Development of In Vitro resistance to fluoroquinolones in Pseudomonas aeruginosa

doi:10.21203/rs.3.rs-19662/v1

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Development of In Vitro resistance to fluoroquinolones in Pseudomonas aeruginosa

https://doi.org/10.21203/rs.3.rs-19662/v1

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published 05 Aug, 2020

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Background: The objectives of this study were to investigate the dynamics of different resistant mechanisms in P.aeruginosa populations that have evolved under fluoroquinolone pressure, and any interactions between these mechanisms in the evolutionary trajectories.

Methods: In this study, bacteria of the strain ATCC27853 were selected under different concentrations of levofloxacin and ciprofloxacin for six parallel lineages. The four target genes in the quinolone-resistance determining region were amplified and then Sanger sequencing was used to find the mutations. The expression of four efflux pump proteins were evaluated by real-time PCR, using the relative quantitation method, and the ATCC27853 was selected as a control.

Results: we found that the P.aeruginosa was killed by ciprofloxacin earlier than levofloxacin. We found five different mutations in three subunits of QRDRs in our study; gyrA was the main mutated gene for conferring resistance to fluoroquinolone. A greater number of mutations appeared at 4mg/L for levofloxacin and at 2mg/L for ciprofloxacin. The main efflux pump that was expressed was MexCD-OprJ, and the first over expressed was evident at 0.5mg/L for levofloxacin and 0.25mg/L for ciprofloxacin.

Conclusions: The mutation of gyrA⁸³and overexpression of MexCD-OprJ were the main mechanisms that conferred resistance of P.aeruginosa to levofloxacin and ciprofloxacin. Ciprofloxacin had a stronger ability to kill the bacteria, while may render bacteria more susceptible to resistance.

General Microbiology

Infectious Diseases

Fluoroquinolone

Pseudomonas aeruginosa

Efflux pumps

Levofloxacin

Ciprofloxacin

Pseudomonas aeruginosa (P.aeruginosa) is a Gram-negative opportunistic human pathogen that can cause patients suffering from cystic fibrosis (CF) to become immune-compromised, occasionally leading to death[1]. Fluoroquinolones (FQ), which have favorable pharmacokinetic/pharmacodynamic properties are one of the major classes of antibiotics used in the treatment of infections caused by P.aeruginosa. P.aeruginosa has a large variety of available resistance mechanisms, and these may act in combination, rendering even the most potent agents useless[2].

The most prevalent mechanisms contributing to FQ resistance in P.aeruginosa involve mutations in the quinolone-resistance determining regions (QRDRs), for example, gyrA and gyrB in DNA gyrase and parC and parE in topoisomerase IV, along with overexpression of Resistance-Nodulation-Division efflux pumps (namely MexAB-OprM, MexCD-OprJ, MexEF-OprN, and MexXY-OprM)[3]. Epidemiological analyses have indicated that high-level resistance to FQ requires QRDR mutations in at least two genes[4], and overexpression of the MexEF-OprN efflux pump represents a major mechanism by which P.aeruginosa may acquire higher resistance levels to FQs[5].

Microbial resistance is an evolutionary response to antibiotic pressure. Evolutionary steps result in the alteration of drug susceptibility to clinical resistance. Microbial evolution experiments comprise a powerful approach to examine pathogen adaptation to antibiotics during the evolutionary process in real-time and under highly controlled laboratory conditions[4]. Recent studies have established causal links between antibiotic deployment therapies and the course and timing of mutations, the cost of resistance and the likelihood of developing compensating mutations[6]. The main limitation of experimental animal models to investigate resistance mechanisms is the risk of spreading the plasmid to the surroundings. In addition, a combination of drugs is usually used in the therapy, which may raise a problem for our experiments investigating the mechanisms of the FQ-resistance of P.aeruginosa. For all of the above reasons, the main purpose of this study was to develop an understanding of chromosome-mediated resistance using an in vitro selection model to block the communication between P.aeruginosa and the environment.

Bacterial strains, growth conditions and MIC determinations

For in vitro resistance selection studies, the P.aeruginosa strain, ATCC 27853, from the American Type Culture Collection was used, bacteria were grown in Mueller-Hinton broth (MHB) or on Mueller-Hinton agar (MHA) plates for18-24 h in a 5% CO₂ atmosphere at 37℃.Antibiotic susceptibility to the levofloxacin and ciprofloxacin was determined by the Agar Dilution Method in accordance with the Clinical & Laboratory Standards Institute (CLSI) approved standards.

Selection of FQ-resistant P.aeruginosa populations by serial passages

Six independent lineages (Lineages 1–6, L1-L6) for levofloxacin and ciprofloxacin were established and propagated by serial passages with increasing concentrations of antibiotics. The selection of 200 µl bacteria was performed in flasks containing 20 mL Mueller-Hinton broth (MHB) with various concentration gradients of levofloxacin and ciprofloxacin. Bacteria were incubated for 24 h at 37℃in the flasks and the resistant bacteria were serially transferred into new flasks containing fresh medium with 2-fold serial antibiotic concentrations. The procedure was repeated until growth was obviously inhibited. At each selection passage, a sample of the bacterial population from each passage was collected with 15% sterilized glycerol and frozen in cryotubes at -80℃.

Bacterial DNA Extraction

Bacterial DNA extracts used as templates in this study were prepared by the heat boiling method. Bacterial suspensions were centrifuged at 12,000 rpm for 10 min to collect bacteria. The collected bacteria were re-suspended in 500 µl sterilized water and incubated at 100 °C for 10 min. The cooling mixture were centrifuged at 12,000 rpm for 5 min. Supernatants were collected and stored at − 20 °C until required for use.

Gene Amplification and DNA gyrase and topoisomerase IV Sanger Sequencing

The QRDRs of the gyrA, gyrB, parC and parE genes of P.aeruginosa populations by serial passages were amplified by PCR using gene-specific primers, as summarized in Table 5. To amplify each gene, 1 µg of the DNA template was subjected to PCR amplification in a 30 µl reaction mixture containing 12 µM of each forward and reverse primers, 7.5 µM of dNTPs, 3 µl 10 × Ex Taq buffer (plus MgCl₂) and 1.5 U of Ex Taq DNA polymerase (TaKaRa BIO INC). PCR amplification was carried out using the following conditions:(1) predenaturation at 94 °C for 5 min, 36 cycles of amplification (94 °C,30sec, 58 °C,30sec, 72 °C,30sec) and a final extension at 72 °C for 7 min for gyrA and gyrB amplification.(2) predenaturation at 94 °C for 5 min, 40 cycles of amplification (94 °C,30sec, 60 °C,30sec, 72 °C,30sec) and a final extension at 72 °C for 7 min for parC amplification.(3) predenaturation at 94 °C for 5 min, 36 cycles of amplification (94 °C,30sec, 70 °C, 30sec, 72 °C, 30sec) and a final extension at 72 °C for 7 min for parE amplification. Amplified PCR products of both genes were visualized on 2% agarose gels containing GoodView ^TM (Beijing SBS Genetech Co.) with a DNA marker (TaKaRa BIO INC)under UV light. Amplified PCR products were sent to the Beijing Genomics Institute to perform a sequence analysis using the Sanger method with the same PCR primers. Nucleotide sequences of genes were compared with the corresponding reference sequence of P.aeruginosa ATCC27853 used in this study.

Evaluation of efflux pump expression

The expression levels of mexA, mexC, mexE, mexX, and the housekeeping gene rpoD were determined by quantitative real-time reverse transcription-PCR (qRT-PCR). RNA was isolated by using the RNAiso reagent (TaKaRa), according to the manufacturer’s instructions. cDNA was synthesized by using the PrimeScript^TMRT reagent Kit with gDNA Eraser (TaKaRa), according to the manufacturer’s instructions. qRT-PCRs were performed in duplicate in a 20 µl volume with 100 ng RNA and a primer concentration of 0.4 µM on a QuantStudio™ DX Flex System (ABI) using the SYBR®Premix Ex Taq^TMⅡ (TaKaRa). The primers for qRT-PCRs are summarized in Table 6.Gene expression was calculated using the ΔΔCt method and a standard curve to measure PCR efficiency. All results were normalized to the expression of the housekeeping gene rpoD and were calibrated relative to expression in P.aeruginosa ATCC27853 used in this study.

Minimum inhibitory concentration (MIC) determination and selection of fluoroquinolone-resistant P.aeruginosa populations

According to the CLSI in 2015, the MIC of P.aeruginosa ATCC27853 used in this study was 0.5 mg/L. Six independent lineages were propagated from P.aeruginosa ATCC27853 by serial passages in liquid medium with increasing concentrations of levofloxacin and ciprofloxacin, until we found that for levofloxacin, L2 was inhibited at 64 mg/L (128MIC) and L6 was inhibited at 32 mg/L (64MIC), while L5 was killed at 32 mg/L (64MIC). We thus defined 32 mg/L (64MIC) as the end point, so each lineage was shown to have eight independent passages (P1-P8) that could survive in different levofloxacin concentrations from 0.5MIC-64MIC. For different passages of six lineages selected from ciprofloxacin, we found that L5 and L6 died at 4 mg/L (8MIC), while L2 and L3 were killed at 8 mg/L (16MIC); L1 and L4 could be significantly suppressed at 32 mg/L (64MIC), which was thus used as the end point of the ciprofloxacin selection. We found that P.aeruginosa is able to survive for longer in levofloxacin selection than ciprofloxacin, which may demonstrate that the ciprofloxacin is more effective in killing the P.aeruginosa.

Determination of DNA gyrase and topoisomerase IV sequences

We sequenced the QRDRs of gyrA, gyrB, parC and parE in each passage of six different lineages of evolved resistant P.aeruginosa. Three different mutations were found in different samples in levofloxacin-resistant lineages (Table 1); firstly, the second position of codon 83 of gyrA (gyrA⁸³), leading to the replacement of the threonine residue 83 by an isoleucine (T83I, ACC→ATC); secondly, the second position of codon 466 of gyrB (gyrB⁴⁶⁶), leading to the replacement of the serine residue 466 by a phenylalanine (S466F, TCC→TTC); thirdly, the first position of codon 457 of parE (parE⁴⁵⁷), leading to the replacement of the serine residue 457 by a cysteine (S457C, AGC→TGC). Regarding ciprofloxacin, the results were different (Table 2). We found four different mutations in different six lineages, but there were three mutations in gyrA and the fourth mutation was the same as the one we found in the experiment of levofloxacin in gyrB⁴⁶⁶. The three mutations in gyrA were gyrA⁸³, which was the same as that found in the passages selected from levofloxacin, the other two, which were found in the ciprofloxacin selection experiment, were at the first position of codon 87 of gyrA(gyrA⁸⁷), leading to the replacement of the aspartic acid residue 87 by an asparagine (D87N, GAC→AAC),and the second position of codon 87 of gyrA(gyrA^87*), leading to the replacement of the aspartic acid residue 87 by glycine (D87G, GAC→GGC). Interestingly, we found that gyrA⁸³ could help P.aeruginosa survive longer under the ciprofloxacin pressure than the lineages with gyrA⁸⁷ mutations only.

Table 1

Mutations in the QRDR genes in the evolved resistant *P.aeruginosa* selected from levofloxacin
Passage	Lineage1	Lineage2	Lineage3	Lineage4	Lineage5	Lineage6
0. (0MIC)	—	—	—	—	—	—
1. (1/2MIC)	—	—	—	—	—	—
2. (1MIC)	A	—	—	A	—	—
3. (2MIC)	A	—	—	A	B	—
4. (4MIC)	A	—	—	A	B	—
5. (8MIC)	A + E	A	A + B	A	B	B
6. (16MIC)	A + E	A	A + B	A	B	B
7. (32MIC)	A + E	A	A	A	B	A + B
8. (64MIC)	A + E	A	A	A	B	A
A: gyrA⁸³ (T83I, ACC→ATC);B: gyrB⁴⁶⁶ (S466F, TCC→TTC);E: parE⁴⁵⁷ (S457C, AGC→TGC)

Table 2

Mutations in the QRDR genes in the evolved resistant *P.aeruginosa* selected from ciprofloxacin
Passage	Lineage1	Lineage2	Lineage3	Lineage4	Lineage5	Lineage6
0. (0MIC)	—	—	—	—	—	—
1. (1/2MIC)	—	—	—	—	—	—
2. (1MIC)	—	A87 + B	A87	—	—	—
3. (2MIC)	A83	A87 + B	A87	—	—	—
4. (4MIC)	A83	A87 + B	A87	A83	—	A87*
5. (8MIC)	A83	A87 + B	A87	A83	—	A87*
6. (16MIC)	A83	A87	A87 + B	A83	/	/
7. (32MIC)	A83	/	/	A83	/	/
8. (64MIC)	A83	/	/	A83	/	/
A83: gyrA⁸³ (T83I, ACC→ATC); A87: gyrA⁸⁷(D87N, GAC→AAC);A87: gyrA⁸⁷(D87G, GAC→GGC);B: gyrB*⁴⁶⁶ (S466F, TCC→TTC)

Here, we found that the gyrA mutations (gyrA⁸³, gyrA⁸⁷, gyrA^87*) were the most important mutations for P.aeruginosa, while the gyrB⁴⁶⁶ mutation and the parE⁴⁵⁷ mutation were just combined with the gyrA mutations. We also discovered an interesting phenomenon that the gyrB⁴⁶⁶ mutation disappeared at a high level of selection concentration. Moreover, in the levofloxacin selection, a large number of genetic mutations were found at 4 mg/L (8MIC), while it was 2 mg/L (4MIC) for ciprofloxacin. This suggests that for P.aeruginosa, ciprofloxacin has a stronger ability to select resistant bacteria.

Evaluation of efflux pump expression

We quantified the transcription of genes encoding efflux pump proteins MexA, MexC, MexE, and MexX. The results of the median of gene expression analysis in eight passages of six lineages selected by levofloxacin are shown in Table 3, while Table 4 display the results of the selections by ciprofloxacin. For P.aeruginosa, overexpression of MexC was the most important mechanism that contributed to the fluoroquinolone resistance, while MexE and MexX played a supplementary role (Fig. 1). For the levofloxacin selection experiment, the first significant overexpression for these three genes were at 0.5 mg/L (1MIC),and 0.25 mg/L (1/2MIC) in the ciprofloxacin selection experiment. Moreover, the second significant overexpression was found at 1 mg/L (2MIC) for MexC in the ciprofloxacin selection experiment and at 2 mg/L (4MIC) for levofloxacin. These results suggest that ciprofloxacin had a stronger effect on inducing resistance in P.aeruginosa than levofloxacin.

Table 3

Efflux pump gene expression in the evolved resistant *P.aeruginosa* selected from levofloxacin*
Passage	mexA	mexC	mexE	mexX
0. (0MIC)	1.0000	1.0000	1.0000	1.0000
1. (1/2MIC)	0.6700	2.6295	1.4200	1.3850
2. (1MIC)	1.2310	8.6650	5.4625	2.2925
3. (2MIC)	0.9325	16.2745	8.3645	1.2030
4. (4MIC)	0.8605	152.1040	2.6225	3.0995
5. (8MIC)	1.2310	52.7525	12.0805	1.3095
6. (16MIC)	1.0385	57.6390	15.4695	2.0900
7. (32MIC)	1.4255	145.8855	10.5065	4.6735
8. (64MIC)	1.5140	74.2490	12.8310	7.5555
* Relative expression quantities were represent by the median of six lineages

Table 4

Efflux pump gene expression in the evolved resistant *P.aeruginosa* selected from ciprofloxacin*
Passage	mexA	mexC	mexE	mexX
0. (0MIC)	1.0000	1.0000	1.0000	1.0000
1. (1/2MIC)	1.3075	9.0280	11.9735	2.3705
2. (1MIC)	0.6615	9.9210	10.1515	1.3515
3. (2MIC)	1.0715	144.4805	6.5980	2.1285
4. (4MIC)	0.8040	11.4565	6.3370	1.4735
5. (8MIC)	0.6125	86.9080	5.0330	1.7635
6. (16MIC)	0.8320	195.6205	8.3245	3.5535
7. (32MIC)	0.6140	132.7665	7.0495	3.5345
8. (64MIC)	0.7175	112.6305	10.0960	4.5755
* Relative expression quantities were represent by the median of six lineages

Correlation between each mutation and efflux pump gene expression

Several significant correlations were found between QRDR mutations and efflux pump expression. Firstly, if there was a gyrA⁸³ mutation in lower selected concentrations, such as L1 and L4 for levofloxacin, and L1 for ciprofloxacin (Fig. 2), we observed that gene expressions of the four different efflux pumps were down-regulated, but the gyrA⁸⁷mutation did not have the same effect. Secondly, the presence of the parE⁴⁵⁷ mutation in L1 for levofloxacin was concomitant with down-regulation in the expression of mexC and mexE. Finally, in the L3, L5 and L6 that were selected from levofloxacin, before the appearance of the gyrA⁸³ mutation or with the gyrB⁴⁶⁶ mutation alone, or with no mutation, such as in L5 for ciprofloxacin (Fig. 3),there was a synergistic interaction between mexC and mexE, which means that when mexC was up-regulated, there was a down-regulation of mexE.

Microbial evolution experiments are powerful tools for investigating bacterial adaptation to antibiotic stress under controlled laboratory conditions and are commonly used to assess the causes and dynamics of the evolution of antibiotic resistance in bacteria[7]. According to our previous experiments in a lung infection model in rats, we were only able to find plasmid-mediated resistance instead of QRDR mutations, which were highly detected in clinical studies[8]. In this study, we developed resistance of P.aeruginosa in liquid medium, in vitro, under sub-lethal concentrations of antibiotics 0.5-fold MIC of the parental strain (ATCC27853) to 64-fold MIC, which is completely different from the stepwise selection of resistant mutants in animal models; we were effectively able to block the communication between P.aeruginosa and the environment, and thus focus on the development of P.aeruginosa resistance under increasing antibiotic pressure. Furthermore, in previous reports, many colleagues used agar plates as their medium and then chose a single colony for subsequent passages and sequencing[9]; this method could exclude the emerging mutations which existed in the minorities[10]. Therefore, we incubated bacteria in liquid medium and extracted DNA and RNA from the collected liquids containing the resistant bacteria obtained from the various resistant mechanisms[4], as previously demonstrated in finding the number of QRDR mutations that increased progressively in Streptococcus pneumoniae. This method also helped us detect the low frequency mutation in the process of evaluation, such as parE^{45 7}(S457C) and gyrB⁴⁶⁶ (S466F).

We found five different mutations in QRDR of which three were consistent with the published results of clinical detection in P.aeruginosa-resistant populations[3, 11], namely gyrA⁸³ (T83I), gyrA⁸⁷ (D87N) and gyrB⁴⁶⁶ (S466F); we detected the replacement of the serine residue 457 by a cysteine in parE, which was similar to the clinical results showing an alteration arginine[12]. However, we did not find a parC mutation, which is different from the clinical results, but this was also demonstrated by a study that sequenced both the clinical isolates and the laboratory-derived mutations of Mycoplasma bovis, showing different abilities in the development of resistance[13]. We speculate that the parC mutation could be related to the drug combination or the hospital environment, which is why we used the in vitro model to investigate the mechanisms of P.aeruginosa resistance to overcome the uncertainty of clinical treatment.

Our results showed that the evolutionary trajectories of fluoroquinolone resistance are more complicated than previously described. Firstly, ciprofloxacin and levofloxacin develop resistance through different mechanisms. In our experiments, mutation in gyrA⁸³ occurred in five lineages for levofloxacin and two lineages for ciprofloxacin, while there were two kinds of gyrA⁸⁷ mutations only under the ciprofloxacin pressure, and the three different gyrA mutations did not appear at the same time. The mutation that occurred in parE was only found in one lineage that evolved under levofloxacin pressure and was found in combination with gyrA⁸³; the mutation in gyrB was always found in combination with the gyrA mutations, and could also disappeared in the evolutionary process. These experiment phenomena were coincident with the clinical results showing that the gyrA mutation is the main resistance mechanism of P.aeruginosa, while the gyrB mutation can reinforce the resistant abilities, and it was rare to find parE mutations[11, 14]. Our study showed that the main mechanism contributing to the resistance of P.aeruginosa, which involved efflux pump expression, was overexpression of MexCD-OprJ, and MexEF-OprN and MexXY-OprM were also up-regulated. These results were in accordance with the clinical detection[12, 15, 16], so our study verified the clinical phenomenon in vitro.

When we analyzed the lineages separately from the perspective of the two different mechanisms, we found a relationship between mutations and overexpression of efflux pumps, in that the gyrA⁸³ mutation could down-regulate the expression of mexC, while gyrA⁸⁷ and gyrB⁴⁶⁶ could not, and mexC and mexE may cooperate with each other.

Although levofloxacin and ciprofloxacin are both FQ, they have different abilities in their bactericidal action and induction of resistance. Our study showed that ciprofloxacin had a stronger ability to kill the bacteria, while the mutations and overexpression of efflux pumps suggest that it may render bacteria more susceptible to resistance.

QRDR: Quinolone-resistance determining region; CF: Cystic fibrosis; FQ: Fluoroquinolones; MHA: Mueller Hinton agar; MHB: Mueller Hinton broth; CLSI: Clinical & Laboratory Standards Institute; MIC: Minimum inhibitory concentration;

Ethics approval and consent to participate

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Consent for publication

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Availability of data and materials

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

Competing interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

This study was funded by Natural Science Foundation of Liaoning Province, China (Funding Number: CA57).

Authors’ contributions

LZ, SW, XL, XH and LJ all contributed to the design and conception of the study. LZ, SW and XL primarily completed data collection. LZ, SW, XH and LJ were involved in the analysis. LZ, SW and XH prepared the initial draft with all authors contributing to critical review. All authors approved the final manuscript.

Acknowledgements

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Development of In Vitro resistance to fluoroquinolones in Pseudomonas aeruginosa

Status:

Journal Publication

Version 1

Abstract

Figures

Background:

Materials And Methods

Bacterial strains, growth conditions and MIC determinations

Selection of FQ-resistant P.aeruginosa populations by serial passages

Bacterial DNA Extraction

Gene Amplification and DNA gyrase and topoisomerase IV Sanger Sequencing

Evaluation of efflux pump expression

Results

Minimum inhibitory concentration (MIC) determination and selection of fluoroquinolone-resistant P.aeruginosa populations

Determination of DNA gyrase and topoisomerase IV sequences

Evaluation of efflux pump expression

Correlation between each mutation and efflux pump gene expression

Discussion

Abbreviations

Declarations

References

Supplementary Files

Status:

Journal Publication

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