG

Peptidoglycan

Author contributions

Conceptualization, Jie Shi; methodology, Jie Shi; software, Danwei Zheng; investigation, Xiaoguang Ma andYankun Zhu; writing—original draft preparation, Jie Shi; writing—review and editing, Jie Shi and Shaohua Wang; visualization, Jie Shi and Wenjing Chang; supervision, Dingyong Sun; project administration, Jie Shi*, Danwei Zheng, Ruyue Su, Xiaoguang Ma, Yankun Zhu, Shaohua Wang and Wenjing Chang. All authors have read and agreed to the published version of the manuscript.” Authorship must be limited to those who have contributed substantially to the work reported.

Funding

Please add: This research was funded by the Natural Science Foundation of Henan Province, grant number “202300420290”.

Data availability

The original contributions presented in the study are included in the article, further inquiries can be directed to the corresponding authors.

Declarations

Ethics approval and consent to participate

The study was conducted in accordance with the Declaration of Helsinki, and approved by the Medical Ethics Committee of the Henan Provincial Center for Disease Control and Prevention(Number: 2023-KY-002-02).Informed consent was obtained from all subjects involved in the study.

Consent for publication

Not applicable.

Clinical trial

Not applicable.

Competing interests

The authors declare no competing interests.