Molecular Epidemiology and Resistance Mechanisms of Tigecycline-Non-Susceptible A. baumannii Isolated from a Tertiary Care Hospital in Chongqing, China

doi:10.21203/rs.3.rs-3769808/v1

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Molecular Epidemiology and Resistance Mechanisms of Tigecycline-Non-Susceptible A. baumannii Isolated from a Tertiary Care Hospital in Chongqing, China

https://doi.org/10.21203/rs.3.rs-3769808/v1

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Objectives

This study aimed to investigate the epidemiology and tigecycline resistance mechanisms of Tigecycline-Non-Susceptible A. baumannii (TNAB) isolates in a large tertiary care hospital in Chongqing, China.

Methods

From July 2019 to June 2022, 34 non-duplicate isolates of A. baumannii determined as tigecycline-non-susceptible by broth microdilution method were collected from a tertiary care hospital in Chongqing. The resistance-related genes were sequenced and the expression level of the resistance-nodulation-cell division (RND) efflux pumps was investigated. The activity of the RND efflux pumps was examined by adding efflux pump inhibitors (EPIs). The molecular epidemiology was analyzed by multi-locus sequence typing (MLST) and goeBURST software.

Results

In this study, the TNAB strains were primarily obtained from ICU and had high resistance rates to commonly used antibiotics. Tigecycline resistance-associated genes adeR, adeS, adeL, adeN, rrf, and rpsJ were detected in most strains. All TNAB strains exhibited 240 nucleotide deletion mutation in the trm gene. The expression level of the RND efflux pumps in TNAB strains was higher than the reference strain ATCC19606. 58.82% of strains had a decrease in the tigecycline minimum inhibitory concentration (MIC) after the addition of carbonyl cyanide 3-chlorophenylhydrazone (CCCP). A total of 8 ST types were identified, including ST195 (35.29%), ST208 (17.65%), and ST369 (14.71%). EBURST analysis found that 8 of the ST types belonged to the Clonal Complex 92.

Conclusion

The TNAB strains in our hospital have a high degree of affinity and antibiotic resistance. Regular surveillance should be conducted to prevent outbreaks of TNAB epidemics.

Bacteriology

tigecycline-non-susceptible

A. baumannii

RND efflux pump

multi-locus sequence typing

A. baumannii is a widely distributed Gram-negative coccobacillus and an opportunistic pathogen that can cause a variety of infections such as pneumonia, meningitis, urethral infection, skin infection, peritonitis, and bacteremia [1, 2]. The considerable adaptability and clonal transmission A. baumannii possesses can lead to the development of multidrug-resistant (MDR), extensively drug-resistant (XDR), and even pan drug-resistant (PDR) phenotypes, thus presenting significant difficulties for clinical treatment[3, 4]. According to the survey data of the China Antibiotic Resistance Surveillance System (CARSS) over the previous three years, the national average resistance rate (53.4%-54.3%) of A. baumannii to carbapenems has been maintained at a high level. Of these, Chongqing has risen from twelfth place (56.1%) to seventh place (58.4%) in the ranking of A. baumannii resistance rates to carbapenems in regional China.

Tigecycline, a derivative of minocycline, belongs to the group of glycyltetracycline antibiotics and functions by interacting with the 30S ribosomal subunit to disrupt bacterial protein synthesis [5, 6]. It effectively overcomes tetracycline resistance mechanisms, thus considered to be one of “the last lines of defense” in the treatment of infections caused by extensively drug-resistant A. baumannii (XDRAB) [7]. The World Health Organization (WHO) classified it as a critically important antimicrobial drug in 2019 (https://www.who.int/foodsafety/publications/antimicrobials-sixth/en/). However, tigecycline-non-susceptible A. baumannii (TNAB) strains have been constantly isolated in the clinic.

The resistance mechanism of TNAB isolates is complex and has not yet been fully elucidated due to the high genomic plasticity and genetic variabilities[8]. The overexpression of resistance-nodulation-cell division (RND) efflux pumps, including AdeABC, AdeFGH, and AdeIJK, which are regulated by their respective upstream regulators adeRS, adeL, and adeN, has been reported to be a critical mechanism for tigecycline resistance in A. baumannii[9, 10]. In addition to chromosome-mediated mechanisms, the plasmid-mediated tet(X) gene variants encoding a flavin-dependent monooxygenase have been reported to be associated with a high-level of tigecycline resistance [11, 12]. Furthermore, mutations in rpsJ, rrf, plsC, and trm genes have been linked to tigecycline resistance [13–16].

The emergence of tigecycline resistance has resulted in fewer clinical options for the treatment of MDR A. baumannii. Therefore, investigations into tigecycline-resistant A. baumannii are essential, while no prior related studies have been conducted in Chongqing, China. The main objective of the current study was to investigate the molecular epidemiology and mechanisms of tigecycline resistance in clinical TNAB isolates in a tertiary care hospital in Chongqing, China from July 2019 to June 2022. This research could potentially provide valuable insights into the rational use of clinical antimicrobial agents and help prevent outbreaks of TNAB in this hospital.

Bacterial isolates and species identification

A total of 34 distinct clinical isolates of A. baumannii were collected from individual patients from July 2019 to June 2022 at a large tertiary teaching hospital in Chongqing, China. All strains were identified as A. baumannii by MALDI-TOF MS (Bruker, Sancordon Inc., Bremen, Germany). This study was granted approval by the medical center's ethical committee. Since no interventions were carried out, the patients' written consent was not obtained.

Antimicrobial susceptibility testing and phenotypic inhibition experiments

The VITEK-2 system (bioMérieux, France) was used to conduct antimicrobial susceptibility tests on antibacterial agents except for tigecycline. The results were interpreted with reference to the Clinical and Laboratory Standards Institute (CLSI) guidelines. The minimum inhibitory concentration (MIC) values (mg/L) of tigecycline were identified by the broth microdilution method using cation‑adjusted Mueller Hinton broth (CAMHB). A MIC of at least 4 mg/L was used to define tigecycline non-susceptibility for A. baumannii, in accordance with the Food and Drug Administration's (FDA) guideline for tigecycline susceptibility breakpoints of Enterobacteriaceae criteria (susceptible MIC, ≤ 2 mg/L; intermediate MIC, > 2 mg/L and < 8 mg/L; resistant MIC, ≥ 8 mg/L). The tigecycline MICs in the presence of efflux pump inhibitors (EPIs) carbonyl cyanide 3-chlorophenylhydrazone (CCCP) (10 mg/L) and phenylalanine-arginine beta-naphthylamide (PAβN) (50mg/L) were calculated to evaluate the efflux pump activity. The inhibition effects were described as having good effect and great effect for at least 4-fold and 16-fold reduction in the MIC value, respectively.

Multi-locus sequence typing analysis

Multi-locus sequence typing (MLST) was performed on all strains in this experiment using the primers of seven housekeeping genes (gltA, gyrB, gdhB, recA, cpn60, gpi, rpoD) listed on the PubMLST website (https://pubmlst.org/abaumannii) according to the scheme (Table S1) [17]. Then the amplified and sequenced genes were analyzed using the MLST database as previously described[18]. The homology analysis of these 34 TNAB strains was performed using the eBURST method by goeBURST software.

Molecular detection of resistance genes

We carried out polymerase chain reaction (PCR) detection of RND efflux pump regulation genes, including adeR, adeS, adeL, and adeN, and potential tigecycline-resistance determinants trm, rrf, and rpsJ were also amplified and sequenced in positive strains. Detection of the tet(X) gene and its variants was accomplished with a pair of universal primers. The cycling condition used for PCR included an initial denaturation step at 94°C for 5 min, followed by 30 cycles of denaturation at 95°C for 1 min, annealing at 55°C for 30 s, extension at 72°C for 1 min, and a final extension at 72°C for 7 min after completion of the 30th cycle. Nucleic acid sequence analysis and mutation sites detection were conducted using SnapGene version 4.1.9 (GSL Biotech LLC, Chicago, IL, USA). The primers used to amplify the coding sequence of each gene are listed in Table S1.

Quantitative real-time PCR (qPCR)

The transcript levels of efflux pump genes adeB, adeG, and adeJ were measured by quantitative real-time PCR (qPCR) assays. RNA was extracted using the E.Z.N.A. Bacterial RNA Kit (Omega, Norcross, USA) according to the manufacturer's instructions. On-column DNA digestion was performed using E.Z.N.A. RNase-Free DNase I Set (Omega, Norcross, USA) to remove DNA. Reverse transcription was performed using a PrimeScript™ RT reagent Kit (TaKaRa, Kyoto, Japan). Real-time PCR performance using TB Green® Premix Ex Taq™ II (TaKaRa, Kyoto, Japan) was run on a CFX96 Real-Time PCR Detection System (Bio-Rad, California, USA). The mRNA of the constitutively expressed single-copy housekeeping gene gyrB was utilized as a control, while the mRNA of the well-characterized susceptible strain ATCC 19606 was employed as a standard reference for the expression level. The qPCR experiments were conducted in triplicate, and the transcript levels were then calculated using the 2^-ΔΔCT method. Significant overexpression was defined as a four-fold or more gene expression.

Data analysis

Statistical analysis was carried out using the GraphPad Prism software V. 8.02 (GraphPad Software Inc., San Diego, CA); the calculation of gene expression differences between groups was based on an Unpaired student t-test. A P value less than 0.05 was considered statistically meaningful.

Bacterial isolates and species identification

All clinical isolates were identified as A. baumannii by MALDI-TOF MS (Bruker, Sancordon Inc., Bremen, Germany). Analysis of the specimen sources revealed that the majority of TNAB isolates originated from respiratory secretions including sputum (55.9%) and Bronchoalveolar lavage (14.7%) (Table 1). Among the 34 isolates, almost three-quarters of the samples were collected from the intensive care unit (ICU) (73.5%), followed by Respiratory Medicine (8.8%), Neurology (5.9%), Hematology (2.9%), Hepatobiliary Surgery (2.9%), Infection Unit (2.9%), and General Practice (2.9%). The average age of patients infected with TNAB strains was 64 years, of whom 21 (61.8%) were males and 13 (38.2%) were females.

Table 1

Clinical Characterization of TNAB Isolates(n=34)
Characteristics		No. (%)
Specimen source	Sputum	19(55.9)
	Bronchoalveolar lavage	5(14.7)
	Catheter	2(5.9)
	Urine	2(5.9)
	Drainage fluid	2(5.9)
	Blood	1(2.9)
	Other	3(8.8)
Department distribution	Intensive Care Unit	25(73.5)
	Respiratory Medicine	3(8.8)
	Neurology	2(5.9)
	Hematology	1(2.9)
	Hepatobiliary Surgery	1(2.9)
	Infection Unit	1(2.9)
	General Practice	1(2.9)
Sex	Male	21(61.8)
Sex	Female	13(38.2)
Age(mean ± SD)		63.59 ± 9.92
Abbreviation: TNAB, tigecycline-non-susceptible A. baumannii

Antimicrobial susceptibility testing and efflux pump activity determination

The 34 study strains showed high resistance rates to ticacillin/clavulanic acid (97.1%), doxycycline (91.2%), cefoperazone/sulbactam (88.2%), tobramycin (82.4%) and minocycline (73.5%) (Fig. 1). They were all resistant to piperacillin/tazobactam, ceftazidime, imipenem, meropenem, ciprofloxacin, and levofloxacin. Whereas, all isolates were revealed to be susceptible to colistin. All isolates were tigecycline non-susceptibility with minimum inhibitory concentrations (MICs) ranging from 4 to 32 mg/L (Fig. 2). After the addition of CCCP, 14 (41.2%) strains had a two-fold decrease, and 6 (17.7%) strains had a four-fold or more decrease in tigecycline MIC values. Nevertheless, 24(70.6%) TNAB strains had a 2-fold increase in tigecycline MIC values with the addition of PAβN, while the remaining 10(29.4%) isolates were not affected by it.

Molecular epidemiology based on MLST

Molecular typing showed that these 34 TNAB strains belonged to 8 distinct sequence types (Table 2). The ST195 (12, 35.3%) contained 12 isolates was the dominant sequence type, followed by ST208 (6, 17.7%), ST369 (5, 14.7%), ST938 (4, 11.8%), ST381 (3, 8.8%), ST191 (2, 5.9%), ST1849 (1, 2.9%) and ST540 (1, 2.9%). In this study, no new ST type was found. The homology analysis found that all of the 8 ST types belonged to the Clonal Complex 92 (CC92) and ST195 (1-3-3-2-2-96-3) was the founder ST type. ST938 and ST381 differ from ST195 in two housekeeping genes and are double-locus variants (DLVs), while the rest of the ST types differ from ST195 in only 1 locus and are single-locus variants (SLVs).

Table 2

Multi-locus sequence typing results of tigecycline-non-susceptible A. baumannii
ST	Allele of gltA	Allele of gyrB	Allele of gdhB	Allele of recA	Allele of cpn60	Allele of gpi	Allele of rpoD	No. (%)
191	1	3	3	2	2	96	3	2(5.88)
195	1	3	3	2	2	97	3	12(35.29)
208	1	3	3	2	2	106	3	6(17.65)
369	1	38	3	2	2	110	3	5(14.71)
381	1	81	3	2	2	16	3	3(8.82)
540	1	3	3	2	2	94	3	1(2.94)
938	1	3	3	2	2	160	3	4(11.76)
1849	1	3	3	2	2	331	3	1(2.94)
Abbreviation: ST, Sequence Type; No., number of isolates

Analysis of resistance determinants in TNAB Strains

As shown in Table 3, the RND efflux pump-related genes adeR, adeS, adeL, and the SAM-dependent methyltransferase encoding gene trm, the 30S ribosomal protein S10 encoding gene rpsJ, the ribosome recycling factor-related gene rrf were presented in all the TNAB strains. AdeN was found in 25 (73.5%) isolates whereas the tet(X) gene and its variants were not found in any of the strains. All TNAB strains had 240 nucleotide deletion mutations in the trm gene, while no mutations in each of the other tigecycline resistance-related genes were found.

Table 3

Tigecycline resistance-related genes detected in this experiment
	adeR	adeS	adeL	adeN	rrf	rpsJ	trm	tetX		adeR	adeS	adeL	adeN	rrf	rpsJ	trm	tetX
TNAB-1	+	+	+	—	+	+	+	—	TNAB-18	+	+	+	+	+	+	+	—
TNAB-2	+	+	+	+	+	+	+	—	TNAB-19	+	+	+	+	+	+	+	—
TNAB-3	+	+	+	+	+	+	+	—	TNAB-20	+	+	+	—	+	+	+	—
TNAB-4	+	+	+	—	+	+	+	—	TNAB-21	+	+	+	+	+	+	+	—
TNAB-5	+	+	+	+	+	+	+	—	TNAB-22	+	+	+	—	+	+	+	—
TNAB-6	+	+	+	+	+	+	+	—	TNAB-23	+	+	+	+	+	+	+	—
TNAB-7	+	+	+	+	+	+	+	—	TNAB-24	+	+	+	+	+	+	+	—
TNAB-8	+	+	+	—	+	+	+	—	TNAB-25	+	+	+	+	+	+	+	—
TNAB-9	+	+	+	+	+	+	+	—	TNAB-26	+	+	+	+	+	+	+	—
TNAB-10	+	+	+	+	+	+	+	—	TNAB-27	+	+	+	+	+	+	+	—
TNAB-11	+	+	+	+	+	+	+	—	TNAB-28	+	+	+	+	+	+	+	—
TNAB-12	+	+	+	+	+	+	+	—	TNAB-29	+	+	+	+	+	+	+	—
TNAB-13	+	+	+	—	+	+	+	—	TNAB-30	+	+	+	+	+	+	+	—
TNAB-14	+	+	+	+	+	+	+	—	TNAB-31	+	+	+	—	+	+	+	—
TNAB-15	+	+	+	+	+	+	+	—	TNAB-32	+	+	+	—	+	+	+	—
TNAB-16	+	+	+	+	+	+	+	—	TNAB-33	+	+	+	+	+	+	+	—
TNAB-17	+	+	+	+	+	+	+	—	TNAB-34	+	+	+	—	+	+	+	—
Abbreviation: +, presence of PCR product; −, absence of PCR product

Relative gene expression of RND efflux systems

The RNA transcription levels of the major part of tripartite RND efflux systems (adeB, adeG, and adeJ) were determined by qPCR relative to that of gyrB and taken ATCC 19606 as a reference. Among the 34 TNAB strains, the expression levels of adeB, adeG, and adeJ were upregulated relative to ATCC19606 in 29 (85.3%), 12 (35.3%), and 34 (100.0%) strains, respectively (Fig. 3). The mean relative expression of adeB, adeG, and adeJ of the TNAB isolates in this experiment were 3.79 ± 0.80, 6.54 ± 3.44, and 4.00 ± 0.48, respectively (Unpaired t-test; p 0.0009 for adeB, p 0.1121 for adeG and p < 0.0001 for adeJ). The highest average expression level of adeB was found in the strains of the MIC16 group, while the highest average expression levels of adeG and adeJ were found in the strains of the MIC8 group. When compared to the MIC4 group, the MIC16 group exhibited a statistically significant increase in adeB (p 0.0302) expression, whereas the MIC8 group showed a statistically significant elevation in adeG (p 0.0254) and adeJ (p 0.0313) expression.

In this research, we collected 34 TNAB isolates from a large tertiary care teaching hospital in Chongqing, China over a three-year period. The review of clinical information showed that TNAB strains in our hospital were primarily isolated from elderly patients and ICU patients with low immunity. According to the findings of antimicrobial susceptibility testing, the TNAB isolates were extremely resistant to the majority of therapeutically used antibiotics, with the exception of colistin. All the strains showed low-level resistance to tigecycline except one demonstrated a tigecycline MIC value of 32 mg/L. EPIs can impair efflux activity and restore the susceptibility to antibiotics. A study by Deng Mei et al. showed that both EPIs PAβN and CCCP were able to partially decrease the MIC of tigecycline[12]. In our study, 17.7% of TNAB strains had a 4-fold or greater decrease in the MIC of tigecycline when CCCP was present. However, the addition of PAβN actually increased the MIC of tigecycline, which contradicts previous reports. This suggests that PAβN and CCCP have different specificities and activities with regard to different efflux pumps. Additionally, there may be an antagonistic effect between PAβN and tigecycline.

Previous studies have proposed that elevated expression of RND efflux pumps contributes to tigecycline resistance in A. baumannii. In comparison to the sensitive reference strain ATCC19606, the relative expression of all three efflux pumps in TNAB of different MIC groups was elevated as shown in Fig. 3. However, none of the strains except TNAB-10 exhibited significant elevation of them simultaneously. This suggests that the three RND efflux pumps may act independently. Whereas a previous study by Damier et al. suggested that tigecycline resistance can be a result of the synergistic contribution of AdeIJK with AdeABC[19], of which AdeABC is considered to have a superior influence[12, 20, 21]. As indicated by Fig. 2, the best inhibitory effect of CCCP was achieved by the strains of MIC16, followed by the strains of MIC8. Among them, the strains of MIC16 were mainly up-regulated by adeB, and the strains of MIC8 had expression of adeG and adeJ elevated compared with other MIC groups. There is only one strain of MIC32, but it can be seen that the expression level of the three efflux pumps in this strain is not very high, and the difference is not statistically significant compared to the other strains. Therefore, we speculate that CCCP may have a better inhibitory response to adeABC. Moreover, the RND efflux pumps primarily mediates low-level tigecycline resistance in our strains. Overall, overexpression of the RND efflux pumps and the reversal of tigecycline resistance by CCCP in this study confirm that tigecycline resistance in A. baumannii is associated with an increase in efflux pump expression activity.

In recent years, mechanisms other than the RND efflux pumps that lead to decreased tigecycline sensitivity have been successively reported. Mutations in the rpsJ gene encoding the 30S ribosomal protein S10 can also lead to tigecycline resistance by modifying the tigecycline binding site in the ribosome [15]. Hua X et al. found that the rrf H33P mutation in tigecycline-treated strain XH1457 resulted in reduced ribosome recycling factor (RRF) expression, which in turn led to tigecycline resistance [16]. However, no mutations of the above related genes have been detected in this experiment. Chen et al. found a deletion mutation in the trm gene by whole genome sequencing of the tigecycline-resistant strain 19606-T8. They verified that the mutation in the trm gene led to a decrease in tigecycline sensitivity, while the wild-type trm gene restored tigecycline sensitivity[14]. All of the TNAB strains in our study had 240 nucleotide deletion mutations in the trm gene, which is in line with the findings of the previous investigation[22]. We hypothesized that this trm deletion mutation may be the result of tigecycline selective pressure in the clinical setting and may not be an independent induction mechanism for tigecycline resistance in strains that do not overexpress efflux pumps. Furthermore, the flavin-dependent monooxygenase encoded by the tet(X) gene and its variants can modify tigecycline, resulting in the development of tigecycline resistance. Recently, the plasmid-mediated tet(X3) and tet(X4) genes, have been reported to be associated with a high-level of tigecycline resistance in A. baumannii[23]. Meanwhile, Yu-Chia Hsieh et al. identified the tet(X6) gene in humans for the first time, which is the first report of tet(X) gene variants in Taiwan[11]. However, no strains in this study were found to harbor the tet(X) gene, and there have been no outbreaks of high-level tigecycline resistance A. baumannii at our hospital within the past three years.

Molecular typing revealed that the dominant sequence type of TNAB isolates in our hospital was ST195 (35.3%), followed by ST208 (17.7%), which aligns with previous study that identified ST208 and ST195 as the prevailing epidemic types of MDRAB in China[24–27]. Our study reports for the first time the clinical infections with TNAB of sequence types ST1849, ST540, ST938, ST381, and ST369. In addition, no outbreaks of the ST1849 clone have been reported in any country or region, except for one case registered at PubMLST in China, and there is no relevant literature. We need to be alert to the prevalence of these rarer sequence types, even though they all belong to CC92, which is the dominant clonal group in China.

The TNAB isolates, which were mainly detected in our hospital's ICU, showed substantial clonal relatedness and antibiotic resistance. Therefore, regular epidemiologic surveillance and rational use of antibiotics should be put into place to prevent the pandemic development of TNAB. The upregulation of the RND efflux pumps is a critical mechanism underlying the low-level tigecycline resistance of A. baumannii in our hospital. Additional investigation is required to identify alternative efflux pumps linked to tigecycline MIC that can be counteracted by CCCP.

Funding This study was supported by the National Natural Science Foundation of China (81572055).

Competing Interests All of the authors declare no conflict of interest in this study.

Author Contributions Yun Xia and Jiajia Liao contributed to the study conception and design. Material preparation, data collection and were performed by Shengli Luo, Shiyu Tang, Yuqiong Li, and Qi Han. Data analysis and curation were conducted by Jinzhu Huang, Peiwen Xia, Bingxue Yang, and Zijun Dang. All authors have read and agreed to the published version of the manuscript.

Data Availability The datasets in the current study are available from the corresponding author upon reasonable request.

Institutional Review Board Statement The study was conducted in accordance with the Declaration of Helsinki and approved by the medical center's ethical committee of the First Affiliated Hospital of Chongqing Medical University.

Consent to participate Not applicable.

Consent for publication Not applicable.

Acknowledgments This study was supported by the National Natural Science Foundation of China (81572055).

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The authors declare no competing interests.

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Molecular Epidemiology and Resistance Mechanisms of Tigecycline-Non-Susceptible A. baumannii Isolated from a Tertiary Care Hospital in Chongqing, China

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Abstract

Objectives

Methods

Results

Conclusion

Figures

Introduction

Materials and Methods

Bacterial isolates and species identification

Antimicrobial susceptibility testing and phenotypic inhibition experiments

Multi-locus sequence typing analysis

Molecular detection of resistance genes

Quantitative real-time PCR (qPCR)

Data analysis

Results

Bacterial isolates and species identification

Antimicrobial susceptibility testing and efflux pump activity determination

Molecular epidemiology based on MLST

Analysis of resistance determinants in TNAB Strains

Relative gene expression of RND efflux systems

Discussion

Conclusion

Declarations

References

Additional Declarations

Supplementary Files

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