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This article discusses the development of cefiderocol resistance in carbapenem-resistant Acinetobacter baumannii (CRAB) bacteria. CRAB is a leading cause of hospital-acquired infections and is often multidrug-resistant, making treatment challenging. Cefiderocol is a promising treatment option for CRAB infections, but resistance has been reported. The study describes the in vivo development of cefiderocol resistance in a CRAB isolate, which was mediated by a mutation in the promoter region of a specific receptor gene. The mutation resulted in decreased expression of the receptor and increased resistance to cefiderocol. The findings highlight the need for cautious use of cefiderocol to preserve its effectiveness. [Extracted from the article]

This article discusses the emergence of multidrug-resistant Gram-negative bacteria, specifically focusing on Escherichia coli (E. coli) strains that are resistant to broad-spectrum cephalosporins. The study examines a particular E. coli strain isolated from a patient in Switzerland and investigates the mechanisms behind its resistance. The researchers found that the resistance was not due to common mechanisms such as β-lactamases, but rather a modification in the penicillin-binding-protein 3 (PBP-3) sequence. The study highlights the importance of monitoring and understanding these resistance mechanisms, especially in high-risk multidrug-resistant clones like the one identified in this study. [Extracted from the article]

Background: Our primary aim was to test whether cattle-associated fluoroquinolone-resistant (FQ-R) Escherichia coli found on dairy farms are closely phylogenetically related to those causing bacteriuria in humans living in the same 50 × 50 km geographical region suggestive of farm-human sharing. Another aim was to identify risk factors for the presence of FQ-R E. coli on dairy farms.Methods: FQ-R E. coli were isolated during 2017-18 from 42 dairy farms and from community urine samples. Forty-two cattle and 489 human urinary isolates were subjected to WGS, allowing phylogenetic comparisons. Risk factors were identified using a Bayesian regularization approach.Results: Of 489 FQ-R human isolates, 255 were also third-generation-cephalosporin-resistant, with strong genetic linkage between aac(6')Ib-cr and blaCTX-M-15. We identified possible farm-human sharing for pairs of ST744 and ST162 isolates, but minimal core genome SNP distances were larger between farm-human pairs of ST744 and ST162 isolates (71 and 63 SNPs, respectively) than between pairs of isolates from different farms (7 and 3 SNPs, respectively). Total farm fluoroquinolone use showed a positive association with the odds of isolating FQ-R E. coli, while total dry cow therapy use showed a negative association.Conclusions: This work suggests that FQ-R E. coli found on dairy farms have a limited impact on community bacteriuria within the local human population. Reducing fluoroquinolone use may reduce the on-farm prevalence of FQ-R E. coli and this reduction may be greater when dry cow therapy is targeted to the ecology of resistant E. coli on the farm. [ABSTRACT FROM AUTHOR]

The temocillin test solution was prepared with 21.3 mg/L of temocillin and 150 µL was added into one well of a 96-well polystyrene plate; 150 µL of test solution without temocillin was added into a second well. Temocillin is a semi-synthetic 6- -methoxy derivative of ticarcillin, first developed in 1981.[1] Despite being developed over 40 years ago, temocillin's use as an antimicrobial agent was largely overlooked as a treatment option for infections caused by Gram-negative bacteria due to its poor activity against non-fermenters, including I Pseudomonas i spp. and I Acinetobacter i spp.[1] Temocillin has been demonstrated to have an affinity for penicillin-binding protein 3 in I Escherichia coli i [2] and remarkable stability against a plethora of -lactamases including ESBLs and AmpCs (both plasmid and chromosomal);[3],[4] however, it is not clinically useful against bacteria producing class B (e.g. NDM) or class D (e.g. OXA-48-type) carbapenemases, because those latter enzymes readily hydrolyse this antibiotic, often leading to high MICs of temocillin.[5] A recent study evaluating the use of temocillin against ESBL- and AmpC-producing Enterobacterales confirmed the excellent activity of temocillin, and the few high MICs observed were linked mostly to the carriage of multiple -lactamases by corresponding isolates rather than any single -lactamase type.[6] In recent years, a number of countries in Europe, including Belgium, France, Luxembourg and the UK, have revived the use of this antibiotic, predominantly for the treatment of urinary tract infections but also for bloodstream and lower respiratory tract infections.[7],[8] Subsequently, the surveillance of resistance to temocillin is essential and can be easily determined using either routine broth microdilution or disc diffusion testing; however, such tests require 18-24 h to achieve a result. [Extracted from the article]

Objectives: To measure the variability in carbapenem susceptibility conferred by different OxaAb variants, characterize the molecular evolution of oxaAb and elucidate the contribution of OxaAb and other possible carbapenem resistance factors in the clinical isolates using WGS and LC-MS/MS.Methods: Antimicrobial susceptibility tests were performed on 10 clinical Acinetobacter baumannii isolates. Carbapenem MICs were evaluated for all oxaAb variants cloned into A. baumannii CIP70.10 and BM4547, with and without their natural promoters. Molecular evolution analysis of the oxaAb variants was performed using FastTree and SplitsTree4. Resistance determinants were studied in the clinical isolates using WGS and LC-MS/MS.Results: Only the OxaAb variants with I129L and L167V substitutions, OxaAb(82), OxaAb(83), OxaAb(107) and OxaAb(110) increased carbapenem MICs when expressed in susceptible A. baumannii backgrounds without an upstream IS element. Carbapenem resistance was conferred with the addition of their natural upstream ISAba1 promoter. LC-MS/MS analysis on the original clinical isolates confirmed overexpression of the four I129L and L167V variants. No other differences in expression levels of proteins commonly associated with carbapenem resistance were detected.Conclusions: Elevated carbapenem MICs were observed by expression of OxaAb variants carrying clinically prevalent substitutions I129L and L167V. To drive carbapenem resistance, these variants required overexpression by their upstream ISAba1 promoter. This study clearly demonstrates that a combination of IS-driven overexpression of oxaAb and the presence of particular amino acid substitutions in the active site to improve carbapenem capture is key in conferring carbapenem resistance in A. baumannii and other mechanisms are not required. [ABSTRACT FROM AUTHOR]

Objectives: To characterize putative AmpC-hyperproducing third-generation cephalosporin-resistant E. coli from dairy farms and their phylogenetic relationships; to identify risk factors for their presence; and to assess evidence for their zoonotic transmission into the local human population.Methods: Proteomics was used to explain differences in antimicrobial susceptibility. WGS allowed phylogenetic analysis. Multilevel, multivariable logistic regression modelling was used to identify risk factors.Results: Increased use of amoxicillin/clavulanate was associated with an increased risk of finding AmpC hyperproducers on farms. Expansion of cephalosporin resistance in AmpC hyperproducers was seen in farm isolates with marR mutations (conferring cefoperazone resistance) or when AmpC was mutated (conferring fourth-generation cephalosporin and cefoperazone resistance). Phylogenetic analysis confirmed the dominance of ST88 amongst farm AmpC hyperproducers but there was no evidence for acquisition of farm isolates by members of the local human population.Conclusions: Clear evidence was found for recent farm-to-farm transmission of AmpC-hyperproducing E. coli and of adaptive mutations to expand resistance. Whilst there was no evidence of isolates entering the local human population, efforts to reduce third-generation cephalosporin resistance on dairy farms must address the high prevalence of AmpC hyperproducers. The finding that amoxicillin/clavulanate use was associated with an increased risk of finding AmpC hyperproducers is important because this is not currently categorized as a highest-priority critically important antimicrobial and so is not currently targeted for specific usage restrictions in the UK. [ABSTRACT FROM AUTHOR]

Objectives: Third-generation cephalosporin-resistant Escherichia coli from community-acquired urinary tract infections are increasingly reported worldwide. We sought to determine and characterize the mechanisms of cefotaxime resistance employed by urinary E. coli obtained from primary care, over 12 months, in Bristol and surrounding counties in South-West England.Methods: Cefalexin-resistant E. coli isolates were identified from GP-referred urine samples using disc susceptibility testing. Cefotaxime resistance was determined by subsequent plating onto MIC breakpoint plates. β-Lactamase genes were detected by PCR. WGS was performed on 225 isolates and analyses were performed using the Center for Genomic Epidemiology platform. Patient information provided by the referring general practices was reviewed.Results: Cefalexin-resistant E. coli (n=900) isolates were obtained from urines from 146 general practices. Following deduplication by patient approximately 69% (576/836) of isolates were cefotaxime resistant. WGS of 225 isolates identified that the most common cefotaxime-resistance mechanism was blaCTX-M carriage (185/225), followed by plasmid-mediated AmpCs (pAmpCs) (17/225), AmpC hyperproduction (13/225), ESBL blaSHV variants (6/225) or a combination of both blaCTX-M and pAmpC (4/225). Forty-four STs were identified, with ST131 representing 101/225 isolates, within which clade C2 was dominant (54/101). Ciprofloxacin resistance was observed in 128/225 (56.9%) of sequenced isolates, predominantly associated with fluoroquinolone-resistant clones ST131 and ST1193.Conclusions: Most cefalexin-resistant E. coli isolates were cefotaxime resistant, predominantly caused by blaCTX-M carriage. The correlation between cefotaxime resistance and ciprofloxacin resistance was largely attributable to the high-risk pandemic clones ST131 and ST1193. Localized epidemiological data provide greater resolution than regional data and can be valuable for informing treatment choices in the primary care setting. [ABSTRACT FROM AUTHOR]

Background: In Klebsiella pneumoniae, loss-of-function mutations in the transcriptional repressors RamR and OqxR both have an impact on the production of efflux pumps and porins relevant to antimicrobial efflux/entry.Objectives: To define, in an otherwise isogenic background, the relative effects of OqxR and RamR loss-of-function mutations on envelope protein production, envelope permeability and antimicrobial susceptibility. We also investigated the clinical relevance of an OqxR loss-of-function mutation, particularly in the context of β-lactam susceptibility.Methods: Envelope permeability was estimated using a fluorescent dye accumulation assay. Antimicrobial susceptibility was measured using disc testing. Total envelope protein production was quantified using LC-MS/MS proteomics and quantitative RT-PCR was used to measure transcript levels.Results: Loss of RamR or OqxR reduced envelope permeability in K. pneumoniae by 45%-55% relative to the WT. RamR loss activated AcrAB efflux pump production ∼5-fold and this reduced β-lactam susceptibility, conferring ertapenem non-susceptibility even in the absence of a carbapenemase. In contrast, OqxR loss specifically activated OqxAB efflux pump production >10 000-fold. This reduced fluoroquinolone susceptibility but had little impact on β-lactam susceptibility even in the presence of a β-lactamase.Conclusions: Whilst OqxR loss and RamR loss are both seen in K. pneumoniae clinical isolates, only RamR loss significantly stimulates AcrAB efflux pump production. This means that only RamR mutants have significantly reduced β-lactamase-mediated β-lactam susceptibility and therefore represent a greater clinical threat. [ABSTRACT FROM AUTHOR]

Objectives: In Klebsiella pneumoniae, overproduction of RamA results in reduced envelope permeability and reduced antimicrobial susceptibility but clinically relevant resistance is rarely observed. Here we have tested whether RamA overproduction can enhance acquired β-lactam resistance mechanisms in K. pneumoniae and have defined the envelope protein abundance changes upon RamA overproduction during growth in low and high osmolarity media.Methods: Envelope permeability was estimated using a fluorescent dye accumulation assay. β-Lactam susceptibility was measured using disc testing. Total envelope protein production was quantified using LC-MS/MS proteomics and transcript levels were quantified using real-time RT-PCR.Results: RamA overproduction enhanced β-lactamase-mediated β-lactam resistance, in some cases dramatically, without altering β-lactamase production. It increased production of efflux pumps and decreased OmpK35 porin production, though micF overexpression showed that OmpK35 reduction has little impact on envelope permeability. A survey of K. pneumoniae bloodstream isolates revealed ramA hyperexpression in 3 of 4 carbapenemase producers, 1 of 21 CTX-M producers and 2 of 19 strains not carrying CTX-M or carbapenemases.Conclusions: Whilst RamA is not a key mediator of antibiotic resistance in K. pneumoniae on its own, it is potentially important for enhancing the spectrum of acquired β-lactamase-mediated β-lactam resistance. LC-MS/MS proteomics analysis has revealed that this enhancement is achieved predominantly through activation of efflux pump production. [ABSTRACT FROM AUTHOR]

Background: Carbapenemase-producing Enterobacteriaceae (CPE), including KPC-producing Klebsiella pneumoniae (KPC-Kpn), are an increasing threat to patient safety.Objectives: To use WGS to investigate the extent and complexity of carbapenemase gene dissemination in a controlled KPC outbreak.Materials and Methods: Enterobacteriaceae with reduced ertapenem susceptibility recovered from rectal screening swabs/clinical samples, during a 3 month KPC outbreak (2013-14), were investigated for carbapenemase production, antimicrobial susceptibility, variable-number-tandem-repeat profile and WGS [short-read (Illumina), long-read (MinION)]. Short-read sequences were used for MLST and plasmid/Tn4401 fingerprinting, and long-read sequence assemblies for plasmid identification. Phylogenetic analysis used IQTree followed by ClonalFrameML, and outbreak transmission dynamics were inferred using SCOTTI.Results: Twenty patients harboured KPC-positive isolates (6 infected, 14 colonized), and 23 distinct KPC-producing Enterobacteriaceae were identified. Four distinct KPC plasmids were characterized but of 20 KPC-Kpn (from six STs), 17 isolates shared a single pKpQIL-D2 KPC plasmid. All isolates had an identical transposon (Tn4401a), except one KPC-Kpn (ST661) with a single nucleotide variant. A sporadic case of KPC-Kpn (ST491) with Tn4401a-carrying pKpQIL-D2 plasmid was identified 10 months before the outbreak. This plasmid was later seen in two other species and other KPC-Kpn (ST14,ST661) including clonal spread of KPC-Kpn (ST661) from a symptomatic case to nine ward contacts.Conclusions: WGS of outbreak KPC isolates demonstrated blaKPC dissemination via horizontal transposition (Tn4401a), plasmid spread (pKpQIL-D2) and clonal spread (K. pneumoniae ST661). Despite rapid outbreak control, considerable dissemination of blaKPC still occurred among K. pneumoniae and other Enterobacteriaceae, emphasizing its high transmission potential and the need for enhanced control efforts. [ABSTRACT FROM AUTHOR]

Objectives: Detection of rarer carbapenemases is challenging, as it requires molecular assays with comprehensive coverage or the use of phenotypic methods for the detection of carbapenemase activity. We describe a new class A carbapenemase, FRI-2, in an Enterobacter cloacae complex isolate following implementation of an in-house multiplex PCR for the detection of 'rare' class A carbapenemases.Methods: MICs were determined by agar dilution. A carbapenem-resistant E. cloacae complex isolate was tested by PCR for the class A carbapenemases blaKPC, blaFRI, blaIMI, blaGES and blaSME. Carbapenemase activity was assessed using Carba NP and the carbapenem inactivation method. Whole genome and plasmid analyses of the clinical isolate and the FRI-2 transformant were performed by WGS, respectively. Typing was carried out by PFGE.Results: The E. cloacae complex isolate showed resistance to imipenem (MIC = 16 mg/L), meropenem (MIC = 8 mg/L) and ertapenem (MIC = 8 mg/L), but remained susceptible to piperacillin/tazobactam (MIC = 8 mg/L). Carbapenemase activity was confirmed in the isolate by both phenotypic methods. A blaFRI-1-like gene was detected by PCR and analysis of WGS data of the clinical isolate identified an ORF of 885 bp, which showed 97% nucleotide identity with blaFRI-1 and was named blaFRI-2. WGS of the transformant indicated blaFRI-2 was located on a 108 kb IncF/IncR plasmid. The FRI-2-positive E. cloacae complex isolate belonged to a novel ST (ST829).Conclusions: The possible circulation of rarer carbapenemases in clinical settings highlights the role of phenotypic tests to detect carbapenemase activity when molecular assays are negative for the 'big 5' carbapenemase families. [ABSTRACT FROM AUTHOR]

Objectives: OXA-48-like carbapenemases have spread worldwide since 2001. We analysed patient and microbiological data for UK isolates with these enzymes as confirmed by the national reference laboratory from November 2007 to December 2014.Methods: MICs were determined using BSAC agar dilution. Isolates with reduced susceptibility or resistance to at least one carbapenem and high-level resistance to both piperacillin/tazobactam (MICs ≥64 mg/L) and temocillin (MICs ≥128 mg/L) were screened by PCR for bla OXA-48-like genes. The genomes of about half of the isolates were sequenced, with MLST types, resistance genes and plasmid replicon types inferred. Patient data provided by sending laboratories were reviewed.Results: Isolates ( n  =   741) with OXA-48-like carbapenemases were submitted from 111 UK laboratories, representing 536 patients. Almost all (99%; 736 of 741) were Enterobacteriaceae, predominantly Klebsiella pneumoniae (55%; 408), and most (80%; 595) were from inpatients. WGS of 351 non-duplicate isolates identified bla OXA-48 as the most common variant, found in two-thirds (235 of 351) of isolates, followed by bla OXA-181 (68), bla OXA-232 (32), bla OXA-244 (10), bla OXA-484 (5) and bla OXA-245 (1). Among K. pneumoniae (163 of 351), Escherichia coli (114 of 351) and Enterobacter cloacae (42 of 351), 119 STs were identified. Mapping analyses revealed that 63% (222 of 351) of isolates harboured plasmids that shared >99% identity to one of four known plasmids [pOXA-48a (44%; 154 of 351), pOXA-232 (10%; 34 of 351), pOXA181 (9%; 30 of 351) and pKP3-A (1%; 4 of 351)]; the remaining 37% of isolates harboured bla OXA-48-like in unknown environments.Conclusions: OXA-48-like carbapenemases are an increasing problem in the UK. This study highlights both the role of successful plasmids and the polyclonal nature of their dissemination. [ABSTRACT FROM AUTHOR]

Objectives: Carbapenemase-producing Enterobacteriaceae (CPE) have been increasingly reported in the UK since 2003. We analysed patient and isolate data for CPE confirmed by the national reference laboratory from laboratories in the West Midlands region from November 2007 to December 2014.Methods: MICs were determined by BSAC agar dilution methodology and isolates exhibiting resistance to one or more carbapenems were screened for carbapenemase genes by PCR. Plasmid analyses were performed after electro-transformation of carbapenemase-encoding plasmids. WGS was performed on both transformants and clinical isolates. Patient data provided by the sending laboratories were reviewed.Results: During the study period, CPE ( n  = 139) were submitted from 13 laboratories in the West Midlands region, originating from 108 patients and including one environmental isolate. CPE submissions increased significantly from 2009 onwards. Isolates were predominantly Klebsiella pneumoniae (89/139) obtained from inpatients. WGS was performed on all clinical isolates and transformants. After deduplication 119 isolates and 96 transformants remained for analysis. Within these, four families of carbapenemase genes were identified: bla NDM (69/119), bla KPC (26/119), bla OXA-48-like (16/119) and bla VIM (7/119); one isolate carried both bla NDM and bla OXA-48-like . Isolates represented diverse STs and plasmid replicon types. Plasmid analyses identified plasmids of different replicon types encoding bla KPC , bla NDM and bla OXA-48-like genes, found across several species and STs.Conclusions: CPE have been reported increasingly in the West Midlands region over a 7 year period. bla NDM , bla KPC and bla OXA-48-like were the dominant carbapenemase genes and were found in a range of diverse genomic/plasmid environments, highlighting their ability to mobilize across different plasmids, often impeding the detection of outbreaks. [ABSTRACT FROM AUTHOR]

Objectives: Klebsiella pneumoniae carbapenemases (KPCs) have been increasingly reported in the UK since 2003. We analysed patient and isolate data for KPC-positive bacteria confirmed by the national reference laboratory from UK laboratories from August 2003 to August 2014, excluding North-West England, where the epidemiology has previously been studied.Methods: MICs were determined by BSAC agar dilution. Carbapenem-resistant isolates lacking imipenem/EDTA synergy were tested by PCR for blaKPC. MLST and blaKPC sequencing were performed on a subset of isolates. Plasmid analysis was performed by transformation, PCR-based replicon typing and, in some cases, whole-plasmid sequencing. Patient data provided by the sending laboratories were reviewed.Results: Two hundred and ten isolates with KPC enzymes were submitted from 71 UK laboratories outside North-West England, representing 160 patients. All were Enterobacteriaceae, predominantly K. pneumoniae (82%; 173/210), and most (91%; 191/210) were from hospitalized patients. Analysis of 100 isolates identified blaKPC-2 (62%), blaKPC-3 (30%) and blaKPC-4 (8%). Clonal group (CG) 258 was dominant among K. pneumoniae (64%; 54/84), but 21 unrelated STs were also identified. Plasmid analysis identified a diverse range of plasmids representing >11 different replicon types and found in multiple STs and species. Most (34/35) plasmids with IncFIB/FIIK replicons exhibited >99% sequence identity to pKpQIL.Conclusions: KPC enzymes are increasingly detected in Enterobacteriaceae in the UK, albeit without the major outbreaks seen in North-West England. K. pneumoniae CG258 are the dominant hosts, but plasmid spread plays a major role in KPC dissemination between other K. pneumoniae STs and enterobacterial species. [ABSTRACT FROM AUTHOR]

Objectives: To assess the performance of three commercial molecular assays for detecting major families of carbapenemases in pure bacterial isolates. Methods A panel of 450 isolates with previously defined carbapenem resistance mechanisms was tested using the Check-Direct CPE kit, the eazyplex® SuperBug complete A kit and the Xpert® Carba-R kit. Isolates included 438 Enterobacteriaceae and 12 Pseudomonas spp. comprising 100 isolates each with KPC, NDM, VIM or OXA-48-like enzymes, two isolates producing both an NDM and an OXA-48-like enzyme, 24 IMP producers and 24 isolates without a known carbapenemase gene. Discordant results (commercial versus in-house) were investigated using in-house PCR and amplicons were sequenced to define the carbapenemase allele present. Results All three commercial assays detected all isolates with KPC, VIM, NDM and classic OXA-48 carbapenemases (no false-negatives). Isolates producing the OXA-181 variant (n = 18) were not detected by the Xpert® Carba-R kit or the eazyplex® SuperBug complete A kit, but were subsequently detected with modified versions of these kits. Only the Xpert® Carba-R kit could detect IMP carbapenemases, although this was limited to the IMP-1 subgroup. Invalid or false-positive results were either not observed when following the manufacturer's protocols or were eliminated by making simple interpretative adjustments to allow use with bacterial isolates rather than clinical samples. Conclusions Commercial assays offer a reliable means of detecting bacteria with clinically significant carbapenemases. Coverage of some assays required expansion to maximize the sensitivity for OXA-48-like carbapenemases. Choice will ultimately depend on preferred gene coverage, intended throughput, cost and ability to fit into local workflows. [ABSTRACT FROM AUTHOR]

The article discusses a research study on an IMI-2 carbapenemase in a Klebsiella variicola strain isolated in Great Britain. The strain was isolated from an intensive therapy unit patient in 2011 from a soft tissue infection of the buttock. It is stated that this is the first report of an IMI carbapenemase outside of the Enterobacter cloacae complex in Britain.

The article discusses a study related to identification of the first serratia marcescens enzymes (SME) positive isolates by Public Health England (PHE) since screening for carbapenemases began in the early 2000s. Topics discussed include resistance shown for ertapenem, meropenem and imipenem by isolates, circulation of carbapenemases in Great Britain at a low level and weak hydrolytic activity of SME producers towards the third generation cephalosporins.

A letter to the editor is presented which discusses the use of oxacillinase (OXA-50) in mediating carbapenem resistance in Pseudomonas aeruginosa.