Accuracy of Different Bioinformatics Methods in Detecting Antibiotic Resistance and Virulence Factors from Staphylococcus aureus Whole-Genome Sequences.

In principle, whole-genome sequencing (WGS) can predict phenotypic resistance directly from a genotype, replacing laboratory-based tests. However, the contribution of different bioinformatics methods to genotype-phenotype discrepancies has not been systematically explored to date. We compared three WGS-based bioinformatics methods (Genefinder [read based], Mykrobe [de Bruijn graph based], and Typewriter [BLAST based]) for predicting the presence/absence of 83 different resistance determinants and virulence genes and overall antimicrobial susceptibility in 1,379 Staphylococcus aureus isolates previously characterized by standard laboratory methods (disc diffusion, broth and/or agar dilution, and PCR). In total, 99.5% (113,830/114,457) of individual resistance-determinant/virulence gene predictions were identical between all three methods, with only 627 (0.5%) discordant predictions, demonstrating high overall agreement (Fleiss' kappa = 0.98, P < 0.0001). Discrepancies when identified were in only one of the three methods for all genes except the cassette recombinase, ccrC(b). The genotypic antimicrobial susceptibility prediction matched the laboratory phenotype in 98.3% (14,224/14,464) of cases (2,720 [18.8%] resistant, 11,504 [79.5%] susceptible). There was greater disagreement between the laboratory phenotypes and the combined genotypic predictions (97 [0.7%] phenotypically susceptible, but all bioinformatic methods reported resistance; 89 [0.6%] phenotypically resistant, but all bioinformatics methods reported susceptible) than within the three bioinformatics methods (54 [0.4%] cases, 16 phenotypically resistant, 38 phenotypically susceptible). However, in 36/54 (67%) cases, the consensus genotype matched the laboratory phenotype. In this study, the choice between these three specific bioinformatic methods to identify resistance determinants or other genes in S. aureus did not prove critical, with all demonstrating high concordance with each other and phenotypic/molecular methods. However, each has some limitations; therefore, consensus methods provide some assurance.

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.

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.

Association of Novel Nonsynonymous Single Nucleotide Polymorphisms in ampD with Cephalosporin Resistance and Phylogenetic Variations in ampC, ampR, ompF, and ompC in Enterobacter cloacae Isolates That Are Highly Resistant to Carbapenems.

InEnterobacter cloacae, the genetic lesions associated with derepression of the AmpC ß-lactamase include diverse single nucleotide polymorphisms (SNPs) and/or indels in theampDandampRgenes and SNPs inampC, while diverse SNPs in the promoter region or SNPs/indels within the coding sequence of outer membrane proteins have been described to alter porin production leading to carbapenem resistance. We sought to define the underlying mechanisms conferring cephalosporin and carbapenem resistance in a collection ofE. cloacaeisolates with unusually high carbapenem resistance and no known carbapenemase and, in contrast to many previous studies, considered the SNPs we detected in relation to the multilocus sequence type (MLST)-based phylogeny of our collection. Whole-genome sequencing was applied on the most resistant isolates to seek novel carbapenemases, expression ofampCwas measured by reverse transcriptase PCR, and porin translation was detected by SDS-PAGE. SNPs occurring inampC,ampR,ompF, andompCgenes (and their promoter regions) were mostly phylogenetic variations, relating to the isolates' sequence types, whereas nonsynonymous SNPs inampDwere associated with derepression of AmpC and cephalosporin resistance. The additional loss of porins resulted in high-level carbapenem resistance, underlining the clinical importance of chromosomal mutations among carbapenem-resistantE. cloacae.

Rise of multidrug-resistant non-vaccine serotype 15A Streptococcus pneumoniae in the United Kingdom, 2001 to 2014.

Conjugate vaccines have reduced pneumococcal disease in vaccinated children and unvaccinated adults, but non-vaccine serotypes are of concern, particularly if antibiotic resistant. We reviewed Streptococcus pneumoniae collected via: (i) the British Society for Antimicrobial Chemotherapy (BSAC) surveillances from 2001-2014; (ii) Public Health England's (PHE) invasive isolate surveillance from 2005-2014 and (iii) referral to PHE for resistance investigation from 2005-2014. Serotype 15A increased in all series, with many representatives showing triple resistance to macrolides, tetracyclines and penicillin. 15A was consistently among the 10 most prevalent serotypes from 2011 in PHE and BSAC invasive isolate/bacteraemia surveillance but never previously; 26-33% of these invasive 15A isolates had triple resistance. BSAC respiratory isolates were only serotyped in 2013/14 and 2014/15 (October to September); 15A was most prevalent serotype in both periods, comprising 9-11% of isolates, 38-48% of them with triple resistance. Serotype 15A represented 0-4% of S. pneumoniae referred to PHE for reference investigation annually until 2008 but rose to 29% (2013) and 32% (2014). Almost all multidrug-resistant 15A isolates were sequence type (ST) 63 variants, whereas susceptible 15A isolates were clonally diverse. The rise of serotype 15A suggests that pneumococcal conjugate vaccines will need ongoing adaptation.

Epidemiology of carbapenemase-producing Enterobacterales in England, May 2015-March 2019: national enhanced surveillance findings and approach.

BACKGROUND: In response to increasing numbers of carbapenemase-producing Enterobacterales (CPE) in England, Public Health England (PHE) launched an electronic reporting system (ERS) for the enhanced surveillance of carbapenemase-producing Gram-negative bacteria. Our study aimed to describe system engagement and the epidemiology of CPE in England. METHODS: Engagement with the ERS was assessed by calculating the proportion of referrals submitted this system. ERS data were extracted and cases defined as patients with CPE isolated from a screening or clinical specimen in England between 1st May 2015 to 31st March 2019. Descriptive summary statistics for each variable were prepared. RESULTS: The ERS processed 12,656 suspected CPE reports. Uptake of the ERS by local microbiology laboratories varied, with approximately 70% of referrals made via the ERS by April 2016; this steadily decreased after March 2018. Six-thousand eight-hundred and fifty-seven cases were included in the analysis. Most cases were from colonised patients (80.6%) rather than infected, and the majority were inpatients in acute hospital settings (87.3%). Carbapenemases were most frequently detected in Klebsiella pneumoniae (39.1%) and Escherichia coli (30.3%). The most frequently identified carbapenemase families were OXA-48-like (45.1%) and KPC (26.4%). Enhanced data variables were poorly completed. CONCLUSIONS: The ERS has provided some insight into the epidemiology of CPE in England. An increasing number of routine diagnostic laboratories have introduced methods to routinely identify acquired carbapenemases and PHE has modified its approach to ensure robust surveillance, which is an essential aspect of an effective response to prevent and control the spread of CPE.

Virulence and competitiveness: testing the relationship during inter- and intraspecific mixed infections.

Understanding the reasons why different parasites cause different degrees of harm to their hosts is an important objective in evolutionary biology. One group of models predicts that if hosts are infected with more than one strain or species of parasite, then competition between the parasites will select for higher virulence. While this idea makes intuitive sense, empirical data to support it are rare and equivocal. We investigated the relationship between fitness and virulence during both inter- and intraspecific competition for a fungal parasite of insects, Metarhizium anisopliae. Contrary to theoretical expectations, competition favored parasite strains with either a lower or a higher virulence depending on the competitor: when in interspecific competition with an entomopathogenic nematode, Steinernema feltiae, less virulent strains of the fungus were more successful, but when competing against conspecific fungi, more virulent strains were better competitors. We suggest that the nature of competition (direct via toxin production when competing against the nematode, indirect via exploitation of the host when competing against conspecific fungal strains) determines the relationship between virulence and competitive ability.