Hindawi BioMed Research International Volume 2018, Article ID 3579832, 8 pages https://doi.org/10.1155/2018/3579832

Research Article In Vitro Activity of �-Lactams in Combination with �-Lactamase Inhibitors against Mycobacterium tuberculosis Clinical Isolates

Fu Li ,1 Li Wan,2 Tongyang Xiao ,1 Haican Liu ,1 Yi Jiang,1 Xiuqin Zhao ,1 Ruibai Wang ,1 and Kanglin Wan 1

1 State Key Laboratory for Infectious Diseases Prevention and Control, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 102206, China 2Department of Physiology, Xiangya School of Medicine, Central South University, Changsha, Hunan 410078, China

Correspondence should be addressed to Ruibai Wang; [email protected] and Kanglin Wan; [email protected]

Received 19 February 2018; Accepted 4 June 2018; Published 2 July 2018

Academic Editor: Isabel Portugal

Copyright © 2018 Fu Li et al. Tis is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Objectives. Evaluating the activity of nineteen �-lactams in combination with diferent �-lactamase inhibitors to determine the most potent combination against Mycobacterium tuberculosis (MTB) in vitro. Methods. Drug activity was examined by drug susceptibility test with 122 clinical isolates from China. Mutations of blaC and drug targets ldtMt1, ldtMt2, dacB2, and crfA were analyzed by nucleotide sequencing. Results. (TBM) in combination with clavulanate (CLA) exhibited the highest anti-TB activity. Te MIC of �-lactam was reduced most evidently in the presence of CLA, compared to (AVI) and (SUB). Eight polymorphism sites were identifed in blaC, which were not associated with �-lactams resistance. Interestingly, one strain carrying G514A mutation in blaC was highly susceptible to �-lactams regardless of the presence of inhibitors. Te transpeptidase encoding genes, ldtMt1, ldtMt2,anddacB2, harboured three mutations, two mutations, and one mutation, respectively, but no correlation was found between these mutations and drug resistance. Conclusion.Teactivityof�-lactams against MTB and diferent synergetic efect of �-lactamase inhibitors were indicated. TBM/CLA exhibited the most activity and has a great prospect in developing novel anti-TB regimen; however, further clinical research is warranted. Moreover, the resistance to the �-lactam antibiotics might not be conferred by single target mutation in MTB and requires further studies.

1. Introduction pretomanid, which are in phase II or III trials [5, 6], long duration and high expenses lead to slow development of Tuberculosis (TB) is a great threat to the global public health new drugs. Repurposing the currently used antibiotics may that caused an estimated 1.3 million deaths in 2016 [1]. Drug contribute to the development of novel anti-TB regimens. resistance has been a major obstacle in the progress of elim- �-lactams disrupt the biosynthesis of bacteria inating TB, especially due to the emergence of multidrug- by preventing the formation of peptide cross-links between resistant (MDR) and extensively drug-resistant (XDR) MTB theadjacentpolysaccharidechainsinthepeptidoglycanlayer worldwide [2–4]. With the rising incidence of drug-resistant [7]. In Mycobacterium tuberculosis (MTB), two kinds of TB, the traditional frst-line and second-line anti-TB drugs transpeptidase, the L,D-transpeptidase (mainly encoded by fail to fulfll the current needs of TB treatments. In such ldtMt1 and ldtMt2) and the D,D-transpeptidase (encoded by cases, developing novel drugs or drug regimens is urgent and dacB2), catalyze the cell wall biosynthesis [8, 9]. Te L,D- necessary for efective control of TB. Although signifcant transpeptidase, which is responsible for the formation of over progress has been made in identifcation of several new 80% transpeptide linkages (3�→ 3 linkages) in MTB, is the anti-TB compounds, such as bedaquiline, delamanid, and target of [10–12]. Te D,D-transpeptidase that 2 BioMed Research International mainly catalyzes the formation of 4�→ 3linkagesisakindof Table 1: Te �-lactams and �-lactamase inhibitors for drug suscep- -binding proteins (PBPs), which can be inhibited by tibility tests. (MEM) [13]. Classifcation Agent Generation Te resistance to �-lactam antibiotics encompasses the activity of �-lactamases. Tousands of diferent �- Tebipenem lactamases have been identifed in various environmental Meropenem and pathogenic species of bacteria [14]. MTB is intrinsically resistant to �-lactams due to the expression of endogenous �-lactamase BlaC active against a broad spectrum of �- lactams. CLA, AVI, and SUB are �-lactamase inhibitors Penicillin � that inhibit the hydrolysis of -lactams. Recent studies � � have reported that a combination of the -lactams and - lactamase inhibitors is efective against MTB in vitro and in vivo [15–17]. Te efcacy, safety, and tolerability of car- bapenems,suchasMEM,,andertapenem(ETP), against MTB have been evaluated in several clinical studies [18]. Amoxicillin/clavulanate (AMX/CLA) combination has beenevaluatedtobeefcientagainstMTBin vitro and is thuscategorizedintoGroup5anti-TBdrugsbyWHO[19, Cephalexin First generation 20]. Tis suggests that combinations of �-lactams and �- Second generation lactamase inhibitors are prospective candidates in developing Cefxime Tird generation more efective treatment against TB. Fourth generation In this study, to determine the most active and applicable � � combination of -lactam and -lactamase inhibitors, the in Oxacephem Moxalactam vitro anti-TB activity of nineteen �-lactams belonging to dif- �-lactamase inhibitor Clavulanate ferent subclasses either alone or in combination with diferent �-lactamase inhibitors was tested using drug susceptibility Avibactam tests. A total of 122 MTB clinical isolates collected from China Sulbactam were used. Mutations in blaC and three main �-lactams targets, ldtMt1, ldtMt2,anddacB2, were analyzed to determine the relationship between drug target mutations and �-lactam was fully ground and adjusted with saline to a cell density × 8 resistance. In addition, T184A mutation in an unannotated of 3 10 cells/mL (1 McFarland standard). Te suspension gene, crfA, reported to confer carbapenem resistance in MTB was diluted 1 : 20 with 7H9 broth (Difco, Detroit, MI, USA) was also detected. containing 10% OADC (BD, Franklin Lakes, NJ, USA). Six groups of serial twofold drug dilutions were prepared 2. Materials and Methods in the 7H9 broth containing either fve diferent constant concentrations of each �-lactamase inhibitor or no inhibitor � 2.1. Strains. A total of 122 identifed clinical MTB isolates, at all. Te fnal reaction mix contained 100 lofdrugsolution and equal volume of bacterial suspension in each well. Te including 47 susceptible (S), 64 MDR, and 11 XDR isolates, ∘ used in this study were collected from seven provincial TB plates were sealed and incubated at 37 Cfor7days.Tenthe � � hospitals of China in Beijing, Anhui, Fujian, Guizhou, Henan, indicator (20 lAlamarBluemixedwith50 l5%Tween- Xinjiang, and Sichuan. All the strains were preserved in the 80) was added to the drug-containing group when the drug- strain bank of National Institute for Communicable Disease free control showed color change (blue to pink). Te lowest Control and Prevention, Chinese Center for Disease Control drug concentration that inhibited the strain growth and and Prevention. prevented color change was recorded as the MIC value. Drug susceptibility tests for each strain were repeated twice. 2.2. �-Lactams and �-Lactamase Inhibitors. MEM was pur- Te fnal concentrations of tebipenem ranged from 0.125 � chased from Sigma-Aldrich Co. (St Louis, MO, USA). Tebi- to 16 g/ml, and for the other carbapenems, 0.25 to 32 � , doripenem, biapenem, ertapenem, amoxicillin, g/ml concentrations were tested in drug susceptibility tests. ampicillin, methicillin, oxacillin, cloxacillin, nafcillin, diclox- Ampicillin (AMP) and cefuroxime (CXM) were used at � acillin, piperacillin, cephalexin, cefuroxime, cefxime, cef- concentrations ranging from 0.5 to 64 g/ml and the other pirome, aztreonam, and moxalactam and three �-lactamase chemicals were tested at concentrations ranging from 1 to 128 � � inhibitors, CLA, AVI, and SUB, used in this study were g/ml. Te fnal concentration of each -lactamase inhibitor � � � � � purchased from MCE Co. (Monmouth Junction, NJ, USA) was 0.625 g/ml, 1.25 g/ml, 2.5 g/ml, 5 g/ml, and 10 g/ml, (Table 1). respectively.

2.3. Drug Susceptibility Test. Drug susceptibility test was 2.4. Polymerase Chain Reaction (PCR) and Sequencing. Ge- performedbybrothmicrodilutionmethodusing96-well nomic DNA was isolated from mycobacterial cultures by microplate. Briefy, a loop of MTB culture in the late log phase boiling method and was used for amplifcation of blaC, BioMed Research International 3

Table 2: Primers and conditions used for amplifcation and sequencing.

� � PCR conditions Primers Sequence (5 -3 ) a ∘ b Product length (bp) Denaturation (s) Annealing ( C, s) Elongation (s) BlaC-F ATGCGCAACAGAGGATTCGGTC 30 61, 30 65 924 BlaC-R CTATGCAAGCACACCGGCAACG

LdtMt1-F ATGCGTCGAGTGGTTCGTTATC 30 58, 30 55 756 LdtMt1-R CTAGCCGACCACCTCAATGG

LdtMt2-F ATGCCAAAGGTGGGGATTGC 30 59, 30 90 1227 LdtMt2-R TTACGCCTTGGCGTTACCGGC DacB2-F ACCAGCAACTGCTGGATTTC 30 60, 30 85 1196 DacB2-R CGTTGATGACCAACGTCTTC CrfA-F ACCCGGCTCACAGAGAATCG 30 60, 30 40 457 CrfA-R TATCACCGGTAGGCCATGC a ∘ b ∘ Note: : denaturation temperature was 94 C; : elongation temperature was 72 C. All PCRs were performed for 31 cycles and each reaction was initialized by ∘ ∘ denaturation at 94 Cfor10minandterminatedwithafnalelongationstepat72Cfor10min.

� � ldtMt1, ldtMt2, dacB2,andcrfA by polymerase chain reaction by AMP/CLA (4 g/ml and 16 g/ml). CXM alone or in (PCR). Te primer sets and conditions for amplifcation combination with any of the inhibitors showed no anti-TB areshowninTable2.TePCRproductsweresequenced activity even when the highest drug concentration was tested. (TSINGKE Biological Technology, Beijing, China) and the Comparison of the synergistic efect of the three diferent gene polymorphisms were identifed by aligning with the �-lactamase inhibitors shows that CLA exhibited the most reference strain H37Rv (GenBank accession no. NC 000962) potent activity in inhibiting the �-lactamase. Overall, the using MEGA 7.0 sofware. MICs of �-lactams were reduced most prominently (4-32 folds) in the presence of CLA, followed by AVI (4-8 folds). 3. Results However, the least activity was observed in case of SUB as MIC50 and MIC90 of all the tested drug combinations with 3.1. Determination of Inhibitors’ Concentrations. Te efective SUB showed lesser reduction (2-8 folds) than that in the concentration of each �-lactamase inhibitor was determined presence of CLA and AVI, especially when used in combi- by drug susceptibility tests against six MTB clinical isolates nation with TBM, BIA, and AMP. with nineteen �-lactams either alone or in combination with fve diferent concentrations of �-lactamase inhibitors. Te 3.3. Relationship between Drug Target Mutation and Resis- results showed that the MICs did not change when 5 �g/ml tance. Since the �-lactamase BlaC, L,D-transpeptidase, and concentration of inhibitors was used (Table 3). Tus, we D,D-transpeptidase are potential targets of �-lactams, the decided to use inhibitors at a fxed fnal concentration of encoding genes, including blaC, ldtMt1, ldtMt2,anddacB2, 5 �g/ml in the subsequent experiments. All the carbapen- were analyzed to illustrate the role of drug target mutations ems, AMX, ampicillin (AMP), and cefuroxime (CXM) were in the emergence of the �-lactams resistance in MTB. In selected to perform further tests, because the other �-lactams addition, mutation at position T184A in an unannotated showed no activity against MTB in the initial screening. protein encoding gene, crfA,whichwasearlierreportedto confer carbapenem resistance in MTB [21], was also analyzed. 3.2. MIC Profles of �-Lactams against Clinical Isolates. Te Mutations in the fve genes are shown in Table 5 and the MIC MIC profles of the clinical isolates are shown in Table 4. In profles of the mutant strains are listed in the supplementary thecaseofcarbapenems,TBMandbiapenem(BIA)showed materials. As observed, 16.4% (20/122) of clinical isolates better activity with MIC90 of 16 �g/ml when they were used carried 8 mutation sites in blaC.Tesilentmutation,C786T alone. Te MICs of carbapenems were greatly reduced (4- (11 isolates), was the most predominant mutation. Tree 8 folds), except for ETP (1-4 folds), in the presence of �- nonsynonymous mutations, T333G, T492A, and G514A, were lactamase inhibitors. TBM in combination with CLA showed observed in blaC, while none of these mutations was related the most inhibitory activity with MIC50 and MIC90 of 1 �g/ml to �-lactam resistance. Interestingly, one strain, GZ10116, and 2 �g/ml, respectively, followed by BIA (2 �g/ml and 4 which carried G514A mutation, was highly susceptible to �- �g/ml) and doripenem (DOR) (2 �g/ml and 8 �g/ml). MEM lactams irrespective of the presence of inhibitors. in combination with CLA also exhibited good activity with Tere were 1, 1, and 2 strains harbouring A111G, G448A, � � MIC50 and MIC90 of 2 g/ml and 8 g/ml, respectively, but and C659T mutations, respectively, on ldtMt1. 1 strain and 4 it was lower than that of TBM and BIA. ETP in combination strains, respectively, harboured C594T and A776G mutation with any of the inhibitors showed the least activity with MIC90 in ldtMt2. Only one strain, which harboured A776G mutation > � � of 16 g/ml. In the case of and , in ldtMt2, was resistant to all the combinations of -lactams AMX/CLA showed highest activity against MTB with MIC50 and inhibitors. However, we did not fnd any mutation � � � ��� and MIC90 of 2 g/ml and 8 g/ml, respectively, followed associated with the -lactam resistance in ��1 and ldtMt2. 4 BioMed Research International > 128 > 128 > 128 > 128 > 128 > 128 > 128 > 128 > 128 > 128 > 128 > 128 > 128 > 128 > 128 > 128 > 128 > 128 > 128 > 128 > 128 > 128 > 128 > 128 > 128 > 128 > 128 > 128 > 128 > 128 > 128 > 128 > 128 > 128 > 128 > 128 > 128 > 128 > 128 > 128 > 128 > 128 > 128 > 128 > 128 > 128 > 128 > 128 128 32- > 128 64-> 128 > 128 32- > > 128 32- > 128 32- > 128 32- > 128 64- > 128 > 128> 128 16- > 128 32- > 128 64- > 128 > 128 32- > 128 64-> 128 > 128> 128 16- > 128 32- > 128 32- > 128 32- > 128 32- > 128 32- > 64 > 64 > 64 > 64 > 64 acillin; CLO, cloxacillin; NAF, nafcillin; DIC, > 128 32-64 > 128 16-32 > 128 16-64 > 128 32- > 128 8-32 > 128 8-32 > 128 8-32 > 128 16-32 > 128 16-32 > 128 32-64 2- > 128> 64 32- 2- > 128 8-32 3 3 > 128 64- > 128 > 128 > 128 32- > 128 32- > 128 32- > 128 32- > 128 32- > 128 64- > 128 32- > 128 > 128 32- > 128 64- > 128 > 128 32- > 128 64- > 128 > 128 32- > 128 32- > 128 32- > 128 > 128 > 128 > 128 > 128 > 128 > 128 > 128 > 128 > 128 64- > 128> 128 16- 4-16 > 128 > 128 > 128 > 128 > 128 64- > 128> 128 16- 4-16 > 128 > 128 > 128 > 128 > 128 > 128 > 128 > 128 > 128 > 128 > 128 > 128 > 128 > 128 > 128 > 128 > 128 > 128 > 128 64- > 128 > 128 > 128 64- > 128 > 128 64- > 128 > 128 64- > 128 > 128 > 128 > 128 > 128 > 128 > 128 MIC range ( � g/ml) > 128 128- > 128 > 128 128- > 128 128- > 128 > 128 > 128 > 128 128- > 128 64- > 128 64- > 128 > 128 64- > 128 64- > 128 > 128 128- > 128 64- > 128 64- > 128 128 64- > 128 128- > 128 128- > 128 128- > 128 64-> 128 128- > > 128 32- > 128 32- > 128 32- > 128 32- > 128 128- > 128 128- > 128 128- rtapenem; AMX, amoxicillin; AMP, ampicillin; MET, methicillin; OXA, ox > 64 > 64 > 64 8-64 32- < 1-32 16- < 1-32 16- � -lactams alone or in combination with inhibitors against six MTB isolates. > 128 > 128 32-64 128- > 128 128- > 128 > 128 > 128 > 32 64- me;CFM,cefxime;CPO,cefpirome;ATM,aztreonam;MOX,moxalactam. Table 3: MIC profles of -4 2-4 1-2 8-16 4-8 2 < 1 2-4 4-8 2-4 8-32 4-32 4-64> 128 32- 64- > 128 128- < 1 4 4-8 4-8 16-32 8-32 16-64 64- > 128 128- < 1 < 1 2-4 2-4 1-2 8-16 4-8 < 1-2 4-8 8-16 8-16 16-32 16-32 32-64 128- < 1-2 4-16 2-4 2-4 8-16 8-32 4-32> 128 32- 64-> 128 128- < 1-2 4-16 2-4 2-4 8-16 8-32 8-32> 128 32- 64- > 128 128- < 1-4 8-16 4-8 4-8 8-32 32-64 8-64 64- > 128 64- > 128 128- TBM MEM DOR BIA ETP AMX AMP MET OXA CLO NAF DIC PIP LEX CXM CFM CPO ATM MOX 5 5 2-8 8-32 4-8 2-8 8-32 16- > 64 4-64 16- 5 10 2-8 8-32 4-8 2-8 8-32> 64 16- 4-64 16- 10 10 2.5 4-8 16-32 4-16 4-8 16-32 32- 2.5 2.5 1.25 4-16 16-32 8-16 4-16 16-32 64- > 128 16-64 64- 1.25 1.25 2-4 8-16 4-16 8-16 16-32 32-128 16-64 128-> 128 128- 0.625 4-8 8-16 8-16 8-16> 32 16- 64-128 32-64 128- 0.625 0.625 8-16 16-32 8-16 8-16> 32 32- 64- Avibactam 0 8-16 16-32 8-16 8-16> 32 32- 64- Inhibitors Concentration Clavulanate 0 8-16 16-32 8-16 8-16 32- Sulbactam 0 8-16 16-32 8-16 8-16> 32 32- 64- dicloxacillin; PIP, piperacillin; LEX, cephalexin; CXM, cefuroxi Note: TBM, tebipenem; MEM, meropenem; DOR, doripenem; BIA, biapenem; ETP, e BioMed Research International 5

� Table 4: MICs of -lactams alone or in combination with a fxed Table 5: Mutations on blaC, ldtMt1, ldtMt2,anddacB2 genes. concentration of 5 �g/ml of the inhibitor against 122 MTB isolates. Gene Substitution Amino acid change Number of mutants MIC range MIC50 MIC90 blaC G183A Leu61Leu 1 Inhibitor � � � ( g/ml) ( g/ml) ( g/ml) T333G Ser111Arg 3 > TBM None 0.5- 16 8 16 C354T Ser118Ser 1 > CLA 0.125- 16 1 2 G486C Ala162Ala 1 AVI 0.125->16 1 2 T492A Phe164Leu 1 SUB 0.5->16 2 4 G514A Gly172Ser 1 BIA None 2->32 8 16 C610T Leu204Leu 1 CLA 0.25->32 2 4 C786T Ile262Ile 11

AVI 0.5->32 2 4 ldtMt1 A111G Pro37Pro 1 SUB 0.5->32 4 8 G448A Ala150Tr 1 DOR None 2->32 16 16 C659T Ala220Val 2 CLA 0.5->32 2 8 ldtMt2 C594T Gly198Gly 1 A776G Asp259Gly 4 AVI 0.5->32 4 8 dacB2 T659A Leu220Gln 4 SUB 0.5->32 4 16 MEM None 4->32 >32 >32 CLA 0.25->32 2 8 bacterial infections [22]. Recently, the combination of �- AVI 1->32 4 16 lactamase inhibitor and �-lactamwasshowntobeefective SUB 4->32 8 16 in restoring the potency of �-lactam against MTB [15– ETP None 8->32 >32 >32 17]. In the present study, nineteen commercially available �-lactams and three �-lactamase inhibitors were screened CLA 2->32 8 16 > in vitro to identify the most potent combination against AVI 4- 32 8 16 MTB.Teresultsshowthatcarbapenems,exceptforETP, SUB 4->32 16 32 exhibited excellent activity against MTB clinical strains. Tis AMX None 8->64 >64 >64 may be due to the inactivation of multiple enzymes by CLA <0.5-32 2 8 carbapenems, whereas the other �-lactams did not inhibit the AVI <0.5-64 8 16 L,D-transpeptidase in MTB [10]. Te activity of AMX was also improved notably in the presence of clavulanate. While SUB 2->64 8 32 > > the efcacy of AMX/CLA against clinical TB remains con- AMP None 32- 64 64 64 troversial, carbapenems may serve as an optimal alternative CLA 0.5->64 4 16 in developing novel anti-TB regimens. Te results showed AVI 0.5->64 8 16 that all the tested cephalosporins in our study were inactive SUB 0.5->64 16 32 against MTB. Dincer et al. [23] reported that in CXM None 16->64 >64 >64 combination with CLA was active against H37Ra. Tis may be due to the diferent strain tested and diferent methodology CLA 1->64 32 >64 > > used. Additionally, the concentration of CLA, that is, 32 AVI 2- 64 32 64 �g/ml, was much higher than the concentration (5 �g/ml) SUB 4->64 64 >64 used in our test. Tis suggests that a more universal drug Note: MIC50/90,MICsthatinhibit50% and 90% of the isolates, respectively. susceptibility testing protocol for determining the MIC of �- lactams against MTB is needed in the future. Carbapenems displayed a variable activity in this study. TBM in combination with CLA exhibited the highest activity Only one mutation, T659A, was found on dacB2 in four againstMTB,followedbyBIAandDOR.MIC90 of TBM/CLA strains, but it was also not associated with the �-lactam was 2 �g/ml, which can be easily obtained in plasma with resistance. In addition, the results showed that no mutation the administration of a single dose of 200 mg TBM pivoxil was detected in crfA among all the strains in our study. We granules [24]. Comparable activity to that of TBM/CLA also did not fnd any strain harbouring mutations in the blaC wasobservedincaseofBIA/CLA.Temaximumplasma and transpeptidase genes simultaneously. concentration (�max) of BIA afer a single intravenous dose was 17.4 �g/ml [25], which exceeded MIC90 (2 �g/ml) of 4. Discussion BIA/CLA. Both of BIA and TBM are stable against renal dehydropeptidase-I and are also safe for central nervous Novel drugs or regimens are urgently needed due to the rise system, since they do not exhibit competitive binding to in the prevalence of multidrug-resistant TB worldwide. �- the �-aminobutyric acid (GABA) receptors [26]. However, lactams are widely used and well-tolerated antibiotics used BIA requires intravenous administration that is not easily for controlling clinical Gram-positive and Gram-negative complied with by the patients. MEM/CLA was previously 6 BioMed Research International

reported to be efective against MDR-TB. Te �max and AUC this study, we only tested four potential targets and none values of MEM were 26 �g/ml and 27.2–32.4 �g⋅h/ml, respec- ofthestrainsharbouredmutationssimultaneouslyonall tively, afer being administered by an intravenous infusion the four genes. It is reported that the �-lactam antibiotics of 500 mg of MEM [27]. In our results also, MEM/CLA have to bind to at least three of the four identifed PBPs exhibited potent activity (MIC90,8�g/ml) against MTB but it to be efective [37]. Considering the multiple targets of �- was weaker than that of TBM/CLA, which is consistent with lactams, we may speculate that the drug resistance may not previous study [28]. Tis may be explained by lower �m and be conferred by mutation in a single target. Further studies less breakdown of the covalent tebipenem-BlaC adduct, in arerequiredtodemonstratetheroleofdrugtargetmutations contrast to that of MEM, thus suggesting a higher activity of in the development of �-lactam resistance in MTB in order to TBM against MTB [17, 29]. Oral administration of the TBM consider the inclusion of �-lactams in the anti-TB treatment pivoxil had a better protective efect than meropenem in all regimens. the tried sepsis models, due to its greater tissue distribution [30]. Hence, as an oral carbapenem, TBM may be a better 5. Conclusions option in developing new anti-TB regimen. MTB harbours a chromosomally encoded �-lactamase, In summary, varied activities of carbapenems, penicillins, which is selectively susceptible to �-lactamase inhibitors. and cephalosporins alone or in combination with �- In this study, varied synergistic efects of three diferent �- lactamase inhibitors against MTB were observed. TBM in lactamase inhibitors were observed. Consistent with previous combination with CLA showed the most remarkable activity study [28], the greatest reduction in MIC was achieved by and has a good prospect in developing novel anti-TB regi- CLA in combination with �-lactams, especially in AMX/CLA mens. Further studies are warranted to investigate the efcacy � (32-fold in MIC50). AVI, a newly developed -lactamase of TBM/CLA in the clinical trials. Additionally, resistance to inhibitor, combining with cefazidime exhibited good activity the �-lactams may not be conferred by single target mutation against Enterobacteriaceae and [31]. in MTB and thus requires further investigation. However, a combination of AVI with �-lactams displayed a lesser reduction in MIC of �-lactams compared to CLA. A recent report has demonstrated that the efciency of AVI Data Availability was lower than that of CLA due to its bulky rings that may Te datasets used to support the fndings of this study are infuence its binding to BlaC [32]. Te unstable covalent available upon request from the corresponding author. adduct of SUB and BlaC may also explain its lesser inhibiting activity than that of CLA [33]. Tus, CLA seems to be the most suitable in developing anti-TB regimens encompassing Conflicts of Interest �-lactams. In addition, the concentration of 5 �g/ml of CLA Te authors declare that there are no conficts of interest used in the current study was lower than the �max of 5.9 �g/ml achieved afer a 250 mg three times a day dosage, suggesting regarding the publication of this manuscript. thatitcanbeeasilyachievedinclinicaluse[34]. For investigating the relationship between mutations in Acknowledgments the �-lactam targets and the drug susceptibility of MTB clin- ical isolates, blaC, ldtMt1, ldtMt2,anddacB2 in 122 MTB clinical Tis work was fnancially supported by the Key project of the isolates were sequenced and analyzed. BlaC hydrolyzes the State Key Laboratory for Infectious Disease Prevention and �-lactam ring, which is essential for the activity of �-lactam Control (2014SKLID104) and the project of the National Key antibiotics, resulting in the �-lactams resistance in MTB. Programs of Mega Infectious Diseases (2013ZX10003002- Eight diferent mutations were observed in blaC.However,we 001). did not fnd any association between these mutations and �- lactam resistance. Interestingly, one of the strains harbouring Supplementary Materials G514A (Gly172Ser) mutation was highly susceptible to �- lactam antibiotics in either the presence or the absence of Te details of mutations and the MIC profle of each mutant the inhibitors. Zhang et al. [35] have reported that the S111R in this study are listed in the supplementary material fle. mutation on BlaC may be associated with an increased sus- (Supplementary Materials) ceptibility of MTB. Considering that the mutation Gly172Ser � may afect the interaction of BlaC and -lactams [36], we References may speculate that this amino change leads to a conformation change in BlaC, which infuences its activity. [1] World Health Organization, Global tuberculosis report, WHO Mutation on target gene ofen confers drug resistance Document, World Health Organization, 2017. to bacteria. Four clinical MTB strains were found to carry [2] L. Wang, H. Zhang, Y. Ruan et al., “Tuberculosis prevalence mutation A776G in ldtMt2 and two of them were susceptible in China, 1990–2010; a longitudinal analysis of national survey to �-lactams. Similarly, among the dacB2 mutants, one data,” Te Lancet,vol.383,no.9934,pp.2057–2064,2014. exhibited resistance, while the other exhibited susceptibility [3]T.Cohen,H.E.Jenkins,C.Lu,M.McLaughlin,K.Floyd,and to �-lactams.However,wedidnotfndanycorrelation M.Zignol,“OnthespreadandcontrolofMDR-TBepidemics: between these mutations and the �-lactams resistance. 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