In vivo evolution of a Klebsiella pneumoniae capsule defect with wcaJ mutation promotes complement-mediated opsono-phagocytosis during recurrent infection.

BACKGROUND: Klebsiella pneumoniae carbapenemase-producing K. pneumoniae (KPC-Kp) bloodstream infections are associated with high mortality. We studied clinical bloodstream KPC-Kp isolates to investigate mechanisms of resistance to complement, a key host defense against bloodstream infection. METHODS: We tested growth of KPC-Kp isolates in human serum. In serial isolates from a single patient, we performed whole genome sequencing and tested for complement resistance and binding by mixing study, direct ELISA, flow cytometry, and electron microscopy. We utilized an isogenic deletion mutant in phagocytosis assays and an acute lung infection model. RESULTS: We found serum resistance in 16 of 59 (27%) KPC-Kp clinical bloodstream isolates. In five genetically-related bloodstream isolates from a single patient, we noted a loss-of-function mutation in the capsule biosynthesis gene, wcaJ. Disruption of wcaJ was associated with decreased polysaccharide capsule, resistance to complement-mediated killing, and surprisingly, increased binding of complement proteins. Furthermore, an isogenic wcaJ deletion mutant exhibited increased opsono-phagocytosis in vitro and impaired in vivo control in the lung after airspace macrophage depletion in mice. CONCLUSIONS: Loss of function in wcaJ led to increased complement resistance, complement binding, and opsono-phagocytosis, which may promote KPC-Kp persistence by enabling co-existence of increased bloodstream fitness and reduced tissue virulence.

In vitro activity of sulbactam-durlobactam against colistin-resistant and/or cefiderocol-non-susceptible, carbapenem-resistant Acinetobacter baumannii collected in U.S. hospitals.

The activity of a novel ß-lactamase inhibitor combination, sulbactam-durlobactam (SUL-DUR), was tested against 87 colistin-resistant and/or cefiderocol-non-susceptible carbapenem-resistant Acinetobacter baumannii clinical isolates collected from U.S. hospitals between 2017 and 2019. Among them, 89% and 97% were susceptible to SUL-DUR and imipenem plus SUL-DUR, with MIC50/MIC90 values of 2 µg/mL/8 µg/mL and 1 µg/mL/4 µg/mL, respectively. The presence of amino acid substitutions in penicillin-binding protein 3, including previously reported A515V or T526S, was associated with SUL-DUR non-susceptibility.

High-level ceftazidime/avibactam resistance in Escherichia coli conferred by the novel plasmid-mediated ß-lactamase CMY-185 variant.

OBJECTIVES: To characterize a blaCMY variant associated with ceftazidime/avibactam resistance from a serially collected Escherichia coli isolate. METHODS: A patient with an intra-abdominal infection due to recurrent E. coli was treated with ceftazidime/avibactam. On Day 48 of ceftazidime/avibactam therapy, E. coli with a ceftazidime/avibactam MIC of >256 mg/L was identified from abdominal drainage. Illumina and Oxford Nanopore Technologies WGS was performed on serial isolates to identify potential resistance mechanisms. Site-directed mutants of CMY ß-lactamase were constructed to identify amino acid residues responsible for ceftazidime/avibactam resistance. RESULTS: WGS revealed that all three isolates were E. coli ST410. The ceftazidime/avibactam-resistant strain uniquely acquired a novel CMY ß-lactamase gene, herein called blaCMY-185, harboured on an IncI-γ/K1 conjugative plasmid. The CMY-185 enzyme possessed four amino acid substitutions relative to CMY-2, including A114E, Q120K, V211S and N346Y, and conferred high-level ceftazidime/avibactam resistance with an MIC of 32 mg/L. Single CMY-2 mutants did not confer reduced ceftazidime/avibactam susceptibility. However, double and triple mutants containing N346Y previously associated with ceftazidime/avibactam resistance in other AmpC enzymes, conferred ceftazidime/avibactam MICs ranging between 4 and 32 mg/L as well as reduced susceptibility to the newly developed cephalosporin, cefiderocol. Molecular modelling suggested that the N346Y substitution confers the reduction of avibactam inhibition due to steric hindrance between the side chain of Y346 and the sulphate group of avibactam. CONCLUSIONS: We identified ceftazidime/avibactam resistance in E. coli associated with a novel CMY variant. Unlike other AmpC enzymes, CMY-185 appears to require an additional substitution on top of N346Y to confer ceftazidime/avibactam resistance.

Genomic classification and antimicrobial resistance profiling of Streptococcus pneumoniae and Haemophilus influenza isolates associated with paediatric otitis media and upper respiratory infection.

Acute otitis media (AOM) is the most common childhood bacterial infectious disease requiring antimicrobial therapy. Most cases of AOM are caused by translocation of Streptococcus pneumoniae or Haemophilus influenzae from the nasopharynx to the middle ear during an upper respiratory tract infection (URI). Ongoing genomic surveillance of these pathogens is important for vaccine design and tracking of emerging variants, as well as for monitoring patterns of antibiotic resistance to inform treatment strategies and stewardship.In this work, we examined the ability of a genomics-based workflow to determine microbiological and clinically relevant information from cultured bacterial isolates obtained from patients with AOM or an URI. We performed whole genome sequencing (WGS) and analysis of 148 bacterial isolates cultured from the nasopharynx (N = 124, 94 AOM and 30 URI) and ear (N = 24, all AOM) of 101 children aged 6-35 months presenting with AOM or an URI. We then performed WGS-based sequence typing and antimicrobial resistance profiling of each strain and compared results to those obtained from traditional microbiological phenotyping.WGS of clinical isolates resulted in 71 S. pneumoniae genomes and 76 H. influenzae genomes. Multilocus sequencing typing (MSLT) identified 33 sequence types for S. pneumoniae and 19 predicted serotypes including the most frequent serotypes 35B and 3. Genome analysis predicted 30% of S. pneumoniae isolates to have complete or intermediate penicillin resistance. AMR predictions for S. pneumoniae isolates had strong agreement with clinical susceptibility testing results for beta-lactam and non beta-lactam antibiotics, with a mean sensitivity of 93% (86-100%) and a mean specificity of 98% (94-100%). MLST identified 29 H. influenzae sequence types. Genome analysis identified beta-lactamase genes in 30% of H. influenzae strains, which was 100% in agreement with clinical beta-lactamase testing. We also identified a divergent highly antibiotic-resistant strain of S. pneumoniae, and found its closest sequenced strains, also isolated from nasopharyngeal samples from over 15 years ago.Ultimately, our work provides the groundwork for clinical WGS-based workflows to aid in detection and analysis of H. influenzae and S. pneumoniae isolates.

Characterization of KPC-82, a KPC-2 Variant Conferring Resistance to Ceftazidime-Avibactam in a Carbapenem-Nonsusceptible Clinical Isolate of Citrobacter koseri.

KPC-82 is a KPC-2 variant identified in a carbapenem-nonsusceptible Citrobacter koseri that confers high-level resistance to ceftazidime-avibactam. Genomic analysis revealed that blaKPC-82 is carried by a chromosomally integrated Tn4401 transposon (disrupting porin gene phoE) and evolved by a 6-nucleotide tandem repeat duplication causing a two-amino-acid insertion (Ser-Asp) within the Ala267-Ser275 loop. Similar to related KPC variants, KPC-82 showed decreased carbapenemase activity when expressed in a heterologous background and remained susceptible to carbapenem/ß-lactamase inhibitor combinations.

Molecular characterization of clinical carbapenem-resistant Enterobacterales from Qatar.

One hundred forty-nine carbapenem-resistant Enterobacterales from clinical samples obtained between April 2014 and November 2017 were subjected to whole genome sequencing and multi-locus sequence typing. Klebsiella pneumoniae (81, 54.4%) and Escherichia coli (38, 25.5%) were the most common species. Genes encoding metallo-ß-lactamases were detected in 68 (45.8%) isolates, and OXA-48-like enzymes in 60 (40.3%). blaNDM-1 (45; 30.2%) and blaOXA-48 (29; 19.5%) were the most frequent. KPC-encoding genes were identified in 5 (3.6%) isolates. Most common sequence types were E. coli ST410 (8; 21.1%) and ST38 (7; 18.4%), and K. pneumoniae ST147 (13; 16%) and ST231 (7; 8.6%).

Clinical Evolution of AmpC-Mediated Ceftazidime-Avibactam and Cefiderocol Resistance in Enterobacter cloacae Complex Following Exposure to Cefepime.

We report 2 independent patients from whom carbapenem and ceftazidime-avibactam-resistant Enterobacter cloacae complex strains were identified. The ceftazidime-avibactam resistance was attributed to a 2-amino acid deletion in the R2 loop of AmpC ß-lactamase, which concurrently caused resistance to cefepime and reduced susceptibility to cefiderocol, a novel siderophore cephalosporin.

Structural Basis of Reduced Susceptibility to Ceftazidime-Avibactam and Cefiderocol in Enterobacter cloacae Due to AmpC R2 Loop Deletion.

Ceftazidime-avibactam and cefiderocol are two of the latest generation ß-lactam agents that possess expanded activity against highly drug-resistant bacteria, including carbapenem-resistant Enterobacterales Here, we show that structural changes in AmpC ß-lactamases can confer reduced susceptibility to both agents. A multidrug-resistant Enterobacter cloacae clinical strain (Ent385) was found to be resistant to ceftazidime-avibactam and cefiderocol without prior exposure to either agent. The AmpC ß-lactamase of Ent385 (AmpCEnt385) contained an alanine-proline deletion at positions 294 and 295 (A294_P295del) in the R2 loop. AmpCEnt385 conferred reduced susceptibility to ceftazidime-avibactam and cefiderocol when cloned into Escherichia coli TOP10. Purified AmpCEnt385 showed increased hydrolysis of ceftazidime and cefiderocol compared to AmpCEnt385Rev, in which the deletion was reverted. Comparisons of crystal structures of AmpCEnt385 and AmpCP99, the canonical AmpC of E. cloacae complex, revealed that the two-residue deletion in AmpCEnt385 induced drastic structural changes of the H-9 and H-10 helices and the R2 loop, which accounted for the increased hydrolysis of ceftazidime and cefiderocol. The potential for a single mutation in ampC to confer reduced susceptibility to both ceftazidime-avibactam and cefiderocol requires close monitoring.

Clinical and Genomic Epidemiology of Carbapenem-Nonsusceptible Citrobacter spp. at a Tertiary Health Care Center over 2 Decades.

Carbapenem-nonsusceptible Citrobacter spp. (CNSC) are increasingly recognized as health care-associated pathogens. Information regarding their clinical epidemiology, genetic diversity, and mechanisms of carbapenem resistance is lacking. We examined microbiology records of adult patients at the University of Pittsburgh Medical Center (UMPC) Presbyterian Hospital (PUH) from 2000 to 2018 for CNSC, as defined by ertapenem nonsusceptibility. Over this time frame, the proportion of CNSC increased from 4% to 10% (P = 0.03), as did daily defined carbapenem doses/1,000 patient days (6.52 to 34.5; R2 = 0.831; P < 0.001), which correlated with the observed increase in CNSC (lag = 0 years; R2 = 0.660). Twenty CNSC isolates from 19 patients at PUH and other UPMC hospitals were available for further analysis, including whole-genome short-read sequencing and additional antimicrobial susceptibility testing. Of the 19 patients, nearly all acquired CNSC in the health care setting and over half had polymicrobial cultures containing at least one other organism. Among the 20 CNSC isolates, Citrobacter freundii was the predominant species identified (60%). CNSC genomes were compared with genomes of carbapenem-susceptible Citrobacter spp. from UPMC and with other publicly available CNSC genomes. Isolates carrying genes encoding carbapenemases (blaKPC-2,blaKPC-3, and blaNDM-1) were also long-read sequenced, and their carbapenemase-encoding plasmid sequences were compared with one another and with publicly available sequences. Phylogenetic analysis of 102 UPMC Citrobacter genomes showed that CNSC from our setting did not cluster together. Similarly, a global phylogeny of 64 CNSC genomes showed a diverse population structure. Our findings suggest that both local and global CNSC populations are genetically diverse and that CNSC harbor carbapenemase-encoding plasmids found in other Enterobacterales.

High-Level Carbapenem Resistance in OXA-232-Producing Raoultella ornithinolytica Triggered by Ertapenem Therapy.

OXA-232 is an OXA-48-group class D ß-lactamase that hydrolyzes expanded-spectrum cephalosporins and carbapenems at low levels. Clinical strains producing OXA-232 are sometimes susceptible to carbapenems, making it difficult to identify them in the clinical microbiology laboratory. We describe the development of carbapenem resistance in sequential clinical isolates of Raoultella ornithinolytica carrying blaOXA-232 in a hospitalized patient, where the ertapenem MIC increased from 0.5 µg/ml to 512 µg/ml and the meropenem MIC increased from 0.125 µg/ml to 32 µg/ml during the course of ertapenem therapy. Whole-genome sequencing (WGS) analysis identified loss-of-function mutations in ompC and ompF in carbapenem-resistant isolates that were not present in the initial carbapenem-susceptible isolate. Complementation of a carbapenem-resistant isolate with an intact ompF gene resulted in 16- to 32-fold reductions in carbapenem MICs, whereas complementation with intact ompC resulted in a 2-fold reduction in carbapenem MICs. Additionally, blaOXA-232 expression increased 2.9-fold in a carbapenem-resistant isolate. Rapid development of high-level carbapenem resistance in initially carbapenem-susceptible OXA-232-producing R. ornithinolytica under selective pressure from carbapenem therapy highlights the diagnostic challenges in detecting Enterobacteriaceae strains producing this inefficient carbapenemase.

Small-Molecule Inhibitor of FosA Expands Fosfomycin Activity to Multidrug-Resistant Gram-Negative Pathogens.

The spread of multidrug or extensively drug-resistant Gram-negative bacteria is a serious public health issue. There are too few new antibiotics in development to combat the threat of multidrug-resistant infections, and consequently the rate of increasing antibiotic resistance is outpacing the drug development process. This fundamentally threatens our ability to treat common infectious diseases. Fosfomycin (FOM) has an established track record of safety in humans and is highly active against Escherichia coli, including multidrug-resistant strains. However, many other Gram-negative pathogens, including the "priority pathogens" Klebsiella pneumoniae and Pseudomonas aeruginosa, are inherently resistant to FOM due to the chromosomal fosA gene, which directs expression of a metal-dependent glutathione S-transferase (FosA) that metabolizes FOM. In this study, we describe the discovery and biochemical and structural characterization of ANY1 (3-bromo-6-[3-(3-bromo-2-oxo-1H-pyrazolo[1,5-a]pyrimidin-6-yl)-4-nitro-1H-pyrazol-5-yl]-1H-pyrazolo[1,5-a]pyrimidin-2-one), a small-molecule active-site inhibitor of FosA. Importantly, ANY1 potentiates FOM activity in representative Gram-negative pathogens. Collectively, our study outlines a new strategy to expand FOM activity to a broader spectrum of Gram-negative pathogens, including multidrug-resistant strains.

A Prospective Study of Acinetobacter baumannii Complex Isolates and Colistin Susceptibility Monitoring by Mass Spectrometry of Microbial Membrane Glycolipids.

Acinetobacter baumannii is a prevalent nosocomial pathogen with a high incidence of multidrug resistance. Treatment of infections due to this organism with colistin, a last-resort antibiotic of the polymyxin class, can result in the emergence of colistin-resistant strains. Colistin resistance primarily occurs via modifications of the terminal phosphate moieties of lipopolysaccharide-derived lipid A, which reduces overall membrane electronegativity. These modifications are readily identified by mass spectrometry (MS). In this study, we prospectively collected Acinetobacter baumannii complex clinical isolates from a hospital system in Pennsylvania over a 3-year period. All isolates were evaluated for colistin resistance using standard MIC testing by both agar dilution and broth microdilution, as well as genospecies identification and lipid A profiling using MS analyses. Overall, an excellent correlation between colistin susceptibility and resistance, determined by MIC testing, and the presence of a lipid A modification, determined by MS, was observed with a sensitivity of 92.9% and a specificity of 94.0%. Additionally, glycolipid profiling was able to differentiate A. baumannii complex organisms based on their membrane lipids. With the growth of MS use in clinical laboratories, a reliable MS-based glycolipid phenotyping method that identifies colistin resistance in A. baumannii complex clinical isolates, as well as other Gram-negative organisms, represents an alternative or complementary approach to existing diagnostics.

Reduced ceftazidime and ertapenem susceptibility due to production of OXA-2 in Klebsiella pneumoniae ST258.

BACKGROUND: OXA-2 is a class D ß-lactamase that confers resistance to penicillins, as well as narrow-spectrum cephalosporins. OXA-2 was recently reported to also possess carbapenem-hydrolysing activity. Here, we describe a KPC-2-encoding Klebsiella pneumoniae isolate that demonstrated reduced susceptibility to ceftazidime and ertapenem due to production of OXA-2. OBJECTIVES: To elucidate the role of OXA-2 production in reduced ceftazidime and ertapenem susceptibility in a K. pneumoniae ST258 clinical isolate. METHODS: MICs were determined by the agar dilution method. WGS was conducted to identify and compare resistance genes between isolates. Expression of KPC-2 was quantified by quantitative RT-PCR and immunoblotting. OXA-2 was expressed in Escherichia coli TOP10, as well as in K. pneumoniae ATCC 13883, to define the relative contribution of OXA-2 in ß-lactam resistance. Kinetic studies were conducted using purified OXA-2 enzyme. RESULTS: K. pneumoniae 1761 belonged to ST258 and carried both blaKPC-2 and blaOXA-2. However, expression of blaKPC-2 was substantially reduced due to an IS1294 insertion in the promoter region. K. pneumoniae 1761, K. pneumoniae ATCC 13883 and E. coli TOP10 carrying blaOXA-2-harbouring plasmids showed reduced susceptibility to ertapenem and ceftazidime, but meropenem, imipenem and cefepime were unaffected. blaOXA-2 was carried on a 2910 bp partial class 1 integron containing aacA4-blaOXA-2-qacEΔ1-sul1 on an IncA/C2 plasmid, which was not present in the earlier ST258 isolates possessing blaKPC-2 with intact promoters. Hydrolysis of ertapenem by OXA-2 was confirmed using purified enzyme. CONCLUSIONS: Production of OXA-2 was associated with reduced ceftazidime and ertapenem susceptibility in a K. pneumoniae ST258 isolate.

Frequency and Mechanisms of Spontaneous Fosfomycin Nonsusceptibility Observed upon Disk Diffusion Testing of Escherichia coli.

Fosfomycin maintains activity against most Escherichia coli clinical isolates, but the growth of E. coli colonies within the zone of inhibition around the fosfomycin disk is occasionally observed upon susceptibility testing. We aimed to estimate the frequency of such nonsusceptible inner colony mutants and identify the underlying resistance mechanisms. Disk diffusion testing of fosfomycin was performed on 649 multidrug-resistant E. coli clinical isolates collected between 2011 and 2015. For those producing inner colonies inside the susceptible range, the parental strains and their representative inner colony mutants were subjected to MIC testing, whole-genome sequencing, reverse transcription-quantitative PCR (qRT-PCR), and carbohydrate utilization studies. Of the 649 E. coli clinical isolates, 5 (0.8%) consistently produced nonsusceptible inner colonies. Whole-genome sequencing revealed the deletion of uhpT encoding hexose-6-phosphate antiporter in 4 of the E. coli inner colony mutants, while the remaining mutant contained a nonsense mutation in uhpA The expression of uhpT was absent in the mutant strains with uhpT deletion and was not inducible in the strain with the uhpA mutation, unlike in its parental strain. All 5 inner colony mutants had reduced growth on minimal medium supplemented with glucose-6-phosphate. In conclusion, fosfomycin-nonsusceptible inner colony mutants can occur due to the loss of function or induction of UhpT but are rare among multidrug-resistant E. coli clinical strains. Considering that these mutants carry high biological costs, we suggest that fosfomycin susceptibility of strains that generate inner colony mutants can be interpreted on the basis of the zone of inhibition without accounting for the inner colonies.

Phylogenomics of colistin-susceptible and resistant XDR Acinetobacter baumannii.

Background: Acinetobacter baumannii is a healthcare-associated pathogen with high rates of carbapenem resistance. Colistin is now routinely used for treatment of infections by this pathogen. However, colistin use has been associated with development of resistance to this agent. Objectives: To elucidate the phylogenomics of colistin-susceptible and -resistant A. baumannii strain pairs from a cohort of hospitalized patients at a tertiary medical centre in the USA. Methods: WGS data from 21 pairs of colistin-susceptible and -resistant, XDR clinical strains were obtained and compared using phylogeny of aligned genome sequences, assessment of pairwise SNP differences and gene content. Results: Fourteen patients had colistin-resistant strains that were highly genetically related to their own original susceptible strain with a median pairwise SNP distance of 5.5 (range 1-40 SNPs), while seven other strain pairs were divergent with ≥84 SNP differences. In addition, several strains from different patients formed distinct clusters on the phylogeny in keeping with closely linked transmission chains. The majority of colistin-resistant strains contained non-synonymous mutations within the pmrAB locus suggesting a central role for pmrAB mutations in colistin resistance. Excellent genotype-phenotype correlation was also observed for carbapenems, aminoglycosides and tetracyclines. Conclusions: The findings suggest that colistin resistance in the clinical setting arises through both in vivo evolution from colistin-susceptible strains and reinfection by unrelated colistin-resistant strains, the latter of which may involve patient-to-patient transmission.

High-Level Fosfomycin Resistance in Vancomycin-Resistant Enterococcus faecium.

Of 890 vancomycin-resistant Enterococcus faecium isolates obtained by rectal screening from patients in Pittsburgh, Pennsylvania, USA, 4 had MICs >1,024 µg/mL for fosfomycin. These isolates had a Cys119Asp substitution in the active site of UDP-N-acetylglucosamine enolpyruvyl transferase. This substitution increased the fosfomycin MIC >4-fold and rendered this drug inactive in biochemical assays.

Inhibition of Fosfomycin Resistance Protein FosA by Phosphonoformate (Foscarnet) in Multidrug-Resistant Gram-Negative Pathogens.

FosA proteins confer fosfomycin resistance to Gram-negative pathogens via glutathione-mediated modification of the antibiotic. In this study, we assessed whether inhibition of FosA by sodium phosphonoformate (PPF) (foscarnet), a clinically approved antiviral agent, would reverse fosfomycin resistance in representative Gram-negative pathogens. The inhibitory activity of PPF against purified recombinant FosA from Escherichia coli (FosA3), Klebsiella pneumoniae (FosAKP), Enterobacter cloacae (FosAEC), and Pseudomonas aeruginosa (FosAPA) was determined by steady-state kinetic measurements. The antibacterial activity of PPF against FosA in clinical strains of these species was evaluated by susceptibility testing and time-kill assays. PPF increased the Michaelis constant (Km ) for fosfomycin in a dose-dependent manner, without affecting the maximum rate (Vmax) of the reaction, for all four FosA enzymes tested, indicating a competitive mechanism of inhibition. Inhibitory constant (Ki ) values were 22.6, 35.8, 24.4, and 56.3 µM for FosAKP, FosAEC, FosAPA, and FosA3, respectively. Addition of clinically achievable concentrations of PPF (∼667 µM) reduced the fosfomycin MICs by ≥4-fold among 52% of the K. pneumoniae, E. cloacae, and P. aeruginosa clinical strains tested and led to a bacteriostatic or bactericidal effect in time-kill assays among representative strains. PPF inhibits FosA activity across Gram-negative species and can potentiate fosfomycin activity against the majority of strains with chromosomally encoded fosA These data suggest that PPF may be repurposed as an adjuvant for fosfomycin to treat infections caused by some FosA-producing, multidrug-resistant, Gram-negative pathogens.

Structural Modification of Lipopolysaccharide Conferred by mcr-1 in Gram-Negative ESKAPE Pathogens.

mcr-1 was initially reported as the first plasmid-mediated colistin resistance gene in clinical isolates of Escherichia coli and Klebsiella pneumoniae in China and has subsequently been identified worldwide in various species of the family Enterobacteriaceaemcr-1 encodes a phosphoethanolamine transferase, and its expression has been shown to generate phosphoethanolamine-modified bis-phosphorylated hexa-acylated lipid A in E. coli Here, we investigated the effects of mcr-1 on colistin susceptibility and on lipopolysaccharide structures in laboratory and clinical strains of the Gram-negative ESKAPE (Enterococcus faecium, Staphylococcus aureus, K. pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) pathogens, which are often treated clinically by colistin. The effects of mcr-1 on colistin resistance were determined using MIC assays of laboratory and clinical strains of E. coli, K. pneumoniae, A. baumannii, and P. aeruginosa Lipid A structural changes resulting from MCR-1 were analyzed by mass spectrometry. The introduction of mcr-1 led to colistin resistance in E. coli, K. pneumoniae, and A. baumannii but only moderately reduced susceptibility in P. aeruginosa Phosphoethanolamine modification of lipid A was observed consistently for all four species. These findings highlight the risk of colistin resistance as a consequence of mcr-1 expression among ESKAPE pathogens, especially in K. pneumoniae and A. baumannii Furthermore, the observation that lipid A structures were modified despite only modest increases in colistin MICs in some instances suggests more sophisticated surveillance methods may need to be developed to track the dissemination of mcr-1 or plasmid-mediated phosphoethanolamine transferases in general.

Structural modification of LPS in colistin-resistant, KPC-producing Klebsiella pneumoniae.

BACKGROUND: Colistin resistance in Klebsiella pneumoniae typically involves inactivation or mutations of chromosomal genes mgrB, pmrAB or phoPQ, but data regarding consequent modifications of LPS are limited. OBJECTIVES: To examine the sequences of chromosomal loci implicated in colistin resistance and the respective LPS-derived lipid A profiles using 11 pairs of colistin-susceptible and -resistant KPC-producing K. pneumoniae clinical strains. METHODS: The strains were subjected to high-throughput sequencing with Illumina HiSeq. The mgrB gene was amplified by PCR and sequenced. Lipid profiles were determined using MALDI-TOF MS. RESULTS: All patients were treated with colistimethate prior to the isolation of colistin-resistant strains (MIC >2 mg/L). Seven of 11 colistin-resistant strains had deletion or insertional inactivation of mgrB. Three strains, including one with an mgrB deletion, had non-synonymous pmrB mutations associated with colistin resistance. When analysed by MALDI-TOF MS, all colistin-resistant strains generated mass spectra containing ions at m/z 1955 and 1971, consistent with addition of 4-amino-4-deoxy-l-arabinose (Ara4N) to lipid A, whereas only one of the susceptible strains displayed this lipid A phenotype. CONCLUSIONS: The pathway to colistin resistance in K. pneumoniae primarily involves lipid A modification with Ara4N in clinical settings.

Coproduction of KPC-18 and VIM-1 Carbapenemases by Enterobacter cloacae: Implications for Newer ß-Lactam-ß-Lactamase Inhibitor Combinations.

Enterobacter cloacae strain G6809 with reduced susceptibility to carbapenems was identified from a patient in a long-term acute care hospital in Kentucky. G6809 belonged to sequence type (ST) 88 and carried two carbapenemase genes, bla(KPC-18) and bla(VIM-1). Whole-genome sequencing localized bla(KPC-18) to the chromosome and bla(VIM-1) to a 58-kb plasmid. The strain was highly resistant to ceftazidime-avibactam. Insidious coproduction of metallo-ß-lactamase with KPC-type carbapenemase has implications for the use of next-generation ß-lactam-ß-lactamase inhibitor combinations.