Activity of RX-04 Pyrrolocytosine Protein Synthesis Inhibitors against Multidrug-Resistant Gram-Negative Bacteria

Pyrrolocytosines RX-04A-D are designed to bind to the bacterial 50S ribosomal subunit differently from currently-used antibiotics. The four analogs had broad anti-Gram-negative activity: RX-04A inhibited 94.7% of clinical Enterobacteriaceae, Acinetobacter baumannii and Pseudomonas aeruginosa at 0.5-4 μg/ml, with no MICs >8 μg/ml. MICs for multi-resistant carbapenemase producers were up to two-fold higher than for control strains, with values ≥8 μg/ml for one Serratia isolate with porin and efflux lesions. mcr-1 did not affect MICs

New insights into the regulatory pathways associated with the activation of the stringent response in bacterial resistance to the PBP2-targeted antibiotics, mecillinam and OP0595/RG6080

Objectives: The diazabicyclooctane β-lactamase inhibitor OP0595 (RG6080) also acts as an antibiotic, targeting PBP2 in Enterobacteriaceae, but this activity is vulnerable to mutational resistance. We used WGS to investigate the basis of this resistance. Methods: Twenty OP0595-selected mutants, comprising four derived from each of five different Escherichia coli strains, were sequenced on Illumina HiSeq. Reads from each mutant were mapped to the assembled genome of the corresponding parent. A variant-calling file generated with Samtools was parsed to determine genetic alterations. Results: Besides OP0595, the mutants consistently showed decreased susceptibility to mecillinam, which likewise targets PBP2, and grew as stable round forms in the presence of subinhibitory concentrations of OP0595. Among the 20 mutants, 18 had alterations in genes encoding tRNA synthase and modification functions liable to induce expression of the RpoS sigma factor through activation of the stringent response or had mutations suppressing inactivators of RpoS or the stringent response signal-degrading enzyme, SpoT. TolB was inactivated in one mutant: this activates RcsBC regulation and was previously associated with mecillinam resistance. The mechanism of resistance remained unidentified in one mutant. Both the RpoS and RcsBC systems regulate genes of cell division, including ftsAQZ that can compensate for loss or inhibition of PBP2, allowing survival of the challenged bacteria as stable round forms, as seen. Conclusions: WGS identified the global stringent response signal, entailing induction of RpoS, as the main mediator of mutational resistance to OP0595 in E. coli

Trends in ExPEC serogroups in the UK and their significance

We thank the British Society for Antimicrobial Chemotherapy for kindly providing E. coli bloodstream isolates from the BSAC Bacteraemia Resistance Surveillance Programme (2011), and all the staff at PHE’s Gastrointestinal Bacteria Reference Unit for their guidance and patience during the serogrouping process. This work was performed as part of a PhD study funded by PHE

Plasmid classification in an era of whole-genome sequencing: application in studies of antibiotic resistance epidemiology

Plasmids are extra-chromosomal genetic elements ubiquitous in bacteria, and commonly transmissible between host cells. Their genomes include variable repertoires of ‘accessory genes,’ such as antibiotic resistance genes, as well as ‘backbone’ loci which are largely conserved within plasmid families, and often involved in key plasmid-specific functions (e.g., replication, stable inheritance, mobility). Classifying plasmids into different types according to their phylogenetic relatedness provides insight into the epidemiology of plasmid-mediated antibiotic resistance. Current typing schemes exploit backbone loci associated with replication (replicon typing), or plasmid mobility (MOB typing). Conventional PCR-based methods for plasmid typing remain widely used. With the emergence of whole-genome sequencing (WGS), large datasets can be analyzed using in silico plasmid typing methods. However, short reads from popular high-throughput sequencers can be challenging to assemble, so complete plasmid sequences may not be accurately reconstructed. Therefore, localizing resistance genes to specific plasmids may be difficult, limiting epidemiological insight. Long-read sequencing will become increasingly popular as costs decline, especially when resolving accurate plasmid structures is the primary goal. This review discusses the application of plasmid classification in WGS-based studies of antibiotic resistance epidemiology; novel in silico plasmid analysis tools are highlighted. Due to the diverse and plastic nature of plasmid genomes, current typing schemes do not classify all plasmids, and identifying conserved, phylogenetically concordant genes for subtyping and phylogenetics is challenging. Analyzing plasmids as nodes in a network that represents gene-sharing relationships between plasmids provides a complementary way to assess plasmid diversity, and allows inferences about horizontal gene transfer to be made

Activity of ceftazidime/avibactam against problem Enterobacteriaceae and Pseudomonas aeruginosa in the UK, 2015-2016

Background: Ceftazidime/avibactam combines an established oxyimino-cephalosporin with the first diazabicyclooctane β-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 β-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 β-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

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 - 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 (MIC ≥ 64 mg/L) and temocillin (MICs ≥ 128 mg/L) were screened by PCR for blaOXA-48-like genes. The genomes of around 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%; 736/741) were Enterobacteriaceae, predominantly Klebsiella pneumoniae (55%; 408), and most (80%; 595) were from inpatients. WGS of 351 non-duplicate isolates identified blaOXA-48 as the most common variant, found in two-thirds (235/351) of isolates, followed by blaOXA-181 (68), blaOXA-232 (32), blaOXA-244 (10), blaOXA-484 (5) and blaOXA-245 (1). Among K. pneumoniae (163/351), E.coli (114/351), and E. cloacae (42/351), 119 STs were identified. Mapping analyses revealed that 63% (222/351) of isolates harboured plasmids that shared >99% identity to one of four known plasmids; pOXA-48a (44%; 154/351), pOXA-232 (10%; 34/351), pOXA181 (9%; 30/351), and pKP3-A (1%; 4/351); the remaining 37% of isolates harboured blaOXA-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 polyclonal nature of their dissemination

OXA-1 β-lactamase and non-susceptibility to penicillin/β-lactamase inhibitor combinations among ESBL-producing Escherichia coli

Background ESBL-producing Escherichia coli have expanded globally since the turn of the century and present a major public health issue. Their in vitro susceptibility to penicillin/inhibitor combinations is variable, and clinical use of these combinations against ESBL producers remains controversial. We hypothesized that this variability related to co-production of OXA-1 penicillinase. Methods During a national study we collected 293 ESBL-producing E. coli from bacteraemias, determined MICs by BSAC agar dilution, and undertook genomic sequencing with Illumina methodology. Results The collection was dominated by ST131 (n = 188 isolates, 64.2%) and bla CTX-M-15 (present in 229 isolates, 78.2%); over half the isolates (159/293, 54.3%) were ST131 with bla CTX-M-15. bla OXA-1 was found in 149 ESBL producers (50.9%) and bla TEM-1/191 in 137 (46.8%). Irrespective of whether all isolates were considered, or ST131 alone, there were strong associations (P < 0.001) between co-carriage of bla OXA-1 and reduced susceptibility to penicillin/inhibitor combinations, whereas there was no significant association with co-carriage of bla TEM-1/191. For piperacillin/tazobactam the modal MIC rose from 2 mg/L in the absence of bla OXA-1 to 8 or 16 mg/L in its presence; for co-amoxiclav the shift was smaller, from 4 or 8 to 16 mg/L, but crossed the breakpoint. bla OXA-1 was strongly associated with co-carriage also of aac(6′)-Ib-cr, which compromises amikacin and tobramycin. Conclusions Co-carriage of OXA-1, a penicillinase with weak affinity for inhibitors, is a major correlate of resistance to piperacillin/tazobactam and co-amoxiclav in E. coli and is commonly associated with co-carriage of aac(6′)-Ib-cr, which narrows aminoglycoside options

Methicillin-resistant Staphylococcus aureus emerged long before the introduction of methicillin into clinical practice.

BACKGROUND: The spread of drug-resistant bacterial pathogens poses a major threat to global health. It is widely recognised that the widespread use of antibiotics has generated selective pressures that have driven the emergence of resistant strains. Methicillin-resistant Staphylococcus aureus (MRSA) was first observed in 1960, less than one year after the introduction of this second generation beta-lactam antibiotic into clinical practice. Epidemiological evidence has always suggested that resistance arose around this period, when the mecA gene encoding methicillin resistance carried on an SCCmec element, was horizontally transferred to an intrinsically sensitive strain of S. aureus. RESULTS: Whole genome sequencing a collection of the first MRSA isolates allows us to reconstruct the evolutionary history of the archetypal MRSA. We apply Bayesian phylogenetic reconstruction to infer the time point at which this early MRSA lineage arose and when SCCmec was acquired. MRSA emerged in the mid-1940s, following the acquisition of an ancestral type I SCCmec element, some 14 years before the first therapeutic use of methicillin. CONCLUSIONS: Methicillin use was not the original driving factor in the evolution of MRSA as previously thought. Rather it was the widespread use of first generation beta-lactams such as penicillin in the years prior to the introduction of methicillin, which selected for S. aureus strains carrying the mecA determinant. Crucially this highlights how new drugs, introduced to circumvent known resistance mechanisms, can be rendered ineffective by unrecognised adaptations in the bacterial population due to the historic selective landscape created by the widespread use of other antibiotics

Complete Genome Sequence of a Colistin-Resistant Uropathogenic Escherichia coli Sequence Type 131 fimH22 Strain Harboring mcr-1 on an IncHI2 Plasmid, Isolated in Riyadh, Saudi Arabia

ABSTRACT We report the complete genome sequence of a colistin-resistant strain of uropathogenic Escherichia coli, isolated in January 2013 at King Abdulaziz Medical City (KAMC), Riyadh, Saudi Arabia. The isolate (named SA186) was sequence type 131 (ST131) and belonged to serotype O25b-H4 and clade B (fimH22).</jats:p