Polymyxin Resistance and Heteroresistance Are Common in Clinical Isolates of Achromobacter Species and Correlate with Modifications of the Lipid A Moiety of Lipopolysaccharide

ABSTRACT The Achromobacter genus includes opportunistic pathogens that can cause chronic infections in immunocompromised patients, especially in people with cystic fibrosis (CF). Treatment of Achromobacter infections is complicated by antimicrobial resistance. In this study, a collection of Achromobacter clinical isolates, from CF and non-CF sources, was investigated for polymyxin B (PmB) resistance. Additionally, the effect of PmB challenge in a subset of isolates was examined and the presence of PmB-resistant subpopulations within the isolates was described. Further, chemical and mass spectrometry analyses of the lipid A of Achromobacter clinical isolates enabled the determination of the most common structures and showed that PmB challenge was associated with lipid A modifications that included the addition of glucosamine and palmitoylation and the concomitant loss of the free phosphate at the C-1 position. This study demonstrates that lipid A modifications associated with PmB resistance are prevalent in Achromobacter and that subresistant populations displaying the addition of positively charged residues and additional acyl chains to lipid A can be selected for and isolated from PmB-sensitive Achromobacter clinical isolates. IMPORTANCE Achromobacter species can cause chronic and potentially severe infections in immunocompromised patients, especially in those with cystic fibrosis. Bacteria cannot be eradicated due to Achromobacter's intrinsic multidrug resistance. We report that intrinsic resistance to polymyxin B (PmB), a last-resort antimicrobial peptide used to treat infections by multiresistant bacteria, is prevalent in Achromobacter clinical isolates; many isolates also display increased resistance upon PmB challenge. Analysis of the lipopolysaccharide lipid A moiety of several Achromobacter species reveals a penta-acylated lipid A, which in the PmB-resistant isolates was modified by the incorporation of glucosamine residues, an additional acyl chain, loss of phosphates, and hydroxylation of acyl chains, all of which can enhance PmB resistance in other bacteria. We conclude that PmB resistance, particularly in Achromobacter isolates from chronic respiratory infections, is a common phenomenon, and that Achromobacter lipid A displays modifications that may confer increased resistance to polymyxins and potentially other antimicrobial peptides.

4-amino-4-deoxy-L-arabinose or other positively charged moieties, which are added to the free phosphates of the lipid A, hence decreasing the negative charge of the lipid A or, alternatively, directly removing free phosphate groups from the lipid A (48).For example, in Bordetella pertussis, which is closely related to Achromobacter species, the lipid A can be modified by the addition of glucosamine (GlcN), resulting in bacterial resistance to CAMPs (49).The number, length, and hydroxylation of acyl chains to lipid A can also be modified, which potentially increases the lipid A packing in the outer membrane, further reducing the permeability of the outer membrane to CAMPs (48,50,51).
The phenomenon of heteroresistance, in which discrete subpopulations of bacteria have a higher level of antimicrobial resistance than the average resistance of the population (52), has been observed across several Gram-negative bacteria, especially in response to polymyxins (53,54).Heteroresistance has not yet been reported in Achromobacter; however, the potential for bacterial isolates to harbor discrete subpopulations primed for resistance could explain therapeutic failures.Research on Enterobacter species has revealed that resistant bacterial subpopulations can also be selected without colistin treatment in response to the host immune system due to upregulation of genes responsible for lipid A modification (53).
CF isolates of A. xylosoxidans have been reported as generally susceptible to colistin at MIC values of the antibiotic below the suggested MIC clinical breakpoint of $4 mg/mL (21,40,43).In contrast, other studies have shown a bimodal colistin resistance in a subset of Achromobacter species from CF isolates and in biofilms and a steady increase in colistin resistance among Achromobacter isolates over time (34,55,56).The increased use of polymyxins as a last-resort treatment has resulted in the emergence of resistance due to a transmissible plasmid carrying the mcr-1 gene encoding a phosphoethanolamine transferase (57,58).However, colistin-resistant CF isolates of Achromobacter do not appear to harbor the mcr-1 plasmid (56), suggesting that other mechanisms of polymyxin resistance operate in Achromobacter species.
In this study, an examination of a collection of Achromobacter clinical isolates from CF and non-CF sources showed that PmB resistance and heteroresistance are common.Characterization of the associated lipid A modifications, by chemical analyses and mass spectrometry of a subset of isolates, helped to correlate the effect of PmB challenge with lipid A modification and to establish the presence of PmB-resistant subpopulations within the isolates.These results indicate that lipid A modifications associated with PmB resistance are prevalent in Achromobacter and that subresistant populations displaying an increased addition of positively charged moieties and additional lipid A acyl chains can be selected for and isolated from PmB-sensitive Achromobacter clinical isolates.

RESULTS
Resistance to PmB is common in Achromobacter sp.clinical isolates.Clinical isolates of Achromobacter species (n = 95) isolated from blood and sputum were examined for resistance to PmB by Etest and broth microdilution (BMD) assays (Fig. 1A and Table 1).The A. xylosoxidans reference strain ATCC 27065 was included as a control.Using BMD, 62 out of the 94 Achromobacter isolates (66%) that grew in cation-adjusted Mueller-Hinton broth (CA-MHB) had MIC values of $4 mg/mL (Fig. 1B).When MIC values from the Etest were considered, 43 out of 95 isolates (45.3%) were found to be resistant (Fig. 1B).Also, the Etest plates of several isolates developed secondary halos between 1 and 6 days after plating, suggesting that these isolates consist of subpopulations of bacterial cells displaying heteroresistance.Comparison of the two tests based on breakpoint indicated disagreement in 16 isolates, with 13 of these showing PmB resistance by BMD but PmB sensitivity by Etest (Table 1).These differences can be explained by heteroresistance, which may be more apparent in broth culture since resistant subpopulations within a given isolate could continue growing in the presence of the antibiotic, resulting in turbidity at higher PmB concentrations relative to the no-antibiotic control.Heteroresistance was determined by population profile analysis as a $8-fold increase in the highest concentration of antibiotic with no inhibition and the lowest concentration with maximum inhibition (52,59).Of the isolates for which resistance was observed by BMD but not by Etest, 54% were classed as heteroresistant.A similar analysis carried out on all the remaining isolates from our collection revealed that 50% had population profiles indicating heteroresistance (Table 1; see Data Set S1 in the supplemental material).Notably, while all the heteroresistant isolates were deemed resistant by BMD, 15% of the heteroresistant isolates were susceptible to PmB by Etest.Only 4 (31%) of the 13 blood isolates (Table 1) were resistant, suggesting that PmB resistance is more prevalent in the isolates obtained from chronic pulmonary infection.Together, our results demonstrate that resistance to PmB is common in clinical isolates of Achromobacter species, especially in those isolates obtained from chronic respiratory infection, and that some isolates display heteroresistance.
The lipid A of Achromobacter species is penta-acylated and can be modified by addition of glucosamine and palmitate.Modifications of the lipid A moiety of LPS are associated with resistance to polymyxins; these include the addition of positively charged residues to the phosphate groups and the alteration in length, number, and hydroxylation state of the acyl chains hypothesized to affect the integrity of the outer membrane (48-50, 60, 61).Because these modifications depend on environmental conditions, we first evaluated the effect of the growth medium (lysogeny broth [LB], CA-MHB, and mineral-tryptone-glycerol [MTG] medium) in the matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (MALDI-MS) lipid A profiles of the isolate AC075 (Achromobacter deleyi), which were obtained by a rapid lysis method (62) and used to standardize the profiles.The most abundant ion peak in all three media during log-phase growth had a mass-to-charge (m/z) ratio of 1,506 (Fig. 2A), corresponding to a penta-acylated lipid A structure with one phosphate at the 49 position and secondary acyl chains at 2 and 29 positions (hydroxylaurate [C12:0 (OH)], and myristate [C14:0(OH)], respectively), giving a 3 1 2 acylation pattern (Fig. 2B).A second peak at m/z 1,586 corresponds to the presence of another phosphate group (Fig. 2A and B).A third ion peak was also present in the lipid A from isolate AC075 grown in CA-MHB and MTG at m/z 1,667 (Fig. 2A), corresponding to a gain of 161 mass units of the m/z 1,506 ion peak; this was consistent with the addition of a HexN residue (61) (Fig. 2B).A relatively small ion peak at m/z 1,756 was interpreted as representing a small quantity of a hexa-acylated lipid A species with hydroxydecanoate [C10:0(OH)] (61).The results suggest that the growth medium influences the lipid A profiles, especially concerning the HexN modification.The lipid A profiles of A. xylosoxidans isolates AC011, AC036, AC061, AC064, and AC080 and Achromobacter insuavis AC054 were also examined from preparations using large-scale cultures in LB and treated to extract the  lipid A as previously described (63) (Fig. S1).These preparations revealed additional peaks indicating variations in the hydroxylation of the acyl chains, but overall, they predicted that the Achromobacter species lipid A is mainly penta-acylated, with modifications resulting in the addition of HexN and in some cases an additional palmitoyl (C16:0) residue.Table S1 summarizes the predicted structural compositions of lipid A forms identified in this work.
To confirm the structural hypotheses deduced by MALDI-TOF MS, a detailed compositional analysis of these lipid A fractions directly extracted from cell pellets of A. xylosoxidans isolates AC011, AC036, AC061, AC064, and AC080 and A. insuavis AC054 was undertaken to establish both the fatty acid (Fig. S2 and Table S2) and sugar (Fig. S3) content.The analysis of acetylated methyl glycoside derivatives showed only the presence of GlcN in all the lipid A preparations (Fig. S3).This was further confirmed by the analysis of the alditol acetates of the unknown HexN properly separated from the lipid A upon dephosphorylation and inspection by means of gas chcromatography (GC)-MS.This analysis showed the presence of glucosaminitol acetate, thus definitively proving that the additional HexN unit decorating the disaccharide backbone of the Achromobacter lipid A molecular species investigated was a GlcN residue.
LPS and lipid A modifications between early and late isolates.Although most of the isolates were from individual patients, our collection included eight pairs of isolates from eight pwCF which were collected at intervals ranging from over 1 month up to more than 1 year.These pairs included isolates classified as A. deleyi, A. xylosoxidans, A. insuavis, and Achromobacter sp.cluster II (Table 2).Of these, one pair of isolates was susceptible to PmB, one pair showed a decrease in MIC when the early and late isolates were compared, and one pair showed an increased MIC from susceptible to resistant (Table 2).For the remaining pairs, the isolates were resistant (Table 2).To gain further insight that could explain the differences in PmB resistance, we investigated in more detail the MALDI-MS lipid A profiles of the isolate pairs that showed an increase in or maintenance of PmB resistance, which included AC036/AC088 (A. xylosoxidans), AC037/ AC058 (A. xylosoxidans), AC046/AC048 (A. xylosoxidans), and AC068/AC075 (A. deleyi) (Table 2).Because no differences in lipid A structure were observed for AC075 in CA-MHB and MTG, we employed overnight cultures in MTG for all subsequent comparisons.The lipid A of the early A. deleyi isolate AC068 exhibited a gain of 16 mass units at each of the main peaks seen in AC075 (Fig. 3A), giving peaks with m/z ratios of 1,522, 1,602, and 1,683.This indicates a gain of a hydroxyl group on the secondary myristate group at the C-29 position (Fig. 3B and Table S1).A low-abundance peak with an m/z ratio of 1,586 was also present, which denotes the lack of the additional hydroxyl and the presence of a second phosphate, showing the potential for variation in lipid A structures even within a single strain.A hexa-acylated, GlcN-modified lipid A (m/z 1,921) (Fig. 3A) was present in AC068 and not in AC075.Variation in hydroxylation was also observed in the A. xylosoxidans AC036/AC088 pair (Fig. 3A).In AC036 lipid A, a monophosphorylated, penta-acylated structure (m/z 1,506) (Fig. 2B) with the gain of either a second phosphate (m/z 1,586) (Fig. 2B) or GlcN (m/z 1,667) (Fig. 2B) was predicted.In contrast, each peak in the lipid A spectrum of the AC088 late isolate corresponded to peaks like those in AC036 with a loss of 16 mass units (Fig. 3A).This suggests that the secondary myristate chains in the AC088 lipid A are not hydroxylated (Fig. 3B and Table 3; Table S1).No differences were observed in the lipid A of the AC037/AC058 and AC046/AC048 pairs of serial isolates, both of which displayed penta-acylated, monophosphorylated lipid A with hydroxylated secondary acyl chains (m/z 1,522), m/z 1,522 with GlcN modification (m/z 1,683), and m/z 1,683 plus a palmitoyl group (m/z 1,921), as with AC068 (Fig. 3B and Table 3; Table S1).Despite the fact that the small sample size precludes us from drawing unequivocal conclusions, our observations suggest that the lipid A of late isolates undergoes modifications involving various levels of hydroxylation of secondary acyl chains and GlcN addition, which could in turn confer high-level PmB resistance or decreased virulence, consistent with bacterial adaptation to chronic airway infection.
The experiments described above were performed in the absence of PmB; therefore, the observed lipid A modifications could not be directly correlated with PmB exposure.We investigated the effect of subinhibitory PmB concentrations (ranging from 0.1 to 10 mg/mL, depending on the specific strain investigated) on the lipid A of the five pairs of isolates under investigation to identify the potential modifications induced in Achromobacter species following exposure to polymyxins.PmB concentrations were initially trialed using the MICs identified by BMD and gradually decreased if insufficient growth was observed overnight.Of the 10 isolates investigated, changes in lipid A structure following overnight culture in PmB were observed in A. xylosoxidans AC036 and AC088 and A. deleyi AC075 (Fig. 3A), while no changes were observed in the remaining strains when cultured in the presence or absence of PmB.MALDI-MS of AC036 lipid A showed penta-acylated structures, one of which contained the GlcN modification (m/z 1,667) (Fig. 2B and Fig. 3A).Upon growth in 0.1 mg/mL PmB, a gain of 238 mass units from m/z 1,667 to m/z 1,905 was observed (Fig. 3), indicating a gain of a palmitoyl group resulting in a hexa-acylated lipid A with a GlcN modification.The peak at m/z 1,586 corresponding to a bisphosphorylated and penta-acylated structure (Fig. 2B) was not observed in AC036 following growth in PmB (Fig. 3A).AC088 cultured in PmB showed a lipid A with a peak at m/z 1,889, an increase in 238 mass units from m/z 1,651, indicating the addition of a palmitate group (Fig. 3A), resulting in a hexaacylated, GlcN-modified lipid A without hydroxylation of the secondary acyl chains (Fig. 3B).While MALDI-MS is not quantitative, the increased relative abundance of the peak at m/z 1,651 (Fig. 3A) and the decrease of the m/z 1,490 ion peak were consistent in both replicates, suggesting a greater proportion of GlcN-modified lipid A after treatment with PmB.Culturing AC075 in PmB revealed a lipid A with a gain of 254 mass units compared to m/z 1,667 (Fig. 3A), giving a peak at m/z 1,921 demonstrating the gain of a palmitate group (236 mass units) and a hydroxyl group (16 mass units) (Fig. 3B).The increased abundance of the GlcN-modified peak in AC088 was also seen in AC075 when treated with PmB (Fig. 3A), with the abundance of m/z 1,506 decreasing and that of m/z 1,667 increasing.The gain of a sixth acyl chain and a high relative abundance of GlcN-modified lipid A were found in isolates with high PmB resistance ($32 mg/mL) without challenge with PmB (AC058, AC046, and AC048).No additional modification to these structures occurred following growth in PmB.Together, our results indicate that Achromobacter species have a heterogenous lipid A structure containing modifications that confer bacterial adaptation to high-level concentrations of PmB.
Achromobacter species isolates can display heteroresistance to PmB.The population profile analysis of all the isolates in our collection indicated that many display heteroresistance (Table 1).To better characterize this phenotype, the growth of every isolate in our collection was observed on a 200-mg/mL PmB LB plate.From this, the isolates that grew most successfully (AC011, AC036, AC061, and AC080) were selected for the PmB challenge assay.The results showed that when the clinical isolates were initially treated with PmB, there was a significant reduction in CFU/mL in comparison to the control group without antibiotic (mean log 10 CFU increased to 4.9, 4.0, 1.6, and 5.7  from the challenge plate to the control plate for AC011, AC036, AC061, and AC080, respectively).The small differences between the challenge and control AC061 can be explained by the already high level of PmB resistance in this isolate (64 mg/mL BMD MIC).However, when the surviving colonies after challenge were challenged a second time, no significant differences were found with the control group of the initial isolate with no antibiotic (mean log CFU differences of 0.9, 0.3, 20.06, and 0 for AC011, AC036, AC061, and AC080, respectively).This indicates that they are resistant to PmB up to a minimum of 200 mg/mL following just two challenges of 2 h from polymyxin B (Fig. 4 and Table 4).Isolates recovered from the challenges were named with a CR-1 suffix (Fig. 4 and Table 4).LPS profiling using silver staining revealed a noticeable reduction in higher-molecularweight bands that represent the O-antigen polysaccharide in isolates AC036, AC061, and AC080 surviving PmB treatment compared to those of the untreated counterparts (Fig. 5).This suggests a switch in the production of O-antigen under PmB stress.In contrast, AC011 and its challenge resistant isolate AC011CR-1 showed no differences in O-antigen production.Loss of O-antigen polysaccharide, as described here, has been reported to confer PmB resistance to Escherichia coli strains even without modification of the phosphate of lipid A (64).Analysis of the lipid A by MALDI-MS was carried out for the four clinical isolates and their challenge resistant isolates (Fig. 6).AC011 lipid A mass spectra predominantly displayed peaks consistent with a bisphosphorylated penta-acylated lipid A structure (m/z 1,586 and 1,602) with one or both secondary acyl chains hydroxylated, respectively.The lipid A of AC011CR-1 shows similar peaks at m/z 1,522 and m/z 1,602 with a drop in incidence of the single hydroxylated structures (m/z 1,586).AC011CR-1 also has the peak m/z 1,683, representative of a penta-acylated, monophosphorylated lipid A with a GlcN modification and both secondary acyl chains hydroxylated (Fig. 3B).The lipid A profile of AC036CR-1 showed the gain of a hexa-acylated, GlcN-modified lipid A structure (m/z 1,905) (Fig. 3B and Fig. 6).The lipid A of AC080 consisted of peaks at m/z 1,522, 1,602, and 1,683 and a peak at m/z 1,840 representative of a palmitoyl group giving a hexa-acylated, bisphosphorylated structure without a GlcN modification.However, AC080CR-1 showed an increase in the relative abundance of the GlcN-modified lipid A (m/z 1,683) (Fig. 3B).Additionally, the gain of a palmitoyl group giving a hexa-acylated structure with a GlcN modification (m/z 1,921) (Fig. 3B) was seen.No differences were found between the lipid A structures of AC061 and AC061CR-1, both displaying peaks at m/z 1,683 and 1,921, as in AC080CR-1, which could be related to the high PmB resistance (16 mg/mL by BMD) initially displayed by this isolate.

DISCUSSION
This study has shown that PmB resistance and heteroresistance are common in clinical isolates of Achromobacter species, especially in respiratory isolates from pwCF, while blood isolates from non-CF patients were mostly PmB sensitive and did not show heteroresistance.Further, this study shows that Achromobacter isolates can modify their lipid A structures by the addition of GlcN, palmitoylation of the penta-acylated lipid A, dephosphorylation, and hydroxylation of secondary acyl chains.The gain of a sixth acyl chain, especially following challenge by PmB, could lead to increased resistance to polymyxins.The palmitoylation of lipid A has been shown to increase resistance to CAMPs, which are a component of the innate immune response (65), and palmitoylation of the lipid A in Salmonella and P. aeruginosa is suggested to increase the permeability barrier of the outer membrane against CAMPs (66)(67)(68).Similarly, disruption of the gene responsible for palmitoylation in Bordetella bronchiseptica resulted in the clearance of infection following the activation of the adaptive immune system, while the palmitoyl-containing wild-type strain persisted (69).
Although ion peaks consistent with bisphosphorylated lipid A structures (m/z 1,586 and 1,602) were present in many isolates, the peaks with the highest relative abundance in each strain were representative of monophosphorylated lipid A, especially in cases where lipid A was modified with GlcN (m/z 1,651, 1,667, 1,683, 1,889, 1,905, and 1,921).This adaptation has been observed in many Gram-negative pathogens, including Salmonella enterica and Helicobacter pylori, in which the loss of the C-1 phosphate group is associated with a higher resistance to PmB (70,71).This confers resistance via  the reduction in the overall negative charge of the lipid A molecule following the loss of a phosphate.Therefore, we propose that the GlcN modification combined with the loss of the C-1 phosphate contribute to PmB resistance in Achromobacter species.Hydroxylation of the secondary acyl chains of lipid A molecules is common in many genera of Gram-negative bacteria and depends on the dioxygenase LpxO.Deletion of the lpxO gene has been associated with various phenotypes in Klebsiella pneumoniae, P. aeruginosa, Acinetobacter baumannii, and Burkholderia pseudomallei strains, including increased virulence and inflammatory responses, reduction of resistance to CAMPs, and increased macrophage-mediated killing across members of the genera (72)(73)(74)(75).A recent study on Enterobacter cloacae complex isolates from neonatal sepsis demonstrated that isolates from asymptomatic patients carried a myristate group at the C-2 position in comparison to a hydroxymyristate at C-2 in sepsis-causing isolates (76).The authors hypothesize that the hydroxyl group at this position increases hydrogen bonding within the outer membrane, conferring resistance to CAMPs produced by the immune system, which would suggest that hydroxylation could be an adaptation to colonizing the lung environment.Both palmitoylation and hydroxylation are controlled by the PhoP/PhoQ regulatory system in other Gram-negative genera (77), but it is currently unknown if Achromobacter species harbor a PhoP/PhoQ regulator.Moreover, within the Alcaligenaceae family, the presence of C14:0 (2-OH) in the fatty acid profiling was reported only for species of Bordetella (78), Pigmentiphaga kullae (79), and representatives of Kerstersia (80).Therefore, the findings in this study extend these observations to Achromobacter species.
We hypothesize that in addition to CAMP resistance, the identified lipid A modifications may have implications in inflammation.Hexa-acylated lipid A is typically considered to be highly proinflammatory due to an enhanced interaction between lipid A, myeloid differentiation factor 2 (MD-2), and Toll-like receptor 4 (TLR4) provided by the presence of the sixth acyl chain, which in turn enhances dimerization of TLR4-MD2, leading to stronger stimulation of inflammatory responses (81).The penta-acylated lipid A structure in most of the Achromobacter isolates examined in this work suggests that their lipid A may be less proinflammatory than the canonical hexa-acylated structures.However, the GlcN modification has been associated with increased dimerization of TLR4 and the enhanced inflammatory response induced by the penta-acylated lipid A of B. pertussis (49).A similar observation has been made for Burkholderia cenocepacia, which produces a penta-acylated lipid A replaced by 4-amino-4-deoxy-L-arabinose (82).In this case, the 4-amino-4-deoxy-L-arabinose residue in the B. cenocepacia lipid A allows exposure of the fifth acyl chain on the surface of MD-2, enabling interactions with TLR4 and its dimerization.The change in lipid A between pairs of early and late isolates from the same patients was consistent with previous studies suggesting that Achromobacter species adapt to the surrounding environment and evolve during chronic infection (36).The chronicity of airway infections in pwCF could exert selective pressure on the pathogen, leading to higher mutation rates.Hypermutable clones causing the formation of genetically diverse subpopulations within the general bacterial population have been observed in Achromobacter ruhlandii (29,30).In E. cloacae and K. pneumoniae, undetected heteroresistance to polymyxins leads to treatment failure in in vivo models of infection (53,54).This can allow infections to reemerge and no longer respond to subsequent treatment after seemingly being eradicated after initial treatment, thus underscoring the potential clinical significance of this phenomenon.
In summary, this study demonstrates that resistance and heteroresistance to PmB in Achromobacter species, particularly in isolates from respiratory sources and chronic infection, are common phenomena.In addition, this study reveals that the Achromobacter lipid A has features resembling the lipid A from the closely related Bordetella genus and displays modifications that are consistent with increased bacterial resistance to polymyxins and potentially other CAMPs.

MATERIALS AND METHODS
Bacterial isolates.The bacterial collection examined consisted of 95 Achromobacter clinical isolates representing different species of the genus, including A. xylosoxidans, A. spanius, A. ruhlandii, A. insuavis, A. marplatensis, A. dolens, A. deleyi, and A. aegrifaciens, which were obtained from sputum and blood and identified by nrdA (ribonucleoside-diphosphate reductase 1 subunit a) and gyrB (DNA gyrase subunit B) PCR gene amplification and DNA sequencing (12) (Table 1).These isolates were obtained from the Antimicrobial Resistance and Healthcare Associated Infections Reference Unit of the UK Health Security Agency and local clinical isolates from the Northern Ireland HSC Microbiology Culture Repository (MicroARK), housed at the Northern Ireland Public Health Laboratory, Belfast City Hospital, Belfast, Northern Ireland.Most of the sputum isolates originated from pwCF and were collected between January 2013 and June 2014 from 96 patients attending 27 UK hospitals, except for two samples taken from a non-CF patient and a patient with chronic obstructive pulmonary disease, while all blood isolates were from non-CF patients (Table 1).
Culture conditions.As the Achromobacter isolates displayed variable growth rates on lysogeny broth (LB) medium (Melford Laboratories, Ltd., Chelsworth, Ipswich, UK) depending on the strain, with some taking up to 4 days to grow, a semidefined mineral-tryptone-glycerol (MTG) medium was developed in our laboratory that provided consistent growth rates in most isolates and exploited the ability of most Achromobacter species to metabolize nitrates, since most species are denitrifying in their natural environment (83).MTG consisted of 15.1 mM (NH 4 ) 2 SO 4 , 16.8 mM Na 2 HPO 4 Á 2H 2 O, 22 mM KH 2 PO 4 , 51.3 mM NaCl, 10 mM KNO 3 , 1% (vol/vol) glycerol as a carbon source, and 0.5% (wt/vol) tryptone, with a final pH of 6.7.A sterile 10Â stock of these salts and glycerol was prepared and added after autoclaving to tryptone for broth and tryptone-1.5% agar for solid medium.All isolates were grown in MTG at 37°C.When indicated, isolates were also grown in cation-adjusted Mueller-Hinton broth (CA-MHB; Sigma-Aldrich) or high-salt LB.
LPS extraction and characterization.LPS was extracted by the hot aqueous-phenol extraction method and characterized using Tricine-SDS-PAGE as previously described, with some minor modifications (84).Overnight incubation in proteinase K was reduced to 3 h with identical results.LPS samples were diluted in loading buffer (188 mM Tris-HCl [pH 6.8], 6% SDS, 15% b-mercaptoethanol, 40% glycerol, and 0.01% bromophenol blue) at a loading dye-to-LPS ratio of 1:2 for storage at 220°C and further diluted at a ratio of 1:1 immediately before being run on a Tricine-SDS-PAGE gel using a 2-mL sample per well.Gels were stained with silver nitrate as previously described (84).
MALDI-TOF mass spectrometry.The characterization of Achromobacter lipid A was performed by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry using a Bruker Autoflex MALDI-TOF spectrometer.Lipid A was isolated from either log-phase-grown bacteria in MTG or from overnight cultures in MTG containing the appropriate subinhibitory concentrations of PmB based on the MIC of each strain.Subinhibitory concentrations were determined by initially trialing 50% MIC and reducing the concentration further if insufficient growth was obtained.The bacteria were centrifuged at 3,220 Â g for 10 min at room temperature before being resuspended in 1 mL of 0.1 M citric acid (62).The samples were centrifuged for 1 min at 14,500 Â g, the supernatant was discarded, and the pellet was resuspended in 1 mL of 0.1 M citric acid.This washing step was repeated once before the performance of lipid A extraction and mass spectrometry as described by Kocsis et al. (62).For validation of lipid A mass spectrometry profiles, lipid A fractions from A. xylosoxidans isolates AC011, AC036, AC061, AC064, and AC080 and A. insuavis AC054 were isolated from cell pellets by the protocol described by El Hamidi et al. (85), with slight modifications.Briefly, an aliquot of each pellet (;1 mg) was washed with 500 mL of chloroform-methanol (1:2, vol/vol) and then with 500 mL of chloroform-methanol-water (3:2:0.25,vol/vol/vol), followed by several steps of centrifugation (1,200 Â g, 30 min).Then, 500 mL of isobutyric acid acid-1 M ammonium hydroxide (5:3, vol/vol) was added to pellets and left at 100°C for 1 h.Upon centrifugation (1,200 Â g, 30 min), the supernatants were collected, lyophilized, and washed several times with methanol, followed by a wash with chloroform-methanol-water (3:1.5:0.25,vol/vol/vol) (63).At this stage, the lipid A fractions were divided into four aliquots, three of which were chemically derivatized to analyze their lipid and sugar contents (see below), whereas the other one was prepared for MALDI-TOF MS analysis and mixed with a matrix solution of 2,4,6-trihydroxyacetophenone (THAP) in methanol-0.1% trifluoroacetic acid-acetonitrile (7:2:1, vol/vol/vol) at a concentration of 75 mg/mL.Lipid A-matrix solutions were spotted in triplicate on a MALDI plate.These MS spectra were recorded in reflectron mode, negative-ion polarity mode, on an ABSCIEX TOF/TOF 5800 Applied Biosystems (Foster City, CA, USA) mass spectrometer equipped with an neodymium-doped yttrium aluminum garnet laser (l = 349 nm), with a 3-ns pulse width and a repetition rate of up to 1,000 Hz and equipped with delayed extraction technology.Each spectrum was a result of the accumulation of 2,000 laser shots.
Chemical analyses of lipid A fractions.To establish the nature of the sugars and lipids composing Achromobacter lipid A, an aliquot of each of the lipid A fractions isolated directly from cell pellets of A. xylosoxidans isolates AC011, AC036, AC061, AC064, and AC080 and A. insuavis AC054 underwent methanolysis (1.25 M hydrochloric acid in methanol, 80°C, 16 h), followed by acetylation (80°C, 20 min) and GC-MS analysis.In parallel, another aliquot of each lipid A was methanolized with 1.25 M hydrochloric acid in methanol (80°C, 2 h) and then extracted three times with hexane.The hexane layer, containing the fatty acids as methyl ester derivatives, was then injected into the GC-MS.Finally, to unequivocally define the nature of the additional hexosamine(s) decorating the Achromobacter lipid A, another aliquot of each lipid A preparation was treated with 50 mL of 48% aqueous hydrofluoric acid (HF) at 4°C for 16 h to remove the phosphate groups.The samples were placed in an ice bath, and the HF was evaporated until dry.Distilled water was added to the HF-treated samples, which were then lyophilized.A mixture of chloroform, methanol, and water (2:1:2, vol/vol/vol) was added to the samples and mixed.

FIG 1
FIG 1 Analysis of the Achromobacter clinical isolate collection (n = 95) and A. xylosoxidans type strain ATCC 27061.(A) Incidence of Achromobacter species within the clinical isolate collection investigated in this study.Species identification was obtained via ribosomal, gyrB, or nrdA PCR.(B) Number of clinical isolates (including ATCC 27061) classed as susceptible, resistant, or heteroresistant to PmB.Isolates with an MIC of #4 mg/mL were deemed susceptible.MICs were established via Etest and broth microdilution (BMD) assay.Heteroresistance was established by population analysis profiling (PAP).

FIG 2
FIG 2 Basic lipid A profile of Achromobacter species.(A) MALDI-TOF spectra of AC075 lipid A from bacteria grown to log phase in lysogeny broth (LB), mineral-tryptone-glycerol (MTG), and cation-adjusted Mueller-Hinton broth (CA-MHB).Spectra were obtained via negative-ion reflectron MALDI-TOF mass spectrometry using a 2,5-dihydroxybenzoic acid matrix and lipid A isolated via citric acid lysis (62).Ion peaks representing the gain of a phosphate group (1P), a hydroxydecanoate acyl chain (1C10), and a glucosamine residue (1GlcN) and the loss of a hydroxyl group (2OH) are indicated by blue dotted lines.(B) Predicted lipid A structures.The additional phosphate group at position 4 (m/z 1,586), the addition of the GlcN residue (tentatively given in its a-anomeric configuration) at position 49 (m/z 1,667), and the addition of a C-10 acyl chain (m/z 1,756) are indicated by dashed boxes.The loss of a 2-OH group on the C-12 acyl chain (m/z 1,491) is indicated by an arrow.

FIG 3
FIG 3 Legend (Continued) group (1P), a palmitate acyl chain (1C16), and a glucosamine residue (1GlcN), as well as the gain (1OH) or loss (2OH) of hydroxyl groups, are indicated by blue dashed lines.(B) Predicted structures of the most abundant ion peaks.Solid boxes indicate hydroxylation or palmitoylation of the lipid A. Dashed boxes indicate the loss of hydroxylation in the secondary acyl chain at position C-3.

FIG 5
FIG 5 LPS profile comparison of the susceptible Achromobacter isolates and PmB-resistant subpopulations following a challenge assay (CR-1) using Tricine-SDS-PAGE and visualized by silver staining.

FIG 6
FIG 6 Lipid A mass spectrometry of A. xylosoxidans clinical isolates and their PmB challenge-resistant subpopulation.Spectra were obtained via negative-ion reflectron MALDI-TOF mass spectrometry using a DHB matrix and lipid A isolated via citric acid lysis following growth in MTG.Structural changes between peaks are indicated by dashed lines and annotated with the component responsible for the change in mass.

TABLE 1
Clinical isolate collection of Achromobacter spp.
(Continued on next page)

TABLE 1 (
Continued) Etest MIC (mg/mL) BMD c MIC (mg/mL) Resistance (S/R d ) PAP HR e a HSCNIMR, Health and Social Care Northern Ireland Microbiology Repository; UKHSA, UK Health and Safety Agency; ATCC, American Type Culture Collection.b BAL, bronchoalveolar lavage; COPD, chronic obstructive pulmonary disease; NCF, Non Cystic Fibrosis.c BMD, broth microdilution assay.d S/R, susceptible or resistant.e PAP HR, heteroresistant by population analysis profile.Y, yes; N, no; NG, no growth.

TABLE 2
PmB susceptibility of early and late isolate pairs from eight pwCF (12)hromobacter sp.cluster II was described by Coward et al.(12).b Y, yes; N, no.

TABLE 3
Peaks indicating loss of a hydroxyl group between early and late isolates from two pwCF

TABLE 4
Comparison of the geometric mean log 10 (CFU/mL) values of the initial clinical isolate (n = 9) and resistant subpopulation (n = 3) of Achromobacter isolates when treated with PmB (200 mg/mL) and a control The upper parts were collected Polymyxin Resistance in Achromobacter Species Microbiology Spectrum Month YYYY Volume XX Issue XX 10.1128/spectrum.03729-22