Nazim Mustafa

In Vitro Selection of Ceftazidime-Avibactam Resistance in Enterobacteriaceae with KPC-3 Carbapenemase

ABSTRACT Ceftazidime-avibactam is active against most Enterobacteriaceae isolates with KPC carbapenemases. We investigated whether this activity could be compromised by mutation. Single-step and multistep selections were attempted using ceftazidime-avibactam (avibactam fixed at 1 or 4 μg/ml) versus two strains each of Enterobacter cloacae and Klebsiella pneumoniae, all with the KPC-3 enzyme. Mutant blaKPC alleles were sequenced, and their parentage was confirmed by typing. Ceftazidime-avibactam selected mutants at up to 16× MIC, with frequencies of ca. 10−9. This contrasted with previous experience for ceftaroline-avibactam, where mutant frequencies under similar conditions were <10−9. The MICs of ceftazidime with 1 μg/ml avibactam for the ceftazidime-avibactam-selected mutants rose from 1 to 8 μg/ml to 16 to >256 μg/ml and those of ceftazidime with 4 μg/ml avibactam from 0.25 to 1 μg/ml to 4 to 128 μg/ml; ceftaroline-avibactam MICs rose less, typically from 0.5 to 1 μg/ml to 1 to 8 μg/ml. The MICs of carbapenems and cephalosporins except ceftazidime and piperacillin-tazobactam were reduced for many mutants. Sequencing of blaKPC revealed point and insertion changes in 12/13 mutants investigated, representing all four parents; one mutant lacked blaKPC changes and possibly had reduced permeability. Amino acid changes commonly involved Ω loop alterations or 1 to 6 amino acid insertions immediately C-terminal to this loop. The most frequent change, seen in four mutants from three strains, was Asp179Tyr, replacing a residue that ordinarily forms a salt bridge to stabilize the Ω loop. Since ceftaroline-avibactam was less affected than ceftazidime-avibactam, we postulate that these mutations increase ceftazidimase specificity rather than conferring avibactam resistance. The clinical relevance remains uncertain.

Clusters of genetically similar isolates of Pseudomonas aeruginosa from multiple hospitals in the UK

Variable number tandem repeat (VNTR) analysis at nine loci of isolates of Pseudomonas aeruginosa submitted to the national reference laboratory from UK hospitals, from over 2000 patients, between June 2010 and June 2012 revealed four widely found types that collectively were received from approximately a fifth of patients, including from those with cystic fibrosis. These types were also prevalent among related submissions from the clinical environment and were received from up to 54 (out of 143) hospitals. Multi-locus sequence typing and blaOXA-50-like sequencing confirmed the clonal relationship within each cluster, and representatives from multiple centres clustered within about 70 % by pulsed-field gel electrophoresis. Illumina sequencing of 12 isolates of cluster A of VNTR profile 8, 3, 4, 5, 2, 3, 5, 2, x (where the repeat number at the last, most discriminatory locus is variable) revealed a large number of variably present targets in the accessory genome and seven of these were sought by PCR among a larger set of isolates. Representatives from patients within a single centre mostly had distinct accessory gene profiles, suggesting that these patients acquired the strain independently, while those with clear epidemiological links shared the same profile. Profiles also varied between representatives from different centres. Epidemiological investigations of widely found types such as these require the use of finer-typing methods, which increasingly will be informed by next generation sequencing.

Molecular epidemiological analysis suggests cross-infection with Pseudomonas aeruginosa is rare in non-cystic fibrosis bronchiectasis

To the Editor: In both cystic fibrosis (CF) and non-CF bronchiectasis (NCFBr) chronic Pseudomonas aeruginosa infection is adversely prognostic [1, 2]. In CF, epidemic infections with specific clones of P. aeruginosa are associated with further adverse outcomes [3, 4]. This cross-infection risk has led to segregation of patients [5]. There are few data on P. aeruginosa cross-infection in NCFBr. As a result, segregation in NCFBr has not been addressed in guidelines [6]. Our aim was to undertake a cross-infection study in NCFBr. This was undertaken in an adult bronchiectasis service in the north-east of England (UK) that is separated from the regional CF unit (sited 2 miles (3 km) away). The service was initiated in 2007 with a weekly specialist clinic without a Pseudomonas -specific clinic. When NCFBr patients are hospitalised, there is a preference for cubicle-based (single-patient room) management, but when cubicles are unavailable, patients are managed in six-bed bays. All patients had computed tomographic confirmation and had predominantly idiopathic or post-infectious bronchiectasis with CF excluded following current guidelines [6]. The study had ethical permission and Caldicott approval (Newcastle and North Tyneside National Research Ethics Service Committee). 56 isolates were selected for analysis. Six were chosen from CF patients as laboratory controls. 50 were NCFBr isolates collected between 2008 and 2011 from …

KPC enzymes in the UK: an analysis of the first 160 cases outside the North-West region

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.

Molecular comparison of isolates of Burkholderia multivorans from patients with cystic fibrosis in the United Kingdom

ABSTRACT Burkholderia multivorans strains from 47 cystic fibrosis (CF) patients in 28 hospitals were compared by pulsed-field gel electrophoresis (PFGE) and flagellin (fliC) PCR-restriction fragment length polymorphism (PCR-RFLP) analysis. A considerable degree of genetic variation was evident, with each patient harboring a strain with a unique PFGE profile. Four sizes of fliC amplicons were produced, and these amplicons gave 13 RFLP types with restriction enzyme MspI. B. multivorans did not appear to spread between patients, suggesting that most CF patients acquire the organism from the natural environment.

Sustained transmission of high-level azithromycin-resistant Neisseria gonorrhoeae in England: an observational study

BACKGROUND Between Nov 3, 2014, and Feb 24, 2017, 70 cases of high-level azithromycin-resistant (HL-AziR; minimum inhibitory concentration [MIC] ≥256 mg/L) Neisseria gonorrhoeae were reported from across England. Whole-genome sequencing was done to investigate this outbreak to determine whether the ongoing outbreak represented clonal spread of an HL-AziR N gonorrhoeae strain identified in Leeds. We also wanted to elucidate the molecular mechanisms of azithromycin resistance in N gonorrhoeae in the UK. METHODS In this observational study, whole-genome sequencing was done on the HL-AziR N gonorrhoeae isolates from England. As comparators, 110 isolates from the UK and Ireland with a range of azithromycin MICs were also sequenced, including eight isolates from Scotland with azithromycin MICs ranging from 0·12 mg/L to 1·00 mg/L that were N gonorrhoeae multi-antigen sequence type 9768 (ST9768), which was the sequence type initially responsible for the outbreak. The presence of mutations or genes associated with azithromycin resistance was also investigated. FINDINGS 37 of the 60 HL-AziR isolates from England belonged to ST9768, and were genetically similar (mean 4·3 single-nucleotide polymorphisms). A 2059A→G mutation was detected in three or all four alleles of the 23S rRNA gene. Five susceptible ST9768 isolates had one mutated 23S rRNA allele and one low-level resistant ST9768 isolate had two mutated alleles. INTERPRETATION Sustained transmission of a successful HL-AziR clone was seen across England. Mutation 2059A→G was found in isolates with lower azithromycin MICs. Azithromycin exposure might have provided the selection pressure for one or two mutated copies of the 23S rRNA gene to recombine with wild-type copies, leading to three or four mutated copies and the HL-AziR phenotype. HL-AziR could emerge in isolates with low azithromycin MICs and eliminate the effectiveness of azithromycin as part of dual therapy for the treatment of gonorrhoea. FUNDING Public Health England.

SPM-1 metallo-β-lactamase-producing Pseudomonas aeruginosa ST277 in the UK.

Metallo-b-lactamase (MBL)-producing Pseudomonas aeruginosa are increasing in incidence in the UK, largely due to the emergence of several internationally recognized ‘high-risk’ clones (Wright et al., 2015). These predominantly produce VIM carbapenemases, but isolates with IMP and NDM MBLs have also been identified, as have occasional isolates with other, rarer, MBLs such as DIM-1 [Antimicrobial Resistance and Healthcare Associated Infections (AMRHAI) Reference Unit, unpublished data].

OXA-48-like carbapenemases in the UK: an analysis of isolates and cases from 2007 to 2014

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.

Carbapenem resistance mediated by blaOXA-181 in Pseudomonas aeruginosa

Sir, The emergence of carbapenemase-producing bacteria worldwide is primarily associated with five acquired carbapenemase families, which belong to Ambler classes A, B and D. Classes A and B are globally dominated by KPC non-metallo-carbapenemases and IMP, NDM and VIM metallo-carbapenemases, respectively. Amongst class D carbapenemases, five subtypes of acquired OXA are restricted mostly to Acinetobacter spp., whereas OXA-48-like enzymes are widespread among the Enterobacteriaceae. OXA-48-like genes have been described in Acinetobacter baumannii, but have not been reported in Pseudomonas aeruginosa. Besides the intrinsic oxacillinase OXA-50, OXA-40 and OXA-198 are the only OXA-type carbapenemases that have been reported in P. aeruginosa to date. OXA-48-like carbapenemases do not confer resistance to third-generation cephalosporins and may confer only low levels of carbapenem resistance or reduced susceptibility, which makes laboratory detection difficult, potentially allowing them to spread undetected. An isolate of P. aeruginosa was submitted for investigation of carbapenem resistance to PHE’s Antimicrobial Resistance and Healthcare Associated Infections (AMRHAI) Reference Unit in 2012. It had been isolated from sputum of a patient who was transferred to the ICU of a UK hospital after a 6 month stay in an ICU in India. MICs were determined by agar dilution and interpreted using BSAC criteria. Carbapenemase genes were sought by commercial microarray (Check-MDR CT102; Check-Points, Wageningen, The Netherlands) and in-house PCRs. Nine-locus variable-number tandem-repeat (VNTR) typing was performed as previously described. WGS was performed using a HiSeq sequencing system (Illumina, Little Chesterford, UK) and data analysed using an in-house bioinformatics pipeline; resistance genes were identified by mapping reads against a library curated in-house from publically accessible databases. The P. aeruginosa isolate was resistant to meropenem (MIC 32 mg/L) and imipenem (MIC 64 mg/L), but without imipenem/ EDTA synergy, ruling out a metallo-carbapenemase. Nevertheless, resistance to carbapenems and piperacillin/tazobactam (MIC 32 mg/L), but with aztreonam relatively spared (MIC 8 mg/L) and susceptibility to ceftazidime (MIC 2 mg/L), raised suspicions that the isolate may harbour a carbapenemase, not least because six other carbapenemase-producing bacteria had been confirmed from the same patient in the preceding 3 weeks, as follows: a blaVIM-positive P. aeruginosa (isolated from sputum); a blaNDM-positive Providencia rettgeri (isolated from a hip wound); and, from rectal swabs, a blaOXA-181-positive Escherichia coli and blaNDM-positive isolates of Citrobacter freundii, Klebsiella pneumoniae and Providencia stuartii. The suspect P. aeruginosa isolate was also screened for acquired carbapenemase genes, with blaOXA-181 detected by microarray, PCR and Sanger sequencing. Carbapenemase activity was detected using a Rapid CARB Screen kit (BioConnections, Knypersley, UK), but the result from the modified Hodge test was negative. Analysis of WGS data identified blaOXA-181 on an 114391 bp contig, where it was located within a 4260 bp transposon that shared 100% identity with nucleotides 2357 to 6616 of pKP3-A (GenBank accession number JN205800.1), indicating that blaOXA-181 was located in Tn2013 as previously described. ISEcp1 was located upstream of blaOXA-181, with truncated lysR, ereA and repA genes located downstream. ISEcp1 has been reported to play a role in the mobilization of blaOXA-181 and other b-lactamase genes via a one-ended transposition process, thus facilitating their wider dissemination.Rather than being located on a ColE-type or IncTplasmid as previously reported in Enterobacteriaceae, blaOXA-181 was integrated into the chromosome between genes encoding a membrane protein and a transcriptional regulator of the PbsX family. Isolation of blaOXA-181-positive P. aeruginosa strains from the same patient in 2014, from a percutaneous endoscopic gastronomy tube insertion site and sputum, indicated that the blaOXA-181 was stably maintained. VNTR typing indicated that the 2012 and 2014 isolates had highly related profiles, which correspond to multilocus ST773, whilst the VIM-positive P. aeruginosa belonged to ST235. Both STs are known as international ‘high-risk clones’, which are commonly associated with multidrug resistance. The gene blaOXA-181 has previously been identified in Enterobacteriaceae originating mainly from the Indian subcontinent, East Asia and the Middle East. Nevertheless, this is the first report of this or any other OXA-48-like carbapenemase in P. aeruginosa and also only the second report of an OXA-48-like carbapenemase outside of the Enterobacteriaceae. The dominant carbapenemases in P. aeruginosa are metallo-enzymes, and most producers can be detected readily using EDTA or dipicolinic acid, which show synergy with carbapenems. However, inferring the presence of a non-metallo-carbapenemase in this species is more difficult owing to its frequent resistance to multiple b-lactams, including carbapenems. Although high-level temocillin resistance (MIC ≥128 mg/L) can serve as a diagnostic marker for OXA-48-like carbapenemases in Enterobacteriaceae, P. aeruginosa is intrinsically resistant to temocillin. This study highlights both the ability of acquired carbapenemase genes to spread beyond their ‘traditional’ host species

Characterization of carbapenemase-producing Enterobacteriaceae in the West Midlands region of England: 2007-14.

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.