Investigating Pseudomonas aeruginosa population structure and frequency of cross-infection in UK cystic fibrosis clinics - a reference laboratory perspective.

BACKGROUND: We aimed to describe the UK Pseudomonas aeruginosa population structure amongst people with cystic fibrosis (PWCF), and to examine evidence for cross-infection. METHODS: Variable Number Tandem Repeat (VNTR) typing was performed on 4640 isolates from 2619 PWCF received from 55 hospital laboratories between 2017 and 2019. A combination of whole genome sequence (WGS)-based analysis of four clusters from one hospital, and epidemiological analysis of shared strains in twelve hospitals evaluated cross-infection. RESULTS: Of 2619 PWCF, 1324 (51%) harboured common clusters or known transmissible strains, while 1295 carried unique strains/those shared among small numbers of patients. Of the former, 9.5% (250 patients) harboured the Liverpool epidemic strain (LES), followed in prevalence by clone C (7.8%; 205 patients), cluster A (5%;130 patients), and cluster D (3.6%; 94 patients). WGS analysis of 10 LES isolates, 9 of cluster D and 6 isolates each of cluster A and clone C from one hospital revealed LES formed the tightest cluster (between 7 and 205 SNPs), and cluster D the loosest (between 53 and 1531 SNPs). Hospital-specific shared strains were found in some centres, although cross-infection was largely historical, with few new acquisitions. Fifty-nine PWCF (2.3%) harboured "high-risk" clones; one ST235 isolate carried a blaIMP-1 allele. CONCLUSION: Of 2619 PWCF who had P. aeruginosa isolates submitted for VNTR, 51% harboured either common clusters or known transmissible strains, of which LES was the most common. Limited evidence of recent patient-to-patient strain transmission was found, suggesting cross-infection prevention measures and surveillance effectively reduce transmission.

Household transmission of non-toxigenic diphtheria toxin gene-bearing Corynebacterium diphtheriae following a cluster of cutaneous cases in a specialist outpatient setting.

Introduction. Combination of PCR and Elek testing to identify toxigenic corynebacteria has revealed organisms described as non-toxigenic toxin-gene bearing (NTTB) Corynebacterium diphtheriae or C. ulcerans (i.e. PCR tox positive; Elek negative). These organisms carry part or all of tox, but are unable to express diphtheria toxin (DT) and present a challenge to clinical and public health case management.Gap analysis/Hypothesis. There are few data on the theoretical risk of NTTB reversion to toxigenicity. This unique cluster and subsequent epidemiologically linked isolates allowed the opportunity to determine any change in DT expression status.Aim. To characterize a cluster of infections due to NTTB in a skin clinic and subsequent cases in two household contacts.Methodology. Epidemiological and microbiological investigations were carried out according to existing national guidance at the time. Susceptibility testing used gradient strips. The tox operon analysis and multi-locus sequence typing (MLST) was derived from whole-genome sequencing. Alignment of the tox operon and phylogenetic analyses were performed using clustalW, mega, the public core-genome MLST (cgMLST) scheme and an in-house bioinformatic single nucleotide polymorphism (SNP) typing pipeline.Results. Isolates of NTTB C. diphtheriae were recovered from four cases (cases 1 to 4) with epidermolysis bullosa attending the clinic. Two further isolates were subsequently recovered from case 4, >18 months later, and from two household contacts (cases 5 and 6) after a further 18 months and 3.5 years, respectively. All eight strains were NTTB C. diphtheriae biovar mitis, belonged to the same sequence type (ST-336) with the same deletion in tox. Phylogenetic analysis showed relatively high diversity between the eight strains with 7-199 SNP and 3-109 cgMLST loci differences between them. The number of SNPs between the three isolates from case 4 and two household contacts (cases 5 and 6) was 44-70 with 28-38 cgMLST loci differences.Conclusions. We report a cluster of NTTB C. diphtheriae cases in a skin clinic and evidence of onward household transmission. We conclude the deletion in the tox was responsible for the non-expression of DT. There was no evidence of reversion to DT expression over the 6.5 year period studied. These data informed revision to guidance in the management of NTTB cases and their contacts in the UK.

Wide distribution of Escherichia coli carrying IncF plasmids containing bla NDM-5 and rmtB resistance genes from hospitalized patients in England.

Introduction. The New Delhi metallo-ß-lactamase (NDM) variant NDM-5 was first described in 2011 in an isolate of Escherichia coli. We noted that a high proportion of isolates of E. coli positive for bla NDM carbapenemase genes submitted to the UK Health Security Agency (formerly Public Health England) between 2019 and mid-2021 carried the bla NDM-5 allele, with many co-harbouring rmtB, rendering them highly resistant to aminoglycosides as well as to most ß-lactams.Hypothesis/Gap Statement. This observation suggested that a common plasmid may be circulating.Aim. To compare these isolates and describe the plasmids carrying these resistance elements.Methodology. All isolates were sequenced on an Illumina platform, with five also subjected to long-read nanopore sequencing to provide complete assemblies. The locations of bla NDM-5, rmtB and other associated genetic elements were identified. Susceptibility testing to a wide range of antibiotics was carried out on representative isolates.Results. The 34 isolates co-harbouring bla NDM-5 and rmtB were from 14 hospital groups and six different regions across England and consisted of 11 distinct sequence types. All carried IncF plasmids. Assembly of the NDM plasmids in five isolates revealed that they carried rmtB and bla NDM-5 in an IncF conjugative plasmid ranging in size from 85.5 to 161 kb. All carried a highly conserved region, previously described in E. coli plasmid pHC105-NDM, that included bla TEM-1B and rmtB followed by sequence bounded by two IS26 elements containing ΔISAba125, bla NDM-5, ble, trpF and tat followed by ISCR1 and an integron with sul1, aadA2 and dfrA12 cassettes. This arrangement has been described in isolates from other countries and continents, suggesting that such plasmids are widely distributed, at least in E. coli, with similar plasmids also found in Klebsiella pneumoniae. Tested isolates were resistant to most antibiotics except colistin, fosfomycin and tigecycline.Conclusion. These observations suggest that conjugative plasmids carrying a highly conserved resistance gene segment have become widespread in England and elsewhere. This study highlights the value of routine whole-genome sequencing in identifying genetic elements responsible for resistance dissemination.

Diversity of carbapenemase-producing Enterobacterales in England as revealed by whole-genome sequencing of isolates referred to a national reference laboratory over a 30-month period.

Introduction. Increasing numbers of carbapenemase-producing Enterobacterales (CPE), which can be challenging to treat, have been referred to the national reference laboratory in England since the early 2000s.Gap Statement/Aim. Previous studies on CPE in the UK have focussed on localized outbreaks. We applied whole-genome sequencing (WGS) to isolates referred to the national reference laboratory over 30 months to inform our understanding of CPE epidemiology in England.Methodology. The first confirmed CPE from each new patient referred by an English diagnostic laboratory between 1 January 2014 and 30 June 2016 was sequenced on an Illumina HiSeq 2500. Multiple isolates from the same patient were included from either different species or the same species with different carbapenemase genes. The data were analysed using an in-house bioinformatics pipeline that determines species identification, multi-locus sequence typing (MLST) profile and antimicrobial resistance gene content.Results. A total of 2658 non-duplicate CPE were sequenced amongst which three host organisms belonging to diverse sequence types (STs) predominated: Klebsiella pneumoniae (1380/2658, 51.9 %; 177 STs), Escherichia coli (723/2658, 27.2 %; 133 STs) and Enterobacter cloacae (294/2658, 11.1 %; 88 STs). Thirty different carbapenemase gene variants were identified, although bla OXA-48-like (1122/2658, 42.2%), bla NDM (692/2658, 26.0 %), bla KPC (571/2658, 21.5 %), bla VIM (100/2658, 3.8 %) and bla IMP (33/2658, 1.2 %) predominated. ST/carbapenemase gene pairings represented widely distributed high-risk clones or clusters at a regional or hospital level.Conclusion. CPE referred to the national reference laboratory are diverse, suggesting multiple introductions to England and a role for horizontal transfer of carbapenemase genes in English CPE epidemiology.

Presence of optrA-mediated linezolid resistance in multiple lineages and plasmids of Enterococcus faecalis revealed by long read sequencing.

Transferable linezolid resistance due to optrA, poxtA, cfr and cfr-like genes is increasingly detected in enterococci associated with animals and humans globally. We aimed to characterize the genetic environment of optrA in linezolid-resistant Enterococcus faecalis isolates from Scotland. Six linezolid-resistant E. faecalis isolated from urogenital samples were confirmed to carry the optrA gene by PCR. Short read (Illumina) sequencing showed the isolates were genetically distinct (>13900 core SNPs) and belonged to different MLST sequence types. Plasmid contents were examined using hybrid assembly of short and long read (Oxford Nanopore MinION) sequencing technologies. The optrA gene was located on distinct plasmids in each isolate, suggesting that transfer of a single plasmid did not contribute to optrA dissemination in this collection. pTM6294-2, BX5936-1 and pWE0438-1 were similar to optrA-positive plasmids from China and Japan, while the remaining three plasmids had limited similarity to other published examples. We identified the novel Tn6993 transposon in pWE0254-1 carrying linezolid (optrA), macrolide (ermB) and spectinomycin [ANT(9)-Ia] resistance genes. OptrA amino acid sequences differed by 0-20 residues. We report multiple variants of optrA on distinct plasmids in diverse strains of E. faecalis. It is important to identify the selection pressures driving the emergence and maintenance of resistance against linezolid to retain the clinical utility of this antibiotic.

Activity of imipenem/relebactam against Pseudomonas aeruginosa producing ESBLs and carbapenemases.

BACKGROUND: ESBL- and carbapenemase-producing Pseudomonas aeruginosa are prevalent in, for example, the Middle East, Eastern Europe and Latin America, though rarer elsewhere. Because P. aeruginosa readily mutate to become carbapenem resistant via loss of OprD, isolates producing ESBLs are often as broadly resistant as those producing carbapenemases. We hypothesized that: (i) relebactam might overcome class A carbapenemases directly in P. aeruginosa; and (ii) relebactam's inhibition of AmpC, which gives a generalized potentiation of imipenem against the species, might restore imipenem susceptibility in OprD-deficient ESBL producers. METHODS: MICs were determined using CLSI agar dilution for P. aeruginosa isolates producing ESBLs, principally VEB types, and for those producing GES-5, KPC and other carbapenemases. RESULTS: Relebactam potentiated imipenem by around 4-8-fold for most P. aeruginosa isolates producing VEB and other ESBLs; however, MICs were typically only reduced to 4-16 mg/L, thus mostly remaining above EUCAST's susceptible range and only partly overlapping CLSI's intermediate range. Strong (approx. 64-fold) potentiation was seen for isolates producing KPC carbapenemases, but only 2-fold synergy for those with GES-5. Predictably, potentiation was not seen for isolates with class B or D carbapenemase activity. CONCLUSIONS: Relebactam did potentiate imipenem against ESBL-producing P. aeruginosa, which are mostly imipenem resistant via OprD loss, but this potentiation was generally insufficient to reduce imipenem MICs to the clinical range. Imipenem resistance owing to KPC carbapenemases was reversed by relebactam in P. aeruginosa, just as for Enterobacterales.

The characterization of mobile colistin resistance (mcr) genes among 33 000 Salmonella enterica genomes from routine public health surveillance in England.

To establish the prevalence of mobile colistin resistance (mcr) genes amongst Salmonella enterica isolates obtained through public health surveillance in England (April 2014 to September 2017), 33 205 S. enterica genome sequences obtained from human, food, animal and environmental isolates were screened for the presence of mcr variants 1 to 8. The mcr-positive genomes were assembled, annotated and characterized according to plasmid type. Nanopore sequencing was performed on six selected isolates with putative novel plasmids, and phylogenetic analysis was used to provide an evolutionary context for the most commonly isolated clones. Fifty-two mcr-positive isolates were identified, of which 32 were positive for mcr-1, 19 for mcr-3 and 1 for mcr-5. The combination of Illumina and Nanopore sequencing identified three novel mcr-3 plasmids and one novel mcr-5 plasmid, as well as the presence of chromosomally integrated mcr-1 and mcr-3. Monophasic S. enterica serovar Typhimurium accounted for 27/52 (52 %) of the mcr-positive isolates, with the majority clustering in clades associated with travel to Southeast Asia. Isolates in these clades were associated with a specific plasmid range and an additional extended-spectrum beta-lactamase genotype. Routine whole-genome sequencing for public health surveillance provides an effective screen for novel and emerging antimicrobial determinants, including mcr. Complementary long-read technologies elucidated the genomic context of resistance determinants, offering insights into plasmid dissemination and linkage to other resistance genes.

Carbapenem-Resistant Enterobacterales, Carbapenem Resistant Organisms, Carbapenemase-Producing Enterobacterales, and Carbapenemase-Producing Organisms: Terminology Past its "Sell-By Date" in an Era of New Antibiotics and Regional Carbapenemase Epidemiology.

Carbapenem resistance in Gram-negative bacteria is a public health concern. Consequently, numerous government and agency reports discuss carbapenem-resistant Enterobacterales (CRE) and carbapenem-resistant organisms (CROs). Unfortunately, these terms are fuzzy. Do they include (1) Proteeae with inherent imipenem resistance; (2) porin-deficient Enterobacterales resistant to ertapenem but not other carbapenems; (3) Enterobacterales with OXA-48-like enzymes that remain "carbapenem susceptible" at breakpoint; and (4) Pseudomonas aeruginosa that merely lack porin OprD? Counting CPE or CPOs is better but still insufficient, because different carbapenemases have differing treatment implications, particularly for new ß-lactam/ß-lactamase inhibitor combinations. At the least, it is essential for authors, journals, and regulatory agencies to specify the carbapenemases meant. The future may demand even greater precision, for mutations can alter hydrolytic activity, and the ability to confer resistance, within carbapenemase families.

Evaluation of the AusDiagnostics MT CRE EU assay for the detection of carbapenemase genes and transferable colistin resistance determinants mcr-1/-2 in MDR Gram-negative bacteria.

Objectives: To evaluate the AusDiagnostics MT CRE EU assay for the detection of carbapenemase and acquired colistin resistance genes in Gram-negative bacteria. Methods: The assay allows the detection of blaKPC, blaOXA-48-like, blaNDM, blaVIM, blaIMP, blaSIM, blaGIM, blaSPM, blaFRI, blaIMI, blaGES (differentiating ESBL and carbapenemase variants), blaSME and mcr-1/-2. It was evaluated against a panel of isolates including Enterobacteriaceae, Pseudomonas spp. and Acinetobacter spp. retrospectively (n = 210) and prospectively (n = 182). Results: The CRE EU assay was able to detect 268/268 carbapenemase genes, with 239 belonging to the 'big five' families (KPC, OXA-48-like, NDM, VIM and IMP) and 29 carbapenemase genes of the SIM, GIM, SPM, FRI, IMI, SME and GES families. It could distinguish between ESBL and carbapenemase variants of GES. It also allowed detection of mcr-1/-2 colistin resistance genes on their own or in isolates co-producing a carbapenemase. Conclusions: The AusDiagnostics MT CRE EU assay offered wide coverage for detection of acquired carbapenemase genes. It required minimal hands-on time and delivered results in less than 4 h from bacterial culture.

Activity of ceftazidime/avibactam against problem Enterobacteriaceae and Pseudomonas aeruginosa in the UK, 2015-16.

Background: Ceftazidime/avibactam combines an established oxyimino-cephalosporin with the first diazabicyclooctane ß-lactamase inhibitor to enter clinical use. We reviewed its activity against Gram-negative isolates, predominantly from the UK, referred for resistance investigation in the first year of routine testing, beginning in July 2015. Methods: Isolates were as received from referring laboratories; there is a bias to submit those with suspected carbapenem resistance. Identification was by MALDI-TOF mass spectroscopy, and susceptibility testing by BSAC agar dilution. Carbapenemase genes were sought by PCR; other resistance mechanisms were inferred using genetic data and interpretive reading. Results: Susceptibility rates to ceftazidime/avibactam exceeded 95% for: (i) Enterobacteriaceae with KPC, GES or other Class A carbapenemases; (ii) Enterobacteriaceae with OXA-48-like enzymes; and (iii) for ESBL or AmpC producers, even when these had impermeability-mediated ertapenem resistance. Almost all isolates with metallo-carbapenemases were resistant. Potentiation of ceftazidime by avibactam was seen for 87% of ceftazidime-resistant Enterobacteriaceae with 'unassigned' ceftazidime resistance mechanisms, including two widely referred groups of Klebsiella pneumoniae where no synergy was seen between cephalosporins and established ß-lactamase inhibitors. Potentiation here may be a diazabicyclooctane/cephalosporin enhancer effect. Activity was seen against Pseudomonas aeruginosa with derepressed AmpC, but not for those with efflux-mediated resistance. Conclusions: Of the available ß-lactams or inhibitor combinations, ceftazidime/avibactam has the widest activity spectrum against problem Enterobacteriaceae, covering all major types except metallo-carbapenemase producers; against P. aeruginosa it has a slightly narrower spectrum than ceftolozane/tazobactam, which also covers efflux-type resistance.

Multicentre evaluation of the BYG Carba v2.0 test, a simplified electrochemical assay for the rapid laboratory detection of carbapenemase-producing Enterobacteriaceae.

Rapid detection of carbapenemase-producing Enterobacteriaceae (CPE) represents a major challenge for microbiology laboratories. We evaluated the BYG Carba v2.0 using a simplified protocol, which detects CPE in less than 30 minutes. This new procedure reduces the hands-on-time from 5 to one minute and only requires a limited amount of material (one to three colonies) thereby preventing the need for subculturing bacterial isolates to reach a larger amount of pure biomass. This multicentre study involved four European reference laboratories. For the 1181 isolates tested across four centres, BYG Carba v2.0 yielded overall sensitivity and specificity of 96.3% (CI95: 94.5-97.5) and 99.7% (CI95: 98.6-100) respectively. Considering only the 670 consecutive isolates tested prospectively, the BYG Carba v2.0 displayed overall positive and negative predictive values of 99.7% (CI95: 95.4-98.9) and 97.5% (CI95: 94.9-98.8). Regarding time to positivity, 85% of CPE detected were positive within ten minutes. The BYG Carba v2.0 is a new highly simplified, rapid and accurate electrochemical assay discriminating between CPE and non-CPE in less than 30 min. The real-time quantified signal allows objective and traceable interpretation of the results.

FRI-2 carbapenemase-producing Enterobacter cloacae complex in the UK.

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.

Activity of ceftolozane/tazobactam against surveillance and 'problem' Enterobacteriaceae, Pseudomonas aeruginosa and non-fermenters from the British Isles.

Background: We assessed the activity of ceftolozane/tazobactam against consecutive isolates collected in the BSAC Bacteraemia Surveillance from 2011 to 2015 and against 'problem' isolates sent to the UK national reference laboratory from July 2015, when routine testing began. Methods: Susceptibility testing was by BSAC agar dilution with resistance mechanisms identified by PCR and interpretive reading. Results: Data were reviewed for 6080 BSAC surveillance isolates and 5473 referred organisms. Ceftolozane/tazobactam had good activity against unselected ESBL producers in the BSAC series, but activity was reduced against ertapenem-resistant ESBL producers, which were numerous among reference submissions. AmpC-derepressed Enterobacter spp. were widely resistant, but Escherichia coli with raised chromosomal AmpC frequently remained susceptible, as did Klebsiella pneumoniae with acquired DHA-1-type AmpC. Carbapenemase-producing Enterobacteriaceae were mostly resistant, except for ceftazidime-susceptible isolates with OXA-48-like enzymes. Ceftolozane/tazobactam was active against 99.8% of the BSAC Pseudomonas aeruginosa isolates; against referred P. aeruginosa it was active against 99.7% with moderately raised efflux, 94.7% with strongly raised efflux and 96.6% with derepressed AmpC. Resistance in P. aeruginosa was largely confined to isolates with metallo-ß-lactamases (MBLs) or ESBLs. MICs for referred Burkholderia spp. and Stenotrophomonas maltophilia were 2-4-fold lower than those of ceftazidime. Conclusions: Ceftolozane/tazobactam is active against ESBL-producing Enterobacteriaceae; gains against other problem Enterobacteriaceae groups were limited. Against P. aeruginosa it overcame the two most prevalent mechanisms (up-regulated efflux and derepressed AmpC) and was active against 51.9% of isolates non-susceptible to all other ß-lactams, rising to 80.9% if ESBL and MBL producers were excluded.

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.