Heteroresistance to Colistin in Klebsiella pneumoniae Associated with Alterations in the PhoPQ Regulatory System

A multidrug-resistant Klebsiella pneumoniae isolate exhibiting heteroresistance to colistin was investigated. The colistin-resistant subpopulation harbored a single amino acid change (Asp191Tyr) in protein PhoP, which is part of the PhoPQ two-component system that activates pmrHFIJKLM expression responsible for l-aminoarabinose synthesis and polymyxin resistance. Complementation assays with a wild-type phoP gene restored full susceptibility to colistin. Then, analysis of the colistin-susceptible subpopulation showed a partial deletion (25 bp) in the phoP gene compared to that in the colistin-resistant subpopulation. That deletion disrupted the reading frame of phoP, leading to a longer and inactive protein (255 versus 223 amino acids long). This is the first report showing the involvement of mutation(s) in PhoP in colistin resistance. Furthermore, this is the first study to decipher the mechanisms leading to colistin heteroresistance in K. pneumoniae.     

Alterations in Two-Component Regulatory Systems of phoPQ and pmrAB Are Associated with Polymyxin B Resistance in Clinical Isolates of Pseudomonas aeruginosa

Polymyxins are often the only option to treat acquired multidrug-resistant Pseudomonas aeruginosa. Polymyxin susceptibility in P. aeruginosa PAO1 is associated with the lipopolysaccharide structure that is determined by arnBCADTEF and modulated by phoPQ and pmrAB. We examined five clonally unrelated clinical isolates of polymyxin B-resistant P. aeruginosa to investigate the molecular basis of polymyxin resistance. All isolates grew with 4 μg/ml polymyxin B (MIC, 8 μg/ml), whereas P. aeruginosa PAO1 grew with 0.25 μg/ml polymyxin B (MIC, 0.5 μg/ml). The resistant isolates were converted to susceptible ones (the MICs fell from 8 to 0.5 μg/ml) following the introduction of phoPQ (four isolates) and pmrAB (one isolate), which had been cloned from strain PAO1. DNA sequence analysis revealed that a single-nucleotide substitution in three isolates replaced a single amino acid of PhoQ, the deletion of 17 nucleotides in one isolate truncated the protein of PhoQ, and two nucleotide substitutions in one isolate replaced two amino acids of PmrB. The involvement of these amino acid substitutions or the truncated protein of PhoQ and PmrB in polymyxin B resistance was confirmed using strain PAO1 lacking phoPQ or pmrAB that was transformed by phoPQ or pmrAB containing the amino acid substitutions or the truncated protein. The resistant clinical isolates were sensitized by the inactivation of arnBCADTEF (the MICs fell from 8 to 0.5 μg/ml). These results suggest that polymyxin B resistance among clinical isolates of P. aeruginosa is associated with alterations in two-component regulatory systems of phoPQ or pmrAB.Pseudomonas aeruginosa is a nosocomial gram-negative opportunistic pathogen that causes a variety of infections (e.g., urinary tract, respiratory, skin, soft tissue, etc.) (3, 12, 18, 19). P. aeruginosa accounts for 11 to 14% of all nosocomial infections and is a major problem for people hospitalized with cancer, cystic fibrosis, or burns (3). Treatment usually involves the use of one or more antibiotics, such as β-lactams, aminoglycosides, or quinolones. Combination therapy usually is recommended for P. aeruginosa infections, as it decreases the risk of antibiotic resistance and enhances the eradication rate. Despite the use of combination therapy, there are numerous reports of the emergence of multidrug-resistant P. aeruginosa. Polymyxins (polymyxin B and colistin) often have been the last resort to treat such isolates (1, 3, 27). However, polymyxin B resistance in multidrug-resistant clinical isolates has been reported (4, 6, 11, 25).The mode of action and the resistance mechanism to polymyxin B has been studied extensively using the reference P. aeruginosa strain PAO1. Polymyxin B is a polycationic lipopeptide antibiotic that interacts with a negatively charged lipid A moiety of the lipopolysaccharide (LPS) of gram-negative bacteria and leads to cell lysis and death (26). Resistance to polymyxin B is caused by the inhibition of the interactions between the antibiotic and the lipid A moiety of the LPS, and the inhibition is based on modifications of lipid A so that it is less negatively charged. LPS modification occurs by the addition of 4-amino-4-deoxy-l-arabinose to lipid A under limiting nutrient conditions, such as 20 μM magnesium or calcium, and is directed by an arnBCADTEF operon modulated by two-component regulatory systems of phoPQ and pmrAB (9, 13, 14, 20). In normal growth conditions, the two-component regulatory systems strictly repress arnBCADTEF, resulting in a phenotype of intrinsic susceptibility to polymyxins (8, 16, 17). Therefore, it is postulated that any interruption of the regulatory systems derepresses arnBCADTEF with resulting resistance to polymyxins. Indeed, in vitro-acquired polymyxin B-resistant P. aeruginosa PAK carried a single-amino-acid substitution in PmrB (20).Although polymyxin-resistant clinical isolates of P. aeruginosa have been reported increasingly worldwide (5, 6, 10, 11, 25), the genetic basis for the resistance in these clinical isolates is unclear. This study aimed to examine the molecular details of polymyxin B resistance among clinical isolates of P. aeruginosa. We characterized five polymyxin B-resistant clinical isolates of P. aeruginosa and found that four isolates carried a single-amino-acid substitution or protein truncation in PhoQ, and one isolate carried dual amino acid substitutions in PmrB. The involvement of the amino acid substitutions or protein truncation in polymyxin B resistance was confirmed by introducing phoPQ or pmrAB containing the amino acid substitutions or protein truncation into P. aeruginosa PAO1 lacking phoPQ or pmrAB.     

Preservation of Acquired Colistin Resistance in Gram-Negative Bacteria

Colistin-resistant mutants were obtained from 17 colistin-susceptible strains of Acinetobacter baumannii, Pseudomonas aeruginosa, Klebsiella pneumoniae, and Escherichia coli. The stability of colistin resistance in these mutants was investigated. Three of four colistin-resistant P. aeruginosa mutants recovered colistin susceptibility in colistin-free medium; however, colistin-susceptible revertants were obtained from only one strain each of A. baumannii and E. coli. No susceptible revertants were obtained from K. pneumoniae mutants.     

Emergence of a carbapenem-resistant and colistin-heteroresistant Enterobacter cloacae clinical isolate in Japan

A carbapenem-resistant and colistin-heteroresistant clinical isolate of Enterobacter cloacae was obtained from an inpatient in Okinawa, Japan. The minimum inhibitory concentrations of both imipenem and meropenem were 32 μg/mL. The isolate showed heteroresistance to colistin using the Etest method and resistance to colistin using the broth microdilution method. It had a disrupted ompC and a mutation in the promoter region of bla_ACT-2, but did not harbor any genes encoding carbapenemase. The disruption of ompC and the mutation in bla_ACT-2 was associated with the carbapenem resistance of this isolate. This isolate also had mutations in pmrAB and phoPQ encoding two-component regulatory systems, which may be associated with colistin heteroresistance.     

Extracellular DNA Acidifies Biofilms and Induces Aminoglycoside Resistance in Pseudomonas aeruginosa

Biofilms consist of surface-adhered bacterial communities encased in an extracellular matrix composed of DNA, exopolysaccharides, and proteins. Extracellular DNA (eDNA) has a structural role in the formation of biofilms, can bind and shield biofilms from aminoglycosides, and induces antimicrobial peptide resistance mechanisms. Here, we provide evidence that eDNA is responsible for the acidification of Pseudomonas aeruginosa planktonic cultures and biofilms. Further, we show that acidic pH and acidification via eDNA constitute a signal that is perceived by P. aeruginosa to induce the expression of genes regulated by the PhoPQ and PmrAB two-component regulatory systems. Planktonic P. aeruginosa cultured in exogenous 0.2% DNA or under acidic conditions demonstrates a 2- to 8-fold increase in aminoglycoside resistance. This resistance phenotype requires the aminoarabinose modification of lipid A and the production of spermidine on the bacterial outer membrane, which likely reduce the entry of aminoglycosides. Interestingly, the additions of the basic amino acid l-arginine and sodium bicarbonate neutralize the pH and restore P. aeruginosa susceptibility to aminoglycosides, even in the presence of eDNA. These data illustrate that the accumulation of eDNA in biofilms and infection sites can acidify the local environment and that acidic pH promotes the P. aeruginosa antibiotic resistance phenotype.     

Resistance to Colistin Associated with a Single Amino Acid Change in Protein PmrB among Klebsiella pneumoniae Isolates of Worldwide Origin

A series of colistin-resistant Klebsiella pneumoniae isolates recovered from different countries was investigated in order to evaluate the involvement of the PmrA/PmrB two-component system in this resistance. Six isolates possessed a mutated PmrB protein, which is encoded by the pmrB gene, part of the pmrCAB operon involved in lipopolysaccharide modification. The same amino acid substitution (Thr157Pro) in PmrB was identified in the six isolates. The six isolates belonged to four distinct clonal groups, recovered in South Africa (sequence type 14 [ST14]), Turkey (ST101), and Colombia (ST258 and ST15). Three out of the four clones produced a carbapenemase, OXA-181, OXA-48, or KPC-3, while a single isolate did not produce any carbapenemase. Expression assays revealed an overexpression of the pmrA (70-fold), pmrB (70-fold), pmrC (170-fold), and pmrK (40-fold) genes in the pmrB-mutated isolate compared to expression of the pmrB wild-type isogenic K. pneumoniae isolate, confirming that the PmrB substitution was responsible for increased expression levels of those genes. Complementation assays leading to the expression of a wild-type PmrB protein restored the susceptibility to colistin in all isolates, confirming that the substitution in PmrB was responsible for the resistance phenotype. This study identified a key amino acid located in the PmrB protein as being responsible for the overexpression of pmrCAB and pmrHFIJKLM operons, leading to resistance to colistin.     

The Two-Component System CprRS Senses Cationic Peptides and Triggers Adaptive Resistance in Pseudomonas aeruginosa Independently of ParRS

Cationic antimicrobial peptides pass across the outer membrane by interacting with negatively charged lipopolysaccharide (LPS), leading to outer membrane permeabilization in a process termed self-promoted uptake. Resistance can be mediated by the addition of positively charged arabinosamine through the action of the arnBCADTEF operon. We recently described a series of two-component regulators that lead to the activation of the arn operon after recognizing environmental signals, including low-Mg²⁺ (PhoPQ, PmrAB) or cationic (ParRS) peptides. However, some peptides did not activate the arn operon through ParRS. Here, we report the identification of a new two-component system, CprRS, which, upon exposure to a wide range of antimicrobial peptides, triggered the expression of the LPS modification operon. Thus, mutations in the cprRS operon blocked the induction of the arn operon in response to several antimicrobial peptides independently of ParRS but did not affect the response to low Mg²⁺. Distinct patterns of arn induction were identified. Thus, the responses to polymyxins were abrogated by either parR or cprR mutations, while responses to other peptides, including indolicidin, showed differential dependency on the CprRS and ParRS systems in a concentration-dependent manner. It was further demonstrated that, following exposure to inducing antimicrobial peptides, cprRS mutants did not become adaptively resistant to polymyxins as was observed for wild-type cells. Our microarray studies demonstrated that the CprRS system controlled a quite modest regulon, indicating that it was quite specific to adaptive peptide resistance. These findings provide greater insight into the complex regulation of LPS modification in Pseudomonas aeruginosa, which involves the participation of at least 4 two-component systems.     

Independent emergence of colistin-resistant Acinetobacter spp. isolates from Korea

High colistin resistance rates in Acinetobacter spp. were recently reported in Korean hospitals (J. Antimicrob. Chemother 2007;60:1163). In this study, we investigated if colistin-resistant Acinetobacter spp. isolates from Korean hospitals disseminated clonally or emerged independently. Multilocus sequence typing (MLST) analysis was performed for 58 colistin-resistant Acinetobacter spp. isolates: 8 isolates of the Acinetobacter baumannii subgroup A, 16 isolates of the A. baumannii subgroup B, and 34 isolates of the genomic species 13TU. A phylogenetic tree inferred from concatenated sequences of 7 MLST loci showed a clear distinction among the 3 Acinetobacter groups. In the MLST analysis, most colistin-resistant Acinetobacter spp. isolates showed different allele profiles at the 7 loci; that is, they belonged to different clones. Despite the clear distinction between the 3 Acinetobacter groups, interrelationships among the 3 groups were not consistent within the gene trees. In addition, some isolates showed clustering incongruent with their species or group identities in some gene trees. MLST analysis indicated that most colistin-resistant Acinetobacter spp. isolates from Korean hospitals arose independently. Considering the increasing use of colistin and the high recombination rate of Acinetobacter spp., independent but frequent emergence of colistin resistance in Acinetobacter spp. isolates is of great concern.     

The Combination of Colistin and Doripenem Is Synergistic against Klebsiella pneumoniae at Multiple Inocula and Suppresses Colistin Resistance in an In Vitro Pharmacokinetic/Pharmacodynamic Model

Multidrug-resistant (MDR) Klebsiella pneumoniae may require combination therapy. We systematically investigated bacterial killing with colistin and doripenem mono- and combination therapy against MDR K. pneumoniae and emergence of colistin resistance. A one-compartment in vitro pharmacokinetic/pharmacodynamic model was employed over a 72-h period with two inocula (～10(6) and ～10(8) CFU/ml); a colistin-heteroresistant reference strain (ATCC 13883) and three clinical isolates (colistin-susceptible FADDI-KP032 [doripenem resistant], colistin-heteroresistant FADDI-KP033, and colistin-resistant FADDI-KP035) were included. Four combinations utilizing clinically achievable concentrations were investigated. Microbiological responses were examined by determining log changes and population analysis profiles (for emergence of colistin resistance) over 72 h. Against colistin-susceptible and -heteroresistant isolates, combinations of colistin (constant concentration regimens of 0.5 or 2 mg/liter) plus doripenem (steady-state peak concentration [C(max)] of 2.5 or 25 mg/liter over 8 h; half-life, 1.5 h) generally resulted in substantial improvements in bacterial killing at both inocula. Combinations were additive or synergistic against ATCC 13883, FADDI-KP032, and FADDI-KP033 in 9, 9, and 14 of 16 cases (4 combinations at 6, 24, 48, and 72 h) at the 10(6)-CFU/ml inoculum and 14, 11, and 12 of 16 cases at the 10(8)-CFU/ml inoculum, respectively. Combinations at the highest dosage regimens resulted in undetectable bacterial counts at 72 h in 5 of 8 cases (4 isolates at 2 inocula). Emergence of colistin-resistant subpopulations in colistin-susceptible and -heteroresistant isolates was virtually eliminated with combination therapy. Against the colistin-resistant isolate, colistin at 2 mg/liter plus doripenem (C(max), 25 mg/liter) at the low inoculum improved bacterial killing. This investigation provides important information for optimization of colistin-doripenem combinations.     

An Amphipathic Undecapeptide with All d-Amino Acids Shows Promising Activity against Colistin-Resistant Strains of Acinetobacter baumannii and a Dual Mode of Action

Multiple strains of Acinetobacter baumannii have developed multidrug resistance (MDR), leaving colistin as the only effective treatment. The cecropin-α–melittin hybrid BP100 (KKLFKKILKYL-NH₂) and its analogs have previously shown activity against a wide array of plant and human pathogens. In this study, we investigated the in vitro antibacterial activities of 18 BP100 analogs (four known and 14 new) against the MDR A. baumannii strain ATCC BAA-1605, as well as against a number of other clinically relevant human pathogens. Selected peptides were further evaluated against strains of A. baumannii that acquired resistance to colistin due to mutations of the lpxC, lpxD, pmrA, and pmrB genes. The novel analogue BP214 showed antimicrobial activity at 1 to 2 μM and a hemolytic 50% effective concentration (EC₅₀) of >150 μM. The lower activity of its enantiomer suggests a dual, specific and nonspecific mode of action. Interestingly, colistin behaved antagonistically to BP214 when pmrAB and lpxC mutants were challenged.     

Real-Time Sequencing To Decipher the Molecular Mechanism of Resistance of a Clinical Pan-Drug-Resistant Acinetobacter baumannii Isolate from Marseille,France

We compare the whole-genome sequences of two multidrug-resistant clinical Acinetobacter baumannii isolates recovered in the same patient before (ABIsac_ColiS susceptible to colistin and rifampin only) and after (ABIsac_ColiR resistant to colistin and rifampin) treatment with colistin and rifampin. We decipher all the molecular mechanisms of antibiotic resistance, and we found mutations in the rpoB gene and in the PmrAB two-component system explaining resistance to rifampin and colistin in ABIsac_ColiR, respectively.     

In vivo emergence of colistin resistance in Acinetobacter baumannii clinical isolates of sequence type 357 during colistin treatment

This study was performed to investigate the mechanisms of in vivo acquisition of colistin resistance in A. baumannii during colistin treatment. Three colistin-susceptible/resistant pairs of A. baumannii were recovered from patients who underwent colistin treatment. All of the 6 isolates included in this study shared an identical sequence type (ST), ST375, and they showed identical SmaI-macrorestriction patterns by pulsed-field gel electrophoresis. The individual colistin-resistant isolates harbored distinct mutations in the pmrB gene. Mutations detected in the pmrB gene were Ala227Val, Pro233Ser, and frame shift from Phe26. In matrix-assisted laser desorption ionization–time of flight analysis, colistin-resistant isolates were different from their colistin-susceptible counterparts, and they showed additional distinct peaks at 1852 m/z, 1937 m/z, 1954 m/z, 1975 m/z, 2034 m/z, and 2157 m/z. In vivo selection of colistin-resistant A. baumannii occurred independently in strains of ST357 during colistin treatment, and the strains acquired colistin resistance via mutations in the pmrB gene resulting in modification of lipid A components.     

Unique Structural Modifications Are Present in the Lipopolysaccharide from Colistin-Resistant Strains of Acinetobacter baumannii

Acinetobacter baumannii is a nosocomial opportunistic pathogen that can cause severe infections, including hospital-acquired pneumonia, wound infections, and sepsis. Multidrug-resistant (MDR) strains are prevalent, further complicating patient treatment. Due to the increase in MDR strains, the cationic antimicrobial peptide colistin has been used to treat A. baumannii infections. Colistin-resistant strains of A. baumannii with alterations to the lipid A component of lipopolysaccharide (LPS) have been reported; specifically, the lipid A structure was shown to be hepta-acylated with a phosphoethanolamine (pEtN) modification present on one of the terminal phosphate residues. Using a tandem mass spectrometry platform, we provide definitive evidence that the lipid A isolated from colistin-resistant A. baumannii MAC204 LPS contains a novel structure corresponding to a diphosphoryl hepta-acylated lipid A structure with both pEtN and galactosamine (GalN) modifications. To correlate our structural studies with clinically relevant samples, we characterized colistin-susceptible and -resistant isolates obtained from patients. These results demonstrated that the clinical colistin-resistant isolate had the same pEtN and GalN modifications as those seen in the laboratory-adapted A. baumannii strain MAC204. In summary, this work has shown complete structure characterization including the accurate assignment of acylation, phosphorylation, and glycosylation of lipid A from A. baumannii, which are important for resistance to colistin.     

Correlation of β-Lactamase Production and Colistin Resistance among Enterobacteriaceae Isolates from a Global Surveillance Program

The increasing use of carbapenems for treating multidrug-resistant (MDR) Gram-negative bacterial infections has contributed to the global dissemination of carbapenem-resistant Enterobacteriaceae (CRE). Serine and metallo-β-lactamases (MBLs) that hydrolyze carbapenems have become prevalent and endemic in some countries, necessitating the use of older classes of agents, such as colistin. A total of 19,719 isolates of Enterobacteriaceae (excluding Proteeae and Serratia spp., which have innate resistance to colistin) were collected from infected patients during 2012 and 2013 in a global surveillance program and tested for antimicrobial susceptibility using CLSI methods. Isolates of CRE were characterized for carbapenemases and extended-spectrum β-lactamases (ESBLs) by PCR and sequencing. Using EUCAST breakpoints, the rate of colistin susceptibility was 98.4% overall, but it was reduced to 88.0% among 482 carbapenemase-positive isolates. Colistin susceptibility was higher among MBL-positive isolates (92.6%) than those positive for a KPC (87.9%) or OXA-48 (84.2%). Of the agents tested, only tigecycline (MIC₉₀, 2 to 4 μg/ml) and aztreonam-avibactam (MIC₉₀, 0.5 to 1 μg/ml) consistently tested with low MIC values against colistin-resistant, ESBL-positive, and carbapenemase-positive isolates. Among the 309 (1.6%) colistin-resistant isolates from 10 species collected in 38 countries, 58 carried a carbapenemase that included KPCs (38 isolates), MBLs (6 isolates), and OXA-48 (12 isolates). These isolates were distributed globally (16 countries), and 95% were Klebsiella pneumoniae. Thirty-nine (67.2%) isolates carried additional ESBL variants of CTX-M, SHV, and VEB. This sample of Enterobacteriaceae demonstrated a low prevalence of colistin resistance overall. However, the wide geographic dispersion of colistin resistance within diverse genus and species groups and the higher incidence observed among carbapenemase-producing MDR pathogens are concerning.