A widespread single amino acid mutation in AcrA reduces tigecycline susceptibility in Klebsiella pneumoniae

ABSTRACT Whole-genome sequencing of a clinical tigecycline non-susceptible Klebsiella pneumoniae strain reveals a novel T188A mutation in AcrA, raising awareness on its role in tigecycline resistance. Through structural analysis of AcrAB-TolC in Escherichia coli that is highly homologous to K. pneumoniae AcrAB-TolC, weaker hydrogen-bond interaction of the AcrAA188 with lipoyl domain than AcrAT188 was found, which may provide greater flexibility for substrate translocation. In order to explore the resistance mechanism of this mutation, wildtype and T188A mutant of acrA were cloned in pACYC184 and transformed into acrA deleted K. pneumoniae. Several experiments, including measurement of MICs for tigecycline, survival assays, and determination of intracellular tigecycline content, were performed and suggested that T188A mutation in AcrA led to increased tigecycline MIC, improved strain survival under tigecycline stress, and decreased intracellular tigecycline concentrations. Further examination of prevalence of this mutation showed its surprisingly wide presence, particularly in hypervirulent multidrug-resistant K. pneumoniae sequence types. These results support the role of T188A as a widespread AcrA mutation reducing tigecycline susceptibility in K. pneumoniae through increasing tigecycline efflux and improving tigecycline tolerance. IMPORTANCE Tigecycline, a glycecycline antibiotic with broad-spectrum activity against almost all Gram-positive and Gram-negative bacteria, is a highly concerned “last-resort” antibiotic. In addition to plasmid-hosted mobile tet(X) conferring high-level resistance to tigecycline, there are many reports suggesting increased expression of AcrAB-TolC efflux pump leads to tigecycline non-susceptibility. However, the role of mutations in AcrAB-TolC on tigecycline resistance has not been identified. This study reports a novel T188A mutation of the AcrA subunit of AcrAB-TolC complex in a clinical tigecycline-resistant Klebsiella pneumoniae strain and reveals the role of AcrA mutation on tigecycline resistance in K. pneumoniae. High prevalence of A188 type AcrA in hypervirulent multidrug-resistant K. pneumoniae indicates that mutations of the AcrAB-TolC complex may play a larger role in determining bacterial pathogenesis and antibiotic susceptibility than previously expected.

T he rapid increase of bacterial resistance has become one of the urgent problems in the field of medicine and health in the 21st century.The effective treatment of serious bacterial infections caused by multidrug-resistant (MDR) bacteria, especially by extensively drug-resistant (XDR) and pandrug-resistant strains, relies on the applica tion of "last-resort" antibiotics.Unfortunately, the presence of resistant genes, such as bla NDM and mcr-1, greatly decreased the efficacy of carbapenems and colistin, two common "last-resort" antibiotics.Tigecycline, a glycylcycline antibiotic with broad-spec trum activity against almost all Gram-positive and Gram-negative bacteria (1), becomes a highly concerned "last-resort" antibiotic for its high frequency of application to treat multidrug-resistant infections.In recent years, resistance to tigecycline has been frequently described and investigated among multidrug-resistant Enterobacteria ceae and Acinetobacter baumannii strains (2)(3)(4).Particularly, two publications reported plasmid-hosted mobile tet(X3) and tet(X4) genes that confer resistance to this antibiotic in 2019, which received worldwide attentions (5,6).It is worrying that the emergence and dissemination of plasmid-mediated high-level tigecycline resistance genes will greatly decrease the efficacy of this "last-resort" antibiotic.
In addition to tet(X) conferring high-level resistance to tigecycline, frequently reported mechanisms associated with resistance to tigecycline include overexpression of AcrAB-TolC, MexXY-OprM, and OqxAB efflux pumps that belong to the resistance-nod ulation-division (RND) family (7)(8)(9).Among them, AcrAB-TolC, as the most common multidrug efflux pump of this category, is composed of three mini-assemblies, the trimeric outer-membrane channel TolC assembly, the trimeric secondary transporter AcrB assembly, and the hexameric periplasmic AcrA assembly linking AcrB and TolC (10).In recent years, the in situ structure of full-length Escherichia coli AcrAB-TolC was determined by electron cryotomography (11)(12)(13).Revealed by the Cryo-EM structure of AcrAB-TolC, the tripartite complex structure can effectively ensure that the antibiotic molecules were pumped out of cell instead of remaining in periplasm and simultane ously avoid their re-entrance into the cell (14,15).It is reported that high-level expres sion of AcrA and AcrB corresponds to lower intracellular tigecycline concentrations in tigecycline resistant strains (14).Furthermore, there have been a number of reports suggesting mutations of transcription factors that lead to increased expression of the AcrAB-TolC pump, which further leads to tigecycline non-susceptibility (9,15,16).
In previous reports, deletion of AcrAB resulted in strains with significantly increased susceptibility to tigecycline and other antibiotics (9).Many mutations of transcription factors, such as ramA, ramR, soxR, and soxS, are also involved in resistance to tigecycline in an AcrAB-dependent manner (17,18).In recent years, many mutations in AcrB were reported to promote resistance to multiple antibiotics, including ampicillin, ciprofloxa cin, fluoroquinolones, erythromycin, and tetracyclines (19,20).It can be explained that AcrB is the substrate-binding domain of the pump system, which is regarded as the drug specificity and energy transduction center for the antibiotic transport process (21).Conversely, there has been few research directly associating mutations of AcrA or TolC with antibiotic resistance.Particularly, no report on the role of mutations on AcrA and AcrB conferring resistance to tigecycline have been published so far.
MDR Klebsiella pneumoniae, particularly carbapenem-resistant and tigecycline-resist ant K. pneumoniae, is a major pathogen causing nosocomial infections and is considered a seriously growing global health threat (16).For a long time, the overproduction of AcrAB-TolC and OqxAB, both of which are nonspecific active RND efflux pumps, is the most common tigecycline-resistance mechanism in K. pneumoniae (16).The role of mutations in AcrAB-TolC on tigecycline resistance, like in other organisms, has not been identified.In this study, a T188A mutation of AcrA in Klebsiella pneumoniae was identified, which leads to the reduction of tigecycline susceptibility.Mechanistic investigations confirmed that this mutation leads to reduced susceptibility to tigecycline by increasing tigecycline efflux.Further in silico surveillance showed that this mutation is widespread in sequenced K. pneumoniae strains.Further correlation analysis of AcrA sequence types and K. pneumoniae sequence types suggests the high prevalence of A 188 type AcrA in hypervirulent multidrug-resistant K. pneumoniae sequence types.The emergence of AcrA mutation in T188A suggested that the periplasmic membrane fusion protein, such as AcrA, may play greater roles in antibiotic efflux than originally expected.

Antibiotic susceptibility and whole-genome sequencing of a tigecyclineresistant Klebsiella pneumoniae strain
A Klebsiella pneumoniae 3-94 strain was originally isolated from the sputum sample of a patient suffering from acute myocardial infarction in the Second Hospital of Shandong University and stored at −80°C as part of the clinical strain stock.During routine antimicrobial resistance screening, it was found that this strain is resistant to tigecycline.More comprehensive antibiotic susceptibility tests following the CLSI guidelines was performed using the K-B disk diffusion method, except for polymyxin E for which only the agar dilution method is available.It was found that K. pneu moniae 3-94 is a multidrug-resistant strain that is resistant to all β-lactams and two β-lactam/β-lactamase inhibitor combos, trimethoprim, trimethoprim-sulfamethoxa zole, a quinolone ciprofloxacin, tetracycline, and tigecycline (Table 1).Additional agar dilution method confirmed K. pneumoniae 3-94 is resistant to tigecycline with an MIC value of 4 µg/mL.Whole-genome sequencing (China National Microbiology Data Center accession: NMDC60064229) and subsequent analysis of antibiotic resistance determinants with the CARD database were performed, leading to the identification of mechanisms of resistance to all antibiotics tested but not tigecycline (Table 1), implying that novel mechanisms of tigecycline non-susceptibility may be present.

A point mutation T188A in AcrA results in decreased tigecycline susceptibility
More in-depth analysis of the genomic sequence of K. pneumoniae 3-94 revealed that AcrA has a T188A point mutation in comparison with AcrA from the tigecycline-sensitive whole-genome sequenced strain K. pneumoniae 2-1 (22) (Genbank accession number: CP031562).This protein is a subunit of the AcrAB-TolC RND-type efflux pump that has been shown to play a major role in tigecycline resistance.
While the structure of K. pneumoniae AcrAB-TolC is unavailable, the structure of AcrAB-TolC complex of Escherichia coli (PDB ID: 5V5S) was previously solved and published (23).Pairwise sequence alignment with EMBOSS needle showed that all three subunits have good sequence identities between K. pneumoniae and E. coli: AcrA 85.1% (Genbank accession: E. coli NP_414996.1,K. pneumoniae WP_267695800.1),AcrB 91.4% (Genbank accession: E. coli NP_414995.1,K. pneumoniae WP_104443262.1),and TolC 83.8% (Genbank accession: E. coli NP_417507.2,K. pneumoniae WP_048334428.1).Therefore, K. pneumoniae AcrAB-TolC is expected to have a similar structure with E. coli AcrAB-TolC.In E. coli, N 188 of AcrA that corresponds to T 188 in K. pneumoniae is located on the lipoyl domain.This periplasmic hexameric AcrA assembly connects the inner membrane-bound trimeric AcrB and the outer membrane-bound trimeric TolC, as seen from the side.The formation of two rings, consisting of lipoyl domains and β-barrel domains, can be observed in the funnel-shaped tunnel that connects AcrB-associated membrane-proximal domains and TolC-associated helical hairpin domains (Fig. 1A).A further bottom-up view (inner membrane to outer membrane) revealed that T 188 is located on the inner surface of the substrate-pumping channel (Fig. 1B).This residue is adjacent to the anti-parallel sheet that forms the major part of the lipoyl domain (Fig. 1C).A closer look at this domain suggests that the hydroxyl group of the T 188 side chain forms two hydrogen bonds (bond length respectively 3.2 Å and 3.4 Å) with L 203 and A 202 , two residues on the β-sheet (Fig. 1D).Upon mutation of T 188 to A 188 , these two hydrogen bonds no long exist, increasing the flexibility of the lipoyl domain.Therefore, it is hypothesized that the AcrA T188A mutation may have an impact on its structure and may lead to the increase of tigecycline export which further decreases tigecycline susceptibility.
To verify this hypothesis, the acrAB gene cluster was deleted from a laboratory-main tained K. pneumoniae S1 strain using CRISPR-Cas9 methods as previously published (24).As expected, the acrAB deletion strain is susceptible to tigecycline.Re-introduc tion of acrA and acrB from K. pneumoniae 3-94 cloned on pACYC184 (K.pneumoniae S1 ΔacrAB + pACYC184acrA A188 acrB) led to resistance to tigecycline (Table 2).When reverting A 188 to T 188 (K.pneumoniae S1 ΔacrAB + pACYC184acrA T188 acrB, Table 2), the MIC values dropped from 4 to 2.5 μg/mL, suggesting improved susceptibility to tigecycline.This is in consistence with the hypothesis that T188A mutation in AcrA led to decreased tigecycline susceptibility.
To further confirm this hypothesis, survival assays were performed, to find out whether T188A mutation in AcrA leads to better tolerance to tigecycline (Fig. 2).When strains were grown in LB medium containing 16 µg/mL of tigecycline, acrAB knockout strains showed poor growth.Introducing acrAB from K. pneumoniae 2-1 (K.pneumoniae S1 ΔacrAB + pACYC184acrA T188 acrB) showed improved growth, while pACYC184 alone cannot, most likely due to increased acrAB levels as a result of multiple plasmid copies.Mutation of T188 to alanine (K.pneumoniae S1 ΔacrAB + pACYC184acrA A188 acrB) further significantly improved tigecycline tolerance and growth, clearly suggesting that T188A leads to reduced tigecycline susceptibility, and improved tolerance.

AcrA T188A mutation leads to reduced tigecycline susceptibility by increas ing tigecycline efflux
In order to find out whether the AcrA T188A mutation reduces tigecycline susceptibility by increasing tigecycline efflux, K. pneumoniae strains were incubated in the presence of tigecycline, and intracellular tigecycline levels were measured with high-perform ance liquid chromatography (HPLC).The K. pneumoniae ΔacrAB strain had significantly higher intracellular tigecycline levels, suggesting impaired tigecycline efflux by deleting acrAB (Fig. 3).Introducing pACYC184 had no impact on intracellular tigecycline levels.
Introducing acrAB with pACYC184 to acrAB knockout strain led to a 45-fold decrease on intracellular tigecycline concentrations.This is in agreement with the findings made with survival assays (Fig. 2).In particular, mutating residue 188 from threonine to alanine led to a 3-fold further reduction of intracellular tigecycline concentration.This is a strong suggestion that AcrA T188A led to significantly increased tigecycline efflux, which further supports the finding that T188A mutation in AcrA led to increase of tigecycline tolerance.

Widespread presence of T188A mutation in K. pneumoniae
The prevalence of T188A mutation in AcrA was investigated in 14,778 K. pneumoniae strains whose genomic sequences are available from Genbank.AcrA-coding genes were found in 14,776 strains.Only 233 different AcrA sequence types were found in all 14,776 K. pneumoniae strains, of which 3 top sequence types account for 96.49% of the sequences (Fig. 4).To our surprise, only 6,059 (41.01%) genomes encode AcrA with the same sequence as tigecycline-sensitive K. pneumoniae ATCC13883 and K. pneumoniae 2-1 (seq4), whereas 7,330 (49.61%) genomes encode AcrA with the same sequence as K. pneumoniae 3-94 reported in this work (seq1).Of all genomes, 8,537 (57.78%) encode AcrA with A 188 , whereas 6,191 (41.91%) encode AcrA with T 188 (Table S1).This finding suggests that the T188A mutation that lead to reduced tigecycline susceptibility is highly prevalent in K. pneumoniae, which may imply that the tigecycline effectiveness in treating K. pneumoniae infections has already been reduced with the high prevalence of a tigecycline tolerant AcrA sequence type.
The sequence types (STs) of all analyzed K. pneumoniae genomes were determined with the MLST algorithm (Table S2), and the distribution of K. pneumoniae STs of genomes encoding K. pneumoniae ATCC13883 type AcrA (seq4, T 188 ) or K. pneumoniae 3-94 type AcrA (seq1, A 188 ) was analyzed.It is to our surprise finding that K. pneumoniae ATCC13883 type AcrA is primarily encoded by only a few STs, whereas K. pneumoniae

DISCUSSION
Tigecycline is considered a "last-resort" antibiotic that is widely used when efficacies of other antibiotics are severely impaired due to antibiotic resistance.The putative devastating consequences of resistance to this antibiotic made its resistance a hot topic for research and discussion.Among these researches, mutations in transcription factors governing the expression of acrAB, such as AcrR and RamR (28), can lead to improved expression of AcrAB-TolC efflux pump, which, in turn, results in tigecycline resistance.However, although reports on AcrAB-TolC mutation that led to antibiotic resistance have been available (21), possible role of AcrAB-TolC sequence polymorphism in tigecycline has not been recognized.This work provides evidence that mutation of T 188 to A 188 in AcrA led to decreased tigecycline susceptibility by increasing tigecycline efflux.This conclusion is supported by analysis of MICs, survival assays under tigecycline stress, and measurements of intracellular tigecycline concentrations following tigecycline treatment.These consistent experimental observations ensure the validity of the discovery made in this work, which appears to be a new mechanism for tigecycline non-susceptibility.By inspection of the structure of AcrAB-TolC homolog in E. coli, we found that this mutation leads to the removal of two hydrogen bonds formed between the hydroxyl group of threonine and a β-sheet that apparently plays a major role in structural rigidity of the lipoyl domain.The removal of hydrogen bonds is predicted to increase the flexibility of the lipoyl domain that is part of the funnel-shaped efflux tunnel of the AcrAB-TolC complex.Weeks et al. also reported that they observed compensatory alterations in the AcrA β-barrel and lipoyl domains when AcrB β-hairpin mutant appears defective in TolC-docking domain.They then explained that the two central domains of AcrA directly interact with AcrB to stabilize the complex and probably permit TolC aperture to transit from closed to open state for drug expulsion by influencing TolC recruitment (20).It is reasonable that the increase of flexibility of the tunnel may, in turn, improve the accommodation of tigecycline molecules, which may be in a variety of orientations and get "stuck" in a rigid tunnel, particularly when a large number of molecular are exported simultaneously.Therefore, although without further experimental structural confirmation, we suspect that the structural basis for increased tigecycline efflux is the improvement of efflux tunnel flexibility.K. pneumoniae is a clinically significant pathogen that is involved in the develop ment of severe life-threatening diseases including pneumonia, bacteremia, pyogenic liver abscess, etc. (29).Its particularly concerning property-extremely strong antibiotic resistance-made it a member of the ESKAPE group that receives special attention for potentials to elicit strong health damage (30).The wide antibiotic resistance spectrum of K. pneumoniae severely limits medical options.It is often a necessity to apply riskier strategies such as relying on "last-resort" antibiotics (31).Therefore, the mechanism found in this work that reduces tigecycline efficacy is more important than it normally is, as it hampers one of the few reliable options to treat K. pneumoniae infections.
The finding that the T188A mutation is widespread in publicly available K. pneu moniae genomes rings an alarming bell that the genotype leading to tigecycline non-susceptibility is already prevalent.This is echoed by previous surveillance studies: 27.8% of studied Enterobacterales were found to be tigecycline resistant in a recent report (32), and meta-analysis of surveillance studies suggests 5.1% of isolated blood stream K. pneumoniae strains are resistant to tigecycline (33).Although genotypes do not necessarily translate into antibiotic resistant phenotypes, it is still worth of con cerns, particularly in the context that tigecycline non-susceptibility genotypes appear expanding or even dominating.The very limited number of sequence types for AcrA also hints that this protein appears to be quite conserved, and genotypes could be quite stable once dominating.Correlation analysis of AcrA sequence type and K. pneumoniae sequence type suggests these two sequence types are closely linked, and hypervirulence K. pneumoniae types usually carry the high-tigecycline-resistance type AcrA.Although the currently prevalent multidrug-resistant sequence types appear to still carry the low-tigecycline-resistance type AcrA, it is likely that hypervirulence K. pneumoniae carrying high-tigecycline-resistance type AcrA will reduce the efficacy of tigecycline and reshapes the K. pneumoniae sequence type landscape in clinical settings when tigecycline is used more and more frequently.In recent years, it is reported that classical XDR K. pneumoniae strains evolved into XDR hypervirulent K. pneumoniae through the acquisition of pLVPK-like virulence plasmids (34).Moreover, K. pneumoniae ST307 was reported to be the predominant clone type among tigecycline-and carbapenem-resist ant K. pneumoniae stains in South Korea (16).The high prevalence of high-tigecyclineresistance AcrA type in hypervirulent K. pneumoniae is particularly worrisome, as these pathogens elicit greater health damage, and reducing efficacy of one of the last medical options could be devastating.
AcrB, rather than AcrA, has commonly been considered a key subunit deciding efflux efficiencies, as it is the substrate-binding subunit that transports substrates, many antibiotics included, across the inner membrane (21).Instead, AcrA is often viewed as a "connector" that shuttles substrates across the periplasm.Considering the diameter of the tunnel in AcrA is generally bigger (~4-5 nm) than the sizes of antibiotics, it appears reasonable to rule AcrA out as a rate-limiting step for substrate efflux.This work suggests otherwise.Although it may be true that AcrB plays a more important role in substrate recognition, potential impacts of AcrA sequence polymorphism should not be neglected.This may also apply to TolC that governs substrate efflux across the outer membrane.
In conclusion, a T188A mutation of the AcrA subunit of AcrAB-TolC complex was found in K. pneumoniae.Evidences support that this mutation leads to reduced tigecycline susceptibility by elevating tigecycline efflux.Further surveillance confirms that this mutation is widespread in sequenced K. pneumoniae genomes.To the best of our knowledge, no report of AcrA mutation on tigecycline resistance in K. pneumoniae has been previously reported.It is suspected that mutations of the AcrAB-TolC complex may play a larger role in determining antibiotic susceptibility than previously expected.

Strains used in this study
K. pneumoniae 3-94 is a strain in the clinical strain stock of Second Hospital of Shandong University.K. pneumoniae S1 and K. pneumoniae 2-1 are laboratory maintained strains.Sequence data of the two strains were deposited in Genbank, with accession numbers of CP103062 and CP031562.

Whole-genome sequencing, assembly, and annotation
Bacterial genomic DNA was extracted using the bacterial genome DNA rapid extraction kit (Mei5 Biotechnology Co., Ltd., Beijing, China) and sequenced at Novogene Technology Co. Ltd, Beijing, China, with an Illumina NovaSeq 6000 platform (Illumina, Inc., San Diego, CA, USA) at PE150 mode and 200 × coverage.SPAdes version 3.15.4 was used for genome assembly (37,38).QUAST version 4.6.0 was used to evaluate the quality of genome assembly (39).RGI version 6.0.1 with CARD version 3.2.6 was used to annotate the resistance genes (40).Assembled genomic sequences are deposited in China National Microbiology Data Center (NMDC) with accession numbers NMDC60064229.

Genetics
Construction of acrAB knockout K. pneumoniae strain was performed using the CRISPR-Cas9 system as previously reported (24).The engineering plasmids used in the knockout process are shown in Table S3.The spacer and single-stranded DNA repair template sequences designed for gene knockout are shown in Table S4.The sequence of the primers required for plasmid construction and the knockout validation are shown in Table S5.
To express acrAB from K. pneumoniae 2-1 and K. pneumoniae 3-84 in K. pneumoniae S1 ΔacrAB, genes were amplified and cloned into pACYC184 whose selection marker was replaced with apramycin-resistant aac (2)IV.Cloning was performed with MultiF Seamless Assembly Mix (ABClonal Inc., China).Ligated plasmids were transformed into E. coli DH5α and selected on LB plates containing 30 mg/L apramycin.Correct constructs were transformed into K. pneumoniae S1 ΔacrAB with electroporation.Primers used for expressing acrAB can be found in Table S5.

Survival assay
K. pneumoniae S1 ΔacrAB, K. pneumoniae S1 ΔacrAB + pACYC184, K. pneumo niae S1 ΔacrAB + pACYC184acrA A188 acrB, and K. pneumoniae S1 ΔacrAB + pACYC184acrA T188 acrB strains were assayed for survivability under TGC stress similar to previous published literature (41).Specifically, cells were grown to exponential phase (OD 600 = 0.5) in LB liquid medium.Four microliters of the culture was diluted by 10 2 -to 10 6 -fold and grew on LB plates for CFU calculation.Tigecycline at a final concentration of 16 µg/mL was added to liquid culture to challenge cells.Culture was removed every 15 min for CFU calculation as mentioned above.The survival rate is calculated by dividing the CFU number post-challenge and pre-challenge.Three biological replicates were performed.

Determination of intracellular TGC content
Determination of intracellular tigecycline levels was performed by the HPLC method similar to previously reported protocol (14).K. pneumoniae S1 ΔacrAB, K. pneumoniae S1 ΔacrAB + pACYC184, K. pneumoniae S1 ΔacrAB + pACYC184acrA A188 acrB, and K. pneumoniae S1 ΔacrAB + pACYC184acrA T188 acrB strains were grown in LB liquid medium to exponential phase (OD 600 = 0.5) and centrifuged at 2000 × g for 5 min.Fifty milligrams of cell pellets was suspended in 5 mL of 0.05 M PBS (pH 7.0) and incubated at 37°C for 10 min.Sterile TGC was added to cell suspensions at a final concentration 50 µg/mL and incubated at 37°C for 5 min.Two hundred and fifty microliters of DMSO was subsequently added, followed by further incubation at 37°C for 5 min.Cells were further pelleted by centrifugation at 2,000 × g for 5 min and washed in 5 mL of pre-chilled 0.05 M PBS (pH 7.0) for 3 times.One milliliter of 0.1 M glycine-hydrochloric acid (pH 3.0) was used to suspend cells, followed by incubation for 16 h.Cells were removed by centrifugation at 10,000 × g for 10 min.The supernatant was further sterilized by passing through 0.22 µm filters.Tigecycline content was assayed with HPLC equipped with a Sepax GP-C18 column.Diammonium hydrogen phosphate-triethylamine-methanol salt solution (50:1:49) was used as the mobile phase at a flow rate of 1.0 mL/min.Column temperature of 30°C and detection wavelength of 248 nm were used.Three replicates were performed.

Statistics
Comparisons of survival rates and intracellular tigecycline levels were made using two-tailed Student's t-test.P < 0.05 was considered statistically significant.

FIG 1
FIG 1 Location of residue 188 on hexameric AcrA assembly.Panel A, side view of E. coli AcrA assembly, red sticks indicates residue 188; Panel B, bottom-up view of E. coli AcrA assembly, red sticks indicates residue 188; Panel C, residue 188 in relation to the lipoyl β-sheet, red sticks indicate residue 188, β-sheet is represented with cartoon representation on the left side and stick representation on the right side; Panel D, residue T 188 forms additional hydrogen bonds with the lipoyl β-sheet; Panel E, loss of hydrogen bonds with T188A mutation.

TABLE 2
Tigecycline susceptibility of constructed strains a a S, sensitive; R, resistant.

TABLE 3
Sequence types (STs) of K. pneumoniae with K. pneumoniae ATCC13883 type AcrA (T 188 ) and K. pneumoniae 3-94 type AcrA (A 188 ) a a Only STs accounting for over 1% of all genomes were listed.