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Influence of PhoPQ and PmrAB two component system alternations on colistin resistance from non-mcr colistin resistant clinical E. Coli strains

BMC Microbiology volume 24, Article number: 109 (2024) Cite this article

Abstract

Background

The current understanding of acquired chromosomal colistin resistance mechanisms in Enterobacterales primarily involves the disruption of the upstream PmrAB and PhoPQ two-component system (TCS) control caused by mutations in the regulatory genes. Interestingly, previous studies have yielded conflicting results regarding the interaction of regulatory genes related to colistin resistance in Escherichia coli, specifically those surrounding PhoPQ and PmrAB TCS.

Results

In our study, we focused on two clinical non-mcr colistin-resistant strains of E. coli, TSAREC02 and TSAREC03, to gain a better understanding of their resistance mechanisms. Upon analysis, we discovered that TSAREC02 had a deletion (Δ27–45) in MgrB, as well as substitutions (G206R, Y222H) in PmrB. On the other hand, TSAREC03 exhibited a long deletion (Δ84–224) in PhoP, along with substitutions (M1I, L14P, P178S, T235N) in PmrB. We employed recombinant DNA techniques to explore the interaction between the PhoPQ and PmrAB two-component systems (TCSs) and examine the impact of the mutated phoPQ and pmrB genes on the minimum inhibitory concentrations (MICs) of colistin. We observed significant changes in the expression of the pmrD gene, which encodes a connector protein regulated by the PhoPQ TCS, in the TSAREC02 wild-type (WT)-mgrB replacement mutant and the TSAREC03 WT-phoP replacement mutant, compared to their respective parental strains. However, the expressions of pmrB/pmrA, which reflect PmrAB TCS activity, and the colistin MICs remained unchanged. In contrast, the colistin MICs and pmrB/pmrA expression levels were significantly reduced in the pmrB deletion mutants from both TSAREC02 and TSAREC03, compared to their parental strains. Moreover, we were able to restore colistin resistance and the expressions of pmrB/pmrA by transforming a plasmid containing the parental mutated pmrB back into the TSAREC02 and TSAREC03 mutants, respectively.

Conclusion

While additional data from clinical E. coli isolates are necessary to validate whether our findings could be broadly applied to the E. coli population, our study illuminates distinct regulatory pathway interactions involving colistin resistance in E. coli compared to other species of Enterobacterales. The added information provided by our study contribute to a deeper understanding of the complex pathway interactions within Enterobacterales.

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Background

The global emergence of colistin-resistant Enterobacterales in humans and animals has been observed in recent years owing to extensive colistin use in human and veterinary medicine [1]. Both chromosome- and plasmid-mediated colistin resistance mechanisms have been reported among colistin-resistant Enterobacterales [2]. While chromosome-encoded resistance mechanisms cannot be transferred between organisms, mutations in the core genome may lead to high genetic stability. This situation can pose a significant health concern when chromosomal mutations accumulate in major human pathogenic bacteria [3, 4]. The current understanding of acquired chromosomal colistin resistance mechanisms in Enterobacterales is primarily associated with the upregulation of the pmrHFIJKLM and pmrCAB operons leading to the enhanced modification of lipopolysaccharides (LPS) and development of colistin resistance [1]. This resistance mechanism is believed to be linked to disruptions in the upstream PmrAB and PhoPQ two-component systems (TCSs) controls. Critical genetic changes in the regulatory genes of these systems play an important role in the development of colistin resistance [5,6,7,8,9,10,11]. Notably, the interaction of regulatory genes surrounding PhoPQ and PmrAB TCSs related to colistin resistance varies across species of Enterobacterales [12, 13]. In the case of Escherichia coli, regulatory networks controlling the PhoPQ and PmrAB TCSs related to colistin resistance still need to be elucidated as previous studies have yielded conflicting results [13, 14].

In our previous study, we collected 11 mcr-negative colistin-resistant E. coli strains to investigate their chromosomal resistance mechanisms. We successfully confirmed that the R81H substitution in PmrA contributed to colistin resistance in three isolates. In contrast, the exact resistance mechanism to colistin is yet to be determined in the remaining eight isolates (TSAREC02, TSAEC03, TSAREC05, TSAREC06, TSAREC07, TSAREC08, TSAREC10, and TSAREC37), all of which harbored substitutions in PmrB [15]. As PhoPQ TCS activation due to mutated regulatory genes conferring colistin resistance has been reported in Klebsiella pneumoniae, we inferred this might also be true for E. coli [10, 11]. After comparing the regulatory genes mgrB, phoP and phoQ from the remaining eight E. coli isolates with those from colistin-susceptible isolates, we uncovered additional unique amino acid deletions in MgrB and PhoP from TSAREC02 and TSAREC03, respectively. To further investigate and validate our hypothesis, we generated different mutants of TSAREC02 and TSAREC03 to assess the impact of the mutations in mgrB and phoP on colistin resistance. This aspect has not been previously addressed yet. Additionally, we re-evaluated the influence of the individual mutated pmrB genes from TSAREC02 and TSAREC03 on colistin resistance using the respective mutants of TSAREC02 and TSAREC03. This approach differed from the previous study which utilized the MG16655 mutant with PmrB deletion as recipients [15].

Methods

Bacterial strains, plasmids, and antimicrobial susceptibility testing

The clinical colistin-resistant strains TSAREC02 and TSAREC03 were obtained from a previously described nationwide surveillance program and their relevant antimicrobial susceptibility data were collected as previously reported [15]. To determine the colistin minimum inhibitory concentrations (MICs) and breakpoints in the current study, we adhered to the broth microdilution method according to the guidelines provided by the international susceptibility testing committee, specifically the European Committee on Antimicrobial Susceptibility Testing 2021. For further information on the E. coli strains, plasmids, and primers used in this study, please refer to Supplementary Table S1, S2, and S3, respectively.

PCR (polymerase chain reaction amplification) and sequencing of genes involved in colistin resistance

In addition to pmrA and pmrB, the genes phoP, phoQ, and mgrB from TSAREC02 and TSAREC03 were amplified and sequenced using the appropriate primers (Supplementary Table S3) [16]. As described previously, the sequences of phoP, phoQ, and mgrB from TSAREC02 and TSAREC03 were compared to those of the wild type (WT) E. coli reference strain MG1655 and eight colistin-sensitive clinical E. coli strains (ECS01, ECS02, ECS03, ECS04, ECS05, ECS06, ECS07, and ECS08) to exclude possible synonymous polymorphisms. This enables the identification of any unique amino acid substitutions that may contribute to colistin resistance in TSAREC02 and TSAREC03. Sequence comparisons was analyzed on the National Center for Biotechnology Information website (www.ncbi.nlm.nih.gov). The Basic Local Alignment Search Tool was used for this analysis [15]. Protein Variation Effect Analyzer (https://www.jcvi.org/research/provean) software was used to predict whether the unique amino acid substitutions found in PhoP, PhoQ, and MgrB would affect the function of these proteins.

Construction of the mgrB or pmrB deletion mutant of TSAREC02, as well as the phoP or pmrB deletion mutants of TSAREC03

The generation of the mgrB deletion mutant of TSAREC02 and the phoP deletion mutant of TSAREC03 were carried out utilizing plasmid-based gene knockout methods with a suicide vector called pUT-KB plasmid [17]. The protocol for generating these mutants followed a similar approach as previously described but with minor modifications [15]. For the creation of the mgrB deletion mutant from TSAREC02, a two-step PCR approach was employed. Two PCR products were initially generated using primer sets KOmgrB_F-1/KOmgrB_R-1 and KOmgrB_F-2/KOmgrB_R-2. These PCR products contained complementary ends that would later be used for overlap PCR. To generate the final deletion fragment, the two PCR products with complementary ends were combined as templates and amplified using KOmgrB_F-1 and KOmgrB_R-2 in an overlap PCR reaction. This resulted in the creation of a 1,667-bp fragment lacking the mgrB gene with 15 nucleotides complementary to a PfoI-digested linear pUT-KB plasmid on both sides. The resulting gel-purified PCR then were cloned into a PfoI-digested linear pUT-KB plasmid with the In-Fusion HD cloning kit (TaKaRa Bio, Japan) according to the manufacturer’ s instructions. Thereafter, the final plasmid was constructed named pUT-KB-KOmgrB. The plasmid pUT-KB-KOmgrB then was transformed into E. coli S17-1 λpir through the process of electroporation. Subsequently, this plasmid was mobilized into TSAREC02 through conjugation. The transconjugants were screened on Luria-Bertani (LB) agar supplemented with kanamycin (50 µg/mL) and colistin (4 µg/mL). The selected transconjugants then were further incubated in 20 mL of brain-heart infusion broth for 6 h without kanamycin. Subsequently, they were spread onto LB agar containing 10% w/v sucrose. After the double-crossover events had occurred, sucrose-resistant and kanamycin sensitive colonies were selected (indicating loss of the plasmid). The mgrB deletion mutants of TSAREC02, hereafter, was referred to as TSAREC02_ΔmgrBΔ43–47. As for phoP deletion mutant construction from TSAREC03, the process is slimier to the previous steps described for the mgrB deletion mutant, with the main difference being the primer sets used. The phoP deletion mutant therefore was referred to as TSAREC03_ΔphoPΔ84–224. The pmrB deletion mutants from TSAREC02 and TSAREC03 were constructed as our reported study with some modification [4]. As for the pmrB deletion mutant of TSAREC02/TSAREC03, the strain TSAREC02/TSAREC03 was used as the template. Two PCR products were created using the primer sets KOpmrB_XbaI-F/ KOpmrB-R and KOpmrB-F/ KOpmrB_BcuI-R. The two PCR products contained complementary ends and were mixed as a template for the subsequent overlap PCR step. Using the primers KOpmrB_XbaI-F and KOpmrB_BcuI-R, the mixed PCR products were amplified again, resulting in a fragment with a 1088 bp deletion in the pmrB gene through overlap PCR. The resulting PCR fragment was subjected to digestion with XbaI and BcuI enzymes. Simultaneously, the pUT-KB plasmid was also digested with XbaI and BcuI. The digested PCR fragment was then inserted into the digested pUT-KB plasmid, resulting in the construction of the pUT-KB-KOpmrB plasmid. Further steps involving the homologous recombination method to construct the pmrB deletion mutant of TSAREC02/TSAREC03 were the same as TSAREC02_ΔmgrBΔ43–47 and TSAREC03_ΔphoPΔ84–224. The pmrB deletion mutants from TSAREC02 and TSAREC03 were referred to as TSAREC02 _ΔpmrBg616a, t618g, t664c and TSAREC03_ΔpmrBg3c, t41c, c532t, c704a, respectively.

Construction of the TSAREC02 mutant with a WT-mgrB replacement and the TSAREC03 mutant with a WT-phoP replacement

The mutated mgrB in TSAEC02 and the mutated phoP in TSAREC03 were replaced with WT-pmrB and phoP from E.coli MG1655, respectively. DNA fragments of the mgrB and phoP genes from E. coli MG1655 were amplified via PCR using the primer sets KOmgrB_F-1/KOmgrB_R-2 for mgrB and KOphoP_F-1/KOphoP_R-2 for phoP. The generated PCR fragments were cloned into pUT-KB, resulting in pUT-KB-WT-mgrB and pUT-KB-WT-phoP, respectively. Using a previously described allelic exchange method with a plasmid pUT-KB-WT-mgrB and a pUT-KB-WT-phoP, a TSAREC02 mutant with WT-mgrB replacement (TSAREC02 WT-mgrB revertant) and TSAREC03 mutant with WT-phoP replacement (TSAREC03 WT-phoP revertant) were created from TSAREC02_ΔmgrBΔ43–47 and TSAREC03_ΔphoPΔ84–224, respectively [4].

Complementation of different TSAREC02 and TSAREC03 mutants

The pmrB genes from TSAREC02 and TSAREC03 were amplified using PCR.

The amplified pmrB genes were then directly inserted into the pCRII-TOPO TA vector (Invitrogen, U.S.A). The insertion was done following the protocol provided by the manufacturer. The ligation reaction between the amplified pmrB genes and the pCRII-TOPO vector resulted in the creation of different pCRII-TOPO-derived plasmids. Each generated plasmid containing a specific variant of the pmrB gene were transformed into different TSAREC02 and TSAREC03 mutants via electroporation method. In the current study, to confirm accuracy the target gene with its franking regions in deletion, revertant and complemented mutants, several experiments were performed. First, we employed PCR method using specific primers (see Table S3) to amplify the region of interest. Subsequently, agarose gel electrophoresis was conducted to visualize the amplicons and confirm their sizes as expected. The expected amplicon sizes using primer pairs mgrB-F and mgrB-R from TSAREC02, TSAREC02_ΔmgrBΔ43–47,and TSAREC02 WT-mgrB revertant were 297 bp, 115 and 297 bp, respectively. The expected amplicon size using primer pairs phoP-F and phoP-R from TSAREC03, TSAREC03_ΔphoPΔ84–224 and TSAREC03 WT-phoP revertant were 920 bp, 250 bp, 920 bp, respectively. The expected amplicon sizes using the primer pairs pmrB-F and pmrB-R from TSAREC02 and TSAREC02 _ΔpmrBg616a, t618g, t664c as well as TSAREC03 and TSAREC03_ΔpmrBg3c, t41c, c532t, c704a were 1397 bp and 302 bp, respectively. Additionally, two amplicons with sizes of 302 and 1397 bp from complemented strains were anticipated. These amplicons serve as indicators: the 302 bp fragment suggests the presence of the deleted pmrB gene in the chromosome, while the 1397 bp fragment indicates the intact cloned pmrB gene in the plasmid transformed. Finally, to ensure the accuracy of the results, the amplicons were subjected to sequencing using sequencing primer for validation (see Table S3). The primer pairs M13-F and M13-R designed based on the sequence of the pCRII-TOPO TA vector (Invitrogen, U.S.A) were used to sequence the cloned pmrB from complemented strains.

Real-time quantitative PCR

An increased expression of the pmrHFIJKLM operon in E. coli is associated with colistin resistance [1]. The expression of the pmrK gene, representative of pmrHFIJKLM operon expression, was measured via real-time quantitative PCR method as described previously [15]. Other genes regulating PmrAB and PhoPQ systems which may be related to colistin resistance were also measured. Briefly, Bacterial RNA and cDNA were obtained using an RNeasy mini kit (Qiagen, U.S.A) and Prime Script RT Master Mix (TaKaRa Bio, Japan) according to the manufacturer’s instructions. Expression levels of pmrK, phoP, pmrD, pmrA and pmrB were estimated using SYBR Green PCR Master Mix (Thermo Fisher Scientific, U.S.A). The gapA gene served as an endogenous reference for normalizing expression levels. Primer pairs used were according to previous studies and were listed in Supplementary Table S3 [8, 14, 18, 19]. Expression levels were calibrated against the baseline expression level of E. coli MG1655, and fold change in expression was calculated using the comparative threshold cycle method [20]. Data are expressed as the mean ± standard deviation of four independent experiments (performed in triplicate). A Student’s t-test was used for statistical analysis. The p values < 0.05 were considered statistically significant.

Results

Detection of mgrB and phoP mutations in colistin-resistant E. Coli strains TSAREC02 and TSAREC03, respectively

In addition to pmrA and pmrB, the genes phoP, phoQ, and mgrB from TSAREC02 and TSAREC03, which are thought to be involved in chromosomally encoded colistin resistance in Enterobacterales, were amplified and sequenced [16]. We firstly determined the full nucleotide sequences of mgrB, phoP, and phoQ from the TSAREC02 and TSAREC03 strains and compared them to E. coli strain MG1655 and eight previously described colistin-susceptible strains (ECS01 to ECS08) [4, 15]. Nucleotide variations in mgrB, phoP, and phoQ that produce amino acid substitutions only in TSAREC02, TSAREC03, as well as pmrB, and pmrA reported previously, were shown in Table 1 [15].

Table 1 Amino acid changes in PmrA/PmrB, PhoP/PhoQa, and MgrB in colistin-resistant E. coli strainsa
Full size table

In addition to the amino acid substitutions in PmrB, a deletion of five amino acid residues (QFIPW) in MgrB and a long deletion of 140 amino acid residues in PhoP were observed in TSAREC02 and TSAREC03, respectively. The deletion mutations in MgrB and PhoP were predicted to affect protein function using the Protein Variation Effect Analyzer software (https://www.jcvi.org/research/provean).

The impact of mgrB mutations in TSAREC02 and phoP mutations in TSAREC03 on colistin resistance

Colistin resistance due to mutations in the regulatory genes controlling PhoPQ TCS has been reported in K. pneumoniae [10, 11]. To investigate the effect of mgrB and phoP mutations in TSAREC02 and TSAREC03 on colistin resistance, TSAREC02 mutants (including TSAREC02_ΔmgrBΔ43–47 and TSAREC02 WT-mgrB revertant) and TSAREC03 mutants (including TSAREC03_ΔphoPΔ84–224 and TSAREC03 WT-phoP revertant) were constructed. The amplicons from each constructs formed a distinct band with the expected size after agarose gel electrophoresis in Fig. S1 (A and B). Compared to TSAREC02, no change in colistin MIC values were observed in TSAREC02_ΔmgrBΔ43–47 and TSAREC02 WT-mgrB revertant (Table 2). Similarly, compared to TSAREC03, no change in colistin MIC values was noted in TSAREC03_ΔphoPΔ84–224 and TSAREC03 WT-phoP revertant (Table 3). The expression levels of pmrD, phoP, pmrK, pmrA, and pmrB in different mutants of TSAREC02 and TSAREC03 were shown in Tables 2 and 3; Figs. 1 and 2. As shown in Fig. 1; Table 2, pmrD and phoP expression levels decreased in the TSAREC02 WT-mgrB revertant compared to the TSAREC02 strain. However, the expression levels of pmrK, pmrB, and pmrA in the TSAREC02 WT-mgrB revertant were comparable to those in TSAREC02. As shown in Fig. 2; Table 3, the TSAREC03 WT-phoP revertant showed higher pmrD and phoP expressions compared to TSAREC03. Comparable expression levels of pmrK, pmrA, and pmrB were observed between TSAREC03 and the TSAREC03 WT-phoP revertant. Based on these results, it appears that mgrB/phoP mutations in TSAREC02/TSAREC03 contributing to PhoPQ alteration did not result in changes in PmrAB TCS activity that further influenced downstream pmrHFIJKLM expression levels and colistin MIC values.

Table 2 Colistin MICs and gene expression levels in different E. coli TSAREC02 mutants
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Table 3 Colistin MICs and gene expression levels in different E. coli TSAREC03 mutants
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Fig. 1
figure 1

The mRNA expression levels of pmrD/phoP (A) and pmrK/pmrB/pmrA (B) in TSAREC02 and TSAREC02 WT-mgrB revertants. Values on the y axis are relative expression levels (fold change) normalized against levels in the colistin susceptible strain MG1655. * Significant differences compared to those in TSAREC02 (Student’s t-test, p < 0.05)

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Fig. 2
figure 2

The mRNA expression levels of pmrD/phoP (A) and pmrK/pmrB/pmrA (B) in TSAREC03 and TSAREC03 WT-phoP revertants. Values on the y axis are relative expression levels (fold change) normalized against levels in the colistin susceptible strain MG1655. * Significant differences compared to those in TSAREC03 (Student’s t-test, p < 0.05)

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The impact of pmrB mutations in TSAREC02 and TSAREC03 on colistin resistance

We re-evaluated the effect of amino acid substitutions in PmrB from TSAREC02 and TSAREC03 on colistin resistance using different TSAREC02 and TSAREC03 mutants. The amplicons from different TSAREC02 and TSAREC03 mutants exhibited a distinct band with the expected sizes following agarose gel electrophoresis as shown in Fig. S1 (C and D). As shown in Table 2, the TSAREC02 mutant with a pmrB deletion showed colistin susceptibility, with an MIC of 0.5 µg/mL. Following transformation with a recombinant plasmid containing a parental mutated pmrB, the complemented strain TSAREC02 _ΔpmrBg616a, t618g, t664c (pCRII-TOPOpmrBg616a, t618g, t664c), exhibited restored colistin resistance (colistin MIC 16 µg/mL). Consistent with the changes in colistin MIC levels, the expression levels of pmrA, pmrB, and pmrK increased in the complemented strains TSAREC02 _ΔpmrBg616a, t618g, t664c (pCRII-TOPOpmrBg616a, t618g, t664c) compared to TSAREC02 _ΔpmrBg616a, t618g, t664c (Table 2; Fig. 3).

Fig. 3
figure 3

The mRNA expression levels of pmrB/pmrK in different TSAREC02 mutants. Values on the y axis are relative expression levels (fold change) normalized against levels in the colistin susceptible strain MG1655. * Significant differences compared to those in TSAREC02 (Student’s t-test, P < 0.05). N.D., non-detectable or expression level < 0.01

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In Table 3, the TSAREC03 mutant with a pmrB deletion showed colistin susceptibility (MIC = 0.25 µg/mL) and restored colistin resistance (MIC = 8 µg/mL) after transformation with a recombinant plasmid containing a parental mutated pmrB. Moreover, the TSAREC03 mutant with a pmrB deletion revealed increased expression levels of pmrA, pmrB, and pmrK after the introduction of a plasmid carrying the parental mutated pmrB, consistent with the increased colistin MIC values (Fig. 4). Taken together, these results confirm that amino acid substitutions in PmrB confer colistin resistance to TSAREC02 and TSAREC03.

Fig. 4
figure 4

The mRNA expression levels of pmrB/pmrK in TSAREC03 mutants. Values on the y axis are relative expression levels (fold change) normalized against levels in the colistin susceptible strain MG1655. * Significant differences compared to those in TSAREC03 (Student’s t-test, P < 0.05). N.D., non-detectable or expression

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Nucleotide sequence accession numbers

The complete nucleotide sequences of mutated mgrB and pmrB from TSAREC02 and mutated phoP and pmrB from TSAREC03 were deposited in the GenBank nucleotide database under accession numbers MT597405, MT597406, MT597417, and MT597407, respectively.

Discussion

Studies have reported differences in regulatory gene interactions involving the PmrA/PmrB and PhoP/PhoQ TCSs which contribute to colistin resistance in different species of Enterobacterales. This suggests a more complex network of interactions than what was previously understood [9, 12, 13, 21]. The PmrA/PmrB TCS in Salmonella enterica and K. pneumoniae is known to responds to a signal that controls the PhoP/PhoQ TCS through the involvement of PmrD [22, 23]. However, it is important to note that while E. coli also encodes a homolog of PmrD, the study of experimentally confirmed mutated genes associated with colistin resistance in E. coli has been primarily limited to pmrAB [4, 6, 8, 15, 24]. Previous studies have reported amino acid substitutions in the PhoPQ and MgrB proteins of E. coli in association with colistin resistance. However, none of these reported substitutions have been experimentally validated for their specific contribution to colistin resistance [25,26,27]. Conflicting results regarding the influence of PmrD on PmrAB activity and colistin resistance have been reported in two studies. A study by Winfield et al. revealed that the PmrAB TCS was not affected by PhoPQ TCS; in contrast, a study by Rubin et al. showed the opposite result that the PmrAB TCS could be indirectly activated by the upregulated PhoPQ TCS via PmrD [13, 14]. The interaction between PmrAB and the PhoPQ TCS was investigated in these two studies using different strains of E. coli. Winfield et al. used E. coli MG1655 whereas Rubin et al. used the closely related K-12 E. coli strain W3110. Both studies examined this interaction under varying Mg2+ concentrations, comparing low versus high Mg2+ environments. Since the phosphatase activity of the PmrB protein plays a major role in PmrAB TCS activity and colistin resistance, differences in the strains or unidentified environmental stimuli that affect PmrB phosphatase activity in the E.coli isolates in the two studies may account for the discrepancy in their results [28]. In the current study, we used clinical strains TSAREC02 and TSAREC03 with mgrB and phoP mutations, respectively, to evaluate the influence of PhoPQ alterations on PmrAB TCS and colistin resistance. Both studied isolates, TSAREC02 and TSAREC03, with colistin resistance were of clinical origin from humans. Moreover, we used colistin MIC levels to indicate colistin resistance phenotypes of studied isolates. Therefore, our study provides insights into the real-world scenarios of colistin resistance in human clinical settings. This enhances the clinical relevance and applicability of our results when compared to previous reports [13, 14]. Considering MgrB as a negative regulator and PhoP as a positive regulator in controlling PhoPQ TCS activity, we can observe increased phoP expressions in TSAREC02 with mutated mgrB and decreased phoP expressions in TSAER03 with mutated phoP [10, 11]. As expected, phoP expressions changed significantly in TSAREC02 revertant with WT-mgrB, and in TSAER03 revertant with WT-phoP. Moreover, pmrD expressions regulated by PhoPQ TCS changing significantly were also observed (Figs. 1 and 2). Nevertheless, expressions of pmrAB and downstream regulatory gene pmrK did not change significantly between strains TSAREC02 and TSAREC02 WT-mgrB revertant, as well as TSAREC03 and TSAREC03 WT-phoP revertant. Consequently, colistin MICs remained unchanged among mutants of TSAREC02 and TSAREC03. The results suggested that PhoPQ TCS alterations due to mutated phoP or mgrB that led to pmrD expressions change do not contribute to PmrAB TCS activity alternation and subsequently colistin MICs changes. No change in colistin MIC values observed in the TSAREC02 mutant, TSAREC02_ΔmgrBΔ43–47, and TSAREC03 mutant, TSAREC03_ΔphoPΔ84–224, further support such speculation. These results may explain why clinical colistin-resistant isolates resulting from PhoPQ activation due to a mutated regulatory gene have not been reported yet.

Current experimentally confirmed results regarding chromosomal colistin resistance from a few clinical or laboratory strains showing that PmrB modification is the primary mechanism for colistin resistance in E. coli [6, 18, 24]. In our previous study, eight colistin-resistant E.coli strains harbored unique amino acid substitutions in PmrB. However, no change in the colistin MICs was observed after a plasmid containing each mutated pmrB from the respective colistin-resistant strain was cloned and expressed in the MG1655 mutant with pmrB deletion. These results showed that mutated pmrB genes from E. coli isolates were not associated with colistin resistance [15]. The surprising result could stem from either the activation of operons pmrHFIJKLM and pmrCAB in a PmrAB-independent manner or an unknown factor in strain MG1655 that interfered with the effect of the cloned pmrB-carrying plasmid. In the current study, we re-evaluated the effect of pmrB mutations in TSAREC02 and TSAREC03 on colistin resistance using different validation methods. The mutated pmrB from TSAREC02 and TSAREC03 was cloned into plasmids and expressed in TSAREC02 and TSAREC03 mutants with pmrB deletion, respectively. Therefore, confounding factors from the genomic backgrounds of different E. coli strains could be eliminated. The results of the present study indicate that pmrB mutation is an independent factor for colistin resistance in TSAREC02 and TSAREC03 via increased PmrAB TCS activity. Moreover, the result support our speculation that some unknown interfering factors in the genomic background of E.coli MG1655 may influence the effect of the cloned mutated pmrB on colistin resistance [15]. Based on the results demonstrated here, we inferred that the colistin resistance mechanism among the remaining six E. coli isolates collected in a previous study may still result from the unique pmrB mutation they harbored [15]. Further investigations of these strains using a method similar to that used in the current study are underway. Moreover, significantly different expression levels of pmrA between TSAREC02 and TSAREC03 strains were observed. Since PmrA (also known as BasR) has interaction with many pathways reported before based up on STRING database (https://version-11-5.string-db.org/network/511145.b4113), different pmrA expression levels between 2 strains may be related to different regulative activity of interactive pathways. Since no mutations in pmrA from both strains and both complemented strains with mutated prmB reached comparable colistin MIC levels to the original clinical strains, we speculated that different pmrA expressions on both strains had no influence on colistin resistance.

Conclusion

Although E. coli is a significant human pathogen within Enterobacterales, studies about chromosome-encoded pathway interactions involving colistin resistance is less described due to its rarity in clinical origin. Our study, based on two clinical colistin resistant E. coli isolates, elucidated the interaction between the PhoPQ and PmrAB TCs and their influence on colistin resistance. Our results demonstrated that alternations in the PhoPQ TC, caused by mutated regulatory genes, did not influence colistin resistance. Instead, mutations in the pmrB gene were identified as the key factor contributing to colistin resistance in both studied clinical E. coli isolates. For new drug development, proper target selection is crucial. Complex regulatory pathway interactions involving colistin resistance control were observed among different species of Enterobacterales. This complexity makes it challenging to select universal targets that cover all olistin resistant Enterobacterales during drug development. Although our study was limited to only two clinical isolates and the results may not be generalizable to the entire E. coli population, it provides valuable insights into the mechanisms underlying colistin resistance. These findings have the potential to contribute significantly to the development of new drugs targeting emerging colistin-resistant Enterobacterales.

Data availability

No datasets were generated or analysed during the current study.

Abbreviations

LPS:

Lipopolysaccharides

TCS:

Two-component system

PCR:

Polymerase chain reaction

MIC:

Minimum inhibitory concentration

WT:

Wild type

LB:

Luria-Bertani

References

  1. Binsker U, Kasbohrer A, Hammerl JA. Global colistin use: a review of the emergence of resistant enterobacterales and the impact on their genetic basis. FEMS Microbiol Rev. 2022;46(1):fuab049.

    Article  CAS  PubMed  Google Scholar 

  2. Aghapour Z, Gholizadeh P, Ganbarov K, Bialvaei AZ, Mahmood SS, Tanomand A, et al. Molecular mechanisms related to colistin resistance in Enterobacteriaceae. Infect Drug Resist. 2019;12:965–75.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Lee JY, Choi MJ, Choi HJ, Ko KS. Preservation of Acquired Colistin Resistance in Gram-negative Bacteria. Antimicrob Agents Chemother. 2016;60(1):609–12.

    Article  PubMed  Google Scholar 

  4. Wang CH, Siu LK, Chang FY, Tsai YK, Lin YT, Chiu SK, et al. A novel deletion mutation in pmrB contributes to Concurrent Colistin Resistance in Carbapenem-Resistant Escherichia coli sequence type 405 of clinical origin. Antimicrob Agents Chemother. 2020;64(6):e00220–20.

    Article  PubMed  PubMed Central  Google Scholar 

  5. Roland KL, Martin LE, Esther CR, Spitznagel JK. Spontaneous pmrA mutants of Salmonella typhimurium LT2 define a new two-component regulatory system with a possible role in virulence. J Bacteriol. 1993;175(13):4154–64.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Sato T, Shiraishi T, Hiyama Y, Honda H, Shinagawa M, Usui M, et al. Contribution of novel amino acid alterations in PmrA or PmrB to Colistin Resistance in mcr-negative Escherichia coli Clinical isolates, including Major Multidrug-resistant lineages O25b:H4-ST131-H30Rx and Non-x. Antimicrob Agents Chemother. 2018;62(9):e00864–18.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Jayol A, Poirel L, Brink A, Villegas MV, Yilmaz M, Nordmann P. Resistance to colistin associated with a single amino acid change in protein PmrB among Klebsiella pneumoniae isolates of worldwide origin. Antimicrob Agents Chemother. 2014;58(8):4762–66.

    Article  PubMed  PubMed Central  Google Scholar 

  8. Cannatelli A, Giani T, Aiezza N, Di Pilato V, Principe L, Luzzaro F, et al. An allelic variant of the PmrB sensor kinase responsible for colistin resistance in an Escherichia coli strain of clinical origin. Sci Rep. 2017;7(1):5071.

    Article  PubMed  PubMed Central  Google Scholar 

  9. Cheng YH, Lin TL, Lin YT, Wang JT. Amino acid substitutions of CrrB responsible for resistance to Colistin through CrrC in Klebsiella pneumoniae. Antimicrob Agents Chemother. 2016;60(6):3709–16.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Cannatelli A, Giani T, D’Andrea MM, Di Pilato V, Arena F, Conte V, et al. MgrB inactivation is a common mechanism of colistin resistance in KPC-producing Klebsiella pneumoniae of clinical origin. Antimicrob Agents Chemother. 2014;58(10):5696–703.

    Article  PubMed  PubMed Central  Google Scholar 

  11. Jayol A, Nordmann P, Brink A, Poirel L. Heteroresistance to colistin in Klebsiella pneumoniae associated with alterations in the PhoPQ regulatory system. Antimicrob Agents Chemother. 2015;59(5):2780–84.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Mitrophanov AY, Jewett MW, Hadley TJ, Groisman EA. Evolution and dynamics of regulatory architectures controlling polymyxin B resistance in enteric bacteria. PLoS Genet. 2008;4(10):e1000233.

    Article  PubMed  PubMed Central  Google Scholar 

  13. Winfield MD, Groisman EA. Phenotypic differences between Salmonella and Escherichia coli resulting from the disparate regulation of homologous genes. Proc Natl Acad Sci U S A. 2004;101(49):17162–7.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Rubin EJ, Herrera CM, Crofts AA, Trent MS. PmrD is required for modifications to Escherichia coli endotoxin that promote antimicrobial resistance. Antimicrob Agents Chemother. 2015;59(4):2051–61.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Wang CH, Siu LK, Chang FY, Chiu SK, Lin JC. A resistance mechanism in Non-mcr colistin-resistant Escherichia coli in Taiwan: R81H substitution in PmrA is an independent factor contributing to Colistin Resistance. Microbiol Spectr. 2021;9(1):e0002221.

    Article  PubMed  Google Scholar 

  16. Quan J, Li X, Chen Y, Jiang Y, Zhou Z, Zhang H, et al. Prevalence of mcr-1 in Escherichia coli and Klebsiella pneumoniae recovered from bloodstream infections in China: a multicentre longitudinal study. Lancet Infect Dis. 2017;17(4):400–10.

    Article  CAS  PubMed  Google Scholar 

  17. Tsai YK, Liou CH, Lin JC, Ma L, Fung CP, Chang FY, et al. A suitable streptomycin-resistant mutant for constructing unmarked in-frame gene deletions using rpsL as a counter-selection marker. PLoS ONE. 2014;9(9):e109258.

    Article  PubMed  PubMed Central  Google Scholar 

  18. Phan MD, Nhu NTK, Achard MES, Forde BM, Hong KW, Chong TM, et al. Modifications in the pmrB gene are the primary mechanism for the development of chromosomally encoded resistance to polymyxins in uropathogenic Escherichia coli. J Antimicrob Chemother. 2017;72(10):2729–36.

    Article  CAS  PubMed  Google Scholar 

  19. Yuan J, Jin F, Glatter T, Sourjik V. Osmosensing by the bacterial PhoQ/PhoP two-component system. Proc Natl Acad Sci U S A. 2017;114(50):E10792–8.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods. 2001;25(4):402–8.

    Article  CAS  PubMed  Google Scholar 

  21. Hjort K, Nicoloff H, Andersson DI. Unstable tandem gene amplification generates heteroresistance (variation in resistance within a population) to colistin in Salmonella enterica. Mol Microbiol. 2016;102(2):274–9.

    Article  CAS  PubMed  Google Scholar 

  22. Cheng HY, Chen YF, Peng HL. Molecular characterization of the PhoPQ-PmrD-PmrAB mediated pathway regulating polymyxin B resistance in Klebsiella pneumoniae CG43. J Biomed Sci. 2010;17(1):60.

    Article  PubMed  PubMed Central  Google Scholar 

  23. Kox LF, Wosten MM, Groisman EA. A small protein that mediates the activation of a two-component system by another two-component system. EMBO J. 2000;19(8):1861–72.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Janssen AB, Bartholomew TL, Marciszewska NP, Bonten MJM, Willems RJL, Bengoechea JA. Nonclonal Emergence of Colistin Resistance Associated with mutations in the BasRS two-component system in Escherichia coli Bloodstream isolates. mSphere. 2020;5(2):e00143–20.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Luo Q, Yu W, Zhou K, Guo L, Shen P, Lu H, et al. Molecular epidemiology and colistin resistant mechanism of mcr-positive and mcr-negative clinical isolated Escherichia coli. Front Microbiol. 2017;8:2262.

    Article  PubMed  PubMed Central  Google Scholar 

  26. Olaitan AO, Thongmalayvong B, Akkhavong K, Somphavong S, Paboriboune P, Khounsy S, et al. Clonal transmission of a colistin-resistant Escherichia coli from a domesticated pig to a human in Laos. J Antimicrob Chemother. 2015;70(12):3402–4.

    CAS  PubMed  Google Scholar 

  27. Nawfal Dagher T, Al-Bayssari C, Chabou S, Baron S, Hadjadj L, Diene SM, et al. Intestinal carriage of colistin-resistant Enterobacteriaceae at Saint Georges Hospital in Lebanon. J Global Antimicrob Resist. 2020;21:386–90.

    Article  Google Scholar 

  28. Chen HD, Jewett MW, Groisman EA. Ancestral genes can control the ability of horizontally acquired loci to confer new traits. PLoS Genet. 2011;7(7):e1002184.

    Article  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

We thank the National Health Research Institute for providing the E. coli strains used in this study. We also thank researcher Shu-Chen Kuo at the National Health Research Institutes for assistance with this study.

Funding

This work was supported by grants from the Tri-Service General Hospital and the National Defense Medical Center (grant numbers TSGH-C107-100, TSGH-C108-138, TSGH-D-109128, TSGH-E-112255, TSGH-D-113143 and TSGH-D-110055) and the Ministry of Science and Technology, Executive Yuan, Taiwan (grand number MOST 112-2314-B-016-043).

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Authors and Affiliations

  1. Division of Infectious Diseases and Tropical Medicine, Department of Internal Medicine, Tri-Service General Hospital, National Defense Medical Center, No. 325, Section 2, Cheng-Kung Road, Neihu 114, Taipei, Taiwan

    Ching-Hsun Wang, Feng-Yee Chang & Jung-Chung Lin

  2. Institute of Infectious Diseases and Vaccinology, National Health Research Institutes, Miaoli, Taiwan

    L. Kristopher Siu, Yu-Kuo Tsai & Li-Yueh Huang

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  1. Ching-Hsun Wang
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  2. L. Kristopher Siu
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  4. Yu-Kuo Tsai
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  5. Li-Yueh Huang
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  6. Jung-Chung Lin
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Contributions

C.W. and L.S.initiate Investigation, Methodology, Writing – original draft. F.C. perform Data analysis. Y.T. Formal analysis, Writing – original draft. L.H. Data analysis. JL. Conceptualization, Writing – review & editing. All authors reviewed and approved the manuscript.

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Correspondence to Jung-Chung Lin.

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Wang, CH., Siu, L.K., Chang, FY. et al. Influence of PhoPQ and PmrAB two component system alternations on colistin resistance from non-mcr colistin resistant clinical E. Coli strains. BMC Microbiol 24, 109 (2024). https://doi.org/10.1186/s12866-024-03259-8

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Keywords

BMC Microbiology

ISSN: 1471-2180

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