Showing papers by "Michelle J Cole published in 2018"

Gonorrhoea treatment failure caused by a Neisseria gonorrhoeae strain with combined ceftriaxone and high-level azithromycin resistance, England, February 2018

Abstract: We describe a gonorrhoea case with combined high-level azithromycin resistance and ceftriaxone resistance. In February 2018, a heterosexual male was diagnosed with gonorrhoea in the United Kingdom following sexual intercourse with a locally resident female in Thailand and failed treatment with ceftriaxone plus doxycycline and subsequently spectinomycin. Resistance arose from two mechanisms combining for the first time in a genetic background similar to a commonly circulating strain. Urgent action is essential to prevent further spread.

Public health surveillance of multidrug-resistant clones of Neisseria gonorrhoeae in Europe: a genomic survey.

Abstract: Summary Background Traditional methods for molecular epidemiology of Neisseria gonorrhoeae are suboptimal. Whole-genome sequencing (WGS) offers ideal resolution to describe population dynamics and to predict and infer transmission of antimicrobial resistance, and can enhance infection control through linkage with epidemiological data. We used WGS, in conjunction with linked epidemiological and phenotypic data, to describe the gonococcal population in 20 European countries. We aimed to detail changes in phenotypic antimicrobial resistance levels (and the reasons for these changes) and strain distribution (with a focus on antimicrobial resistance strains in risk groups), and to predict antimicrobial resistance from WGS data. Methods We carried out an observational study, in which we sequenced isolates taken from patients with gonorrhoea from the European Gonococcal Antimicrobial Surveillance Programme in 20 countries from September to November, 2013. We also developed a web platform that we used for automated antimicrobial resistance prediction, molecular typing ( N gonorrhoeae multi-antigen sequence typing [NG-MAST] and multilocus sequence typing), and phylogenetic clustering in conjunction with epidemiological and phenotypic data. Findings The multidrug-resistant NG-MAST genogroup G1407 was predominant and accounted for the most cephalosporin resistance, but the prevalence of this genogroup decreased from 248 (23%) of 1066 isolates in a previous study from 2009–10 to 174 (17%) of 1054 isolates in this survey in 2013. This genogroup previously showed an association with men who have sex with men, but changed to an association with heterosexual people (odds ratio=4·29). WGS provided substantially improved resolution and accuracy over NG-MAST and multilocus sequence typing, predicted antimicrobial resistance relatively well, and identified discrepant isolates, mixed infections or contaminants, and multidrug-resistant clades linked to risk groups. Interpretation To our knowledge, we provide the first use of joint analysis of WGS and epidemiological data in an international programme for regional surveillance of sexually transmitted infections. WGS provided enhanced understanding of the distribution of antimicrobial resistance clones, including replacement with clones that were more susceptible to antimicrobials, in several risk groups nationally and regionally. We provide a framework for genomic surveillance of gonococci through standardised sampling, use of WGS, and a shared information architecture for interpretation and dissemination by use of open access software. Funding The European Centre for Disease Prevention and Control, The Centre for Genomic Pathogen Surveillance, Orebro University Hospital, and Wellcome.

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

Abstract: Summary 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.

Stably high azithromycin resistance and decreasing ceftriaxone susceptibility in Neisseria gonorrhoeae in 25 European countries, 2016

Abstract: The European Gonococcal Antimicrobial Surveillance Programme (Euro-GASP) performs annual sentinel surveillance of Neisseria gonorrhoeae susceptibility to therapeutically relevant antimicrobials across the European Union/European Economic Area (EU/EEA). We present the Euro-GASP results from 2016 (25 countries), linked to patient epidemiological data, and compared with data from previous years. Agar dilution and minimum inhibitory concentration (MIC) gradient strip methodologies were used to determine the antimicrobial susceptibility (using EUCAST breakpoints) of 2660 N. gonorrhoeae isolates from 25 countries across the EU/EEA. Significance of differences compared with Euro-GASP results in previous years was analysed using Z-tests. No isolates with resistance to ceftriaxone (MIC > 0.125 mg/L) were detected in 2016 (one in 2015). However, the proportion of isolates with decreased susceptibility to ceftriaxone (MICs from 0.03 mg/L to 0.125 mg/L) increased significantly (p = 0.01) from 2015 to 2016. There were 14 (0.5%) isolates with ceftriaxone MICs 0.125 mg/L (on the resistance breakpoint), of which one isolate was resistant to azithromycin and four showed intermediate susceptibility to azithromycin. Cefixime resistance was detected in 2.1% of isolates in 2016 compared with 1.7% in 2015 (p = 0.26) and azithromycin resistance in 7.5% in 2016 compared with 7.1% in 2015 (p = 0.74). Seven (0.3%) isolates from five countries displayed high-level azithromycin resistance (MIC≥256 mg/L) in 2016 compared with five (0.2%) isolates in 2015. Resistance rate to ciprofloxacin was 46.5% compared with 49.4% in 2015 (p = 0.06). No isolates were resistant to spectinomycin and the MICs of gentamicin remained stable compared with previous years. Overall AMR rates in gonococci in EU/EEA remained stable from 2015 to 2016. However, the ceftriaxone MIC distribution shifted away from the most susceptible (≤0.016 mg/L) and the proportion of isolates with decreased susceptibility to ceftriaxone increased significantly. This development is of concern as current European gonorrhoea management guideline recommends ceftriaxone 500 mg plus azithromycin 2 g as first-line therapy. With azithromycin resistance at 7.5%, the increasing ceftriaxone MICs might soon threaten the effectiveness of this therapeutic regimen and requires close monitoring.

Detection of markers predictive of macrolide and fluoroquinolone resistance in Mycoplasma genitalium from patients attending sexual health services

Abstract: Objectives Resistance to both macrolides and fluoroquinolones has been reported in Mycoplasma genitalium; however, due to limited diagnostics, studies are often small and confined to specific geographical areas. This study sought to determine the rate of predicted resistance in M. genitalium -positive specimens referred for diagnostic testing. Methods Seventy-four M. genitalium -positive specimens, referred to the national reference laboratory (2010-2013) from 19 centres across England, were blinded and anonymised. Specimens were examined for markers predictive of resistance to macrolides and fluoroquinolones using PCR followed by sequence analysis of 23 S rRNA gene, or gyrA and parC , respectively. Results 23 S rRNA gene PCR sequencing revealed that 82.4% (61/74) of specimens harboured a single nucleotide polymorphism (SNP) associated with macrolide resistance. Differences were observed between the rates of predicted macrolide resistance in male (95.1% (58/61)) and female (23.1% (3/13)) patients (P = gyrA sequences; and 58/61 (95.1%) had wild-type parC genes. Three specimens (3/61 4.9%) had SNPs in the parC gene associated with fluoroquinolone treatment failure, and all three also had predicted resistance to macrolides. Conclusions Eighty-two per cent and 4.9% of M. genitalium specimens had SNPs associated with macrolide and fluoroquinolone resistance, respectively. Due to lack of widespread availability of testing for M. genitalium in the UK, this study sample was likely to be sourced from patients who may have already failed first-line macrolide therapy. Nevertheless, this study highlights the need for both greater access to M. genitalium diagnostics and genetic antimicrobial resistance testing.

Prevalence of and factors associated with MDR Neisseria gonorrhoeae in England and Wales between 2004 and 2015: analysis of annual cross-sectional surveillance surveys.

Abstract: Objectives To describe trends in prevalence, susceptibility profile and risk factors for MDR Neisseria gonorrhoeae (MDR-NG) in England and Wales. Methods Isolates from 16 242 gonorrhoea episodes at sexual health clinics within the Gonococcal Resistance to Antimicrobials Surveillance Programme (GRASP) underwent antimicrobial susceptibility testing. MDR-NG was defined as resistance to ceftriaxone, cefixime or azithromycin, plus at least two of penicillin, ciprofloxacin and spectinomycin. Trends in resistance are presented for 2004-15; prevalence and logistic regression analyses for MDR-NG cover the period of the most recent treatment guideline (ceftriaxone plus azithromycin), 2011-15. Results Between 2004 and 2015, the proportion of N. gonorrhoeae isolates fully susceptible to all antimicrobial classes fell from 80% to 46%, with the proportion resistant to multiple (two or more) classes increasing from 7.3% to 17.5%. In 2011-15, 3.5% of isolates were MDR-NG, most of which were resistant to cefixime (100% in 2011, decreasing to 36.9% in 2015) and/or azithromycin (4.2% in 2011, increasing to 84.3% in 2015). After excluding azithromycin-resistant isolates, modal azithromycin MICs were higher in MDR versus non-MDR isolates (0.5 versus 0.125 mg/L), with similar results for ceftriaxone (modal MICs 0.03 versus ≤0.002 mg/L). After adjustment for confounders, MDR-NG was more common among isolates from heterosexual men, although absolute differences in prevalence were small [4.6% versus 3.3% (MSM) and 2.5% (women)]. Conclusions N. gonorrhoeae is becoming less susceptible to available antimicrobials. Since 2011, a minority of isolates were MDR-NG; however, MICs of azithromycin or ceftriaxone (first-line therapies) for many of these were elevated. These findings highlight the importance of continued antimicrobial stewardship for gonorrhoea.

Cysteamine, an Endogenous Aminothiol, and Cystamine, the Disulfide Product of Oxidation, Increase Pseudomonas aeruginosa Sensitivity to Reactive Oxygen and Nitrogen Species and Potentiate Therapeutic Antibiotics against Bacterial Infection

Abstract: Cysteamine is an endogenous aminothiol produced in mammalian cells as a consequence of coenzyme A metabolism through the activity of the vanin family of pantetheinase ectoenzymes. It is known to have a biological role in oxidative stress, inflammation, and cell migration. There have been several reports demonstrating anti-infective properties targeting viruses, bacteria, and even the malarial parasite. We and others have previously described broad-spectrum antimicrobial and antibiofilm activities of cysteamine. Here, we go further to demonstrate redox-dependent mechanisms of action for the compound and how its antimicrobial effects are, at least in part, due to undermining bacterial defenses against oxidative and nitrosative challenges. We demonstrate the therapeutic potentiation of antibiotic therapy against Pseudomonas aeruginosa in mouse models of infection. We also demonstrate potentiation of many different classes of antibiotics against a selection of priority antibiotic-resistant pathogens, including colistin (often considered an antibiotic of last resort), and we discuss how this endogenous antimicrobial component of innate immunity has a role in infectious disease that is beginning to be explored and is not yet fully understood.

Evaluation of the Mycoplasma genitalium Resistance Plus kit for the detection of M. genitalium and mutations associated with macrolide resistance.

Abstract: Objectives To compare performance of the ResistancePlus kit (SpeeDx, Australia) with in-house methods for the detection of Mycoplasma genitalium- specific DNA and mutations associated with resistance to macrolide antimicrobials, directly from clinical specimens. Methods Assay specificity and sensitivity was analysed using DNA from 46 non- M. genitalium organisms and standard curve analysis, respectively. A panel of archived DNA extracted from 97 M. genitalium -positive clinical specimens, for which the macrolide susceptibility genotype had been previously determined, were tested on the assay and results compared. Results Final analytical specificity was 100%. Sensitivity was detected to at least 140 genome copies/µL. The assay detected M. genitalium in 92/97 (94.9%, 95% CI 88.4% to 98.3%) previously positive specimens. The genetic macrolide susceptibility assigned was concordant with previous results in 85/92 (92.4%, 95% CI 85.0% to 96.9%) specimens or 85/97 (87.6%, 95% CI: 79.4% to 93.4%) when the false-negative specimens were included. On seven (7/92, 7.6%) occasions, resistant specimens were called susceptible. Further testing resolved discrepancies for all but five (5.2%) specimens. Conclusions The ResistancePlus assay generally performed well in comparison to methods currently employed at the reference laboratory. It detected a range of different mutations; however, a small number of specimens that were genotyped as macrolide resistant by Sanger sequencing were either not detected by the assay or were genotyped as susceptible. This could impact on treatment outcomes if assay results were used for patient management.

Is previous azithromycin treatment associated with azithromycin resistance in Neisseria gonorrhoeae? A cross-sectional study using national surveillance data in England

Abstract: Objectives It has been suggested that treatment of STIs with azithromycin may facilitate development of azithromycin resistance in Neisseria gonorrhoeae (NG) by exposing the organism to suboptimal doses. We investigated whether treatment history for non-rectal Chlamydia trachomatis (CT), non-gonococcal urethritis (NGU) or NG (proxies for azithromycin exposure) in sexual health (GUM) services was associated with susceptibility of NG to azithromycin. Methods Azithromycin susceptibility data from the Gonococcal Resistance to Antimicrobials Surveillance Programme (GRASP 2013–2015, n=4606) and additional high-level azithromycin-resistant isolates (HL-AziR) identified by the Public Health England reference laboratory (2013–2016, n=54) were matched to electronic patient records in the national GUMCAD STI surveillance dataset (2012–2016). Descriptive and regression analyses were conducted to examine associations between history of previous CT/NGU/NG and subsequent susceptibility of NG to azithromycin. Results Modal azithromycin minimum inhibitory concentration (MIC) was 0.25 mg/L (one dilution below the resistance breakpoint) in those with and without history of previous CT/NGU/NG (previous 1 month/6 months). There were no differences in MIC distribution by history of CT/NGU (P=0.98) or NG (P=0.85) in the previous 1 month/6 months or in the odds of having an elevated azithromycin MIC (>0.25 mg/L) (Adjusted OR for CT/NGU 0.97 (95% CI 0.76 to 1.25); adjusted OR for NG 0.82 (95% CI: 0.65 to 1.04)) compared with those with no CT/NGU/NG in the previous 6 months. Among patients with HL-AziR NG, 3 (4%) were treated for CT/NGU and 2 (3%) for NG in the previous 6 months, compared with 6% and 8%, respectively for all GRASP patients. Conclusions We found no evidence of an association between previous treatment for CT/NGU or NG in GUM services and subsequent presentation with an azithromycin-resistant strain. As many CT diagnoses occur in non-GUM settings, further research is needed to determine whether azithromycin-resistant NG is associated with azithromycin exposure in other settings and for other conditions.

Performance characteristics of newer MIC gradient strip tests compared with the Etest for antimicrobial susceptibility testing of Neisseria gonorrhoeae

Abstract: For Neisseria gonorrhoeae susceptibility testing, Etest, comparable to agar dilution, is frequently used In recent years, newer MIC gradient strip tests have been commercialized However, these tests have not been appropriately evaluated for gonococci We evaluated the sensitivity, specificity, accuracy, quality, availability of antimicrobials and cost of the MIC Test Strip (Liofilchem), MICEvaluator (Oxoid) and Ezy MIC Strip (HiMedia), compared to the reference Etest (bioMerieux), for gonococcal susceptibility testing The MICs of eight antimicrobials in 103 gonococcal international reference strains (n = 29) and clinical isolates (n = 74) were examined Coefficient of determination (R2 ), complete agreement, essential agreement, SIR categorical agreement, sensitivity, specificity and accuracy were calculated R2 of the MICs for the antimicrobials ranged between 0674-0996, 0617-0993, and 0643-0994 for the MIC Test Strip, MICEvaluator strips and Ezy MIC Strips respectively The essential agreement (SIR categorical agreement) was 996% (886%), 100% (871%) and 930% (831%) respectively MICEvaluator strips for gonococcal key antimicrobials were lacking and the Ezy MIC Strips showed an inconsistent accuracy, quality and some strips were contaminated The Liofilchem MIC Test Strips had limitations, but might be relatively accurate alternatives to Etest for gonococci Strict quality assurance (at manufacturing and testing laboratory), including quality controls, are required

Phenotypic antimicrobial susceptibility testing of Chlamydia trachomatis isolates from patients with persistent or successfully treated infections.

Abstract: Objectives Antimicrobial susceptibility data for Chlamydia trachomatis are lacking. Methodologies for susceptibility testing in C. trachomatis are not well-defined, standardized or performed routinely owing to its intracellular growth requirements. We sought to develop an assay for the in vitro susceptibility testing of C. trachomatis isolates from two patient cohorts with different clinical outcomes. Methods Twenty-four clinical isolates (11 from persistently infected and 13 from successfully treated patients) were overlaid with media containing two-fold serial dilutions of azithromycin or doxycycline. After incubation, aliquots were removed from the stock inoculum (SI) and each antimicrobial concentration for total RNA extraction, complementary DNA generation and real-time PCR. The MIC was defined as the lowest antimicrobial concentration where a 95% reduction in transcription was evident in comparison with the SI for each isolate. Results MICs of azithromycin were comparable for isolates from the two patient groups (82% ≤ 0.25 mg/L for persistently infected and 100% ≤ 0.25 mg/L for successfully treated patients). Doxycycline MICs were at least two-fold lower for isolates from the successfully treated patients (53.9% ≤ 0.064 mg/L) than for the persistently infected patients (100% ≥ 0.125 mg/L) (P = 0.006, Fisher's exact test). Overall, 96% of isolates gave reproducible MICs when re-tested. Conclusions A reproducible assay was developed for antimicrobial susceptibility testing of C. trachomatis. MICs of azithromycin were generally comparable for the two different patient groups. MICs of doxycycline were significantly higher in the persistently infected patients. However, interpretation of elevated MICs in C. trachomatis is extremely challenging in the absence of breakpoints, or wild-type and treatment failure MIC distribution data.

Chlamydia trachomatis in Cervical Lymph Node of Man with Lymphogranuloma Venereum, Croatia, 20141.

Abstract: We report an HIV-infected person who was treated for lymphogranuloma venereum cervical lymphadenopathy and proctitis in Croatia in 2014. Infection with a variant L2b genovar of Chlamydia trachomatis was detected in a cervical lymph node aspirate. A prolonged course of doxycycline was required to cure the infection.

Transfer of a gonococcal β-lactamase plasmid into Neisseria gonorrhoeae belonging to the globally distributed ST1407 lineage.

Abstract: Sir, Neisseria gonorrhoeae isolates belonging to multi-antigen ST (NG-MAST) ST1407 have been detected globally, with the proportion ranging from 16.1% in the Far East to 23% in Europe. ST1407 gonococcal isolates are associated with cefixime and ciprofloxacin resistance, show raised MICs of ceftriaxone and azithromycin and are responsible for many cefixime or ceftriaxone treatment failures. Despite occurring in great numbers and having global distribution, ST1407 isolates have not been reported to harbour the b-lactamase-encoding plasmids that confer high-level penicillin resistance in N. gonorrhoeae and the reasons for this are not known. We sought to establish whether a panel of ST1407 isolates could acquire, express and stably maintain a gonococcal b-lactamase plasmid via conjugation. We selected seven ST1407 gonococci isolated from patients attending UK clinics during 2010–12 (Table 1). Conjugation experiments were performed using a filter-mating procedure with strain WHO-M, which contains an ‘African’ b-lactamase plasmid, as the donor. As a ‘control’ recipient, we used strain WHO-K (NG-MAST ST1424), which is b-lactamase negative and also has ciprofloxacin and cefixime resistance, similar to that of the ST1407 isolates. For matings between the WHO-K recipient and WHO-M, GC base (BD, Oxford, UK) supplemented with 1% Vitox (Oxoid, Basingstoke, UK) and both penicillin (8 mg/L) and ciprofloxacin (16 mg/L) was used for selection of transconjugants. For conjugations between ST1407 isolates and WHO-M, a range of penicillin and ciprofloxacin concentrations was used to establish optimal selection (Table 1). Suspected transconjugants were confirmed to be Gramnegative cocci and oxidase positive. Nitrocefin (Oxoid, Basingstoke, UK) was used to detect b-lactamase production and thus confirm the acquisition of the b-lactamase plasmids. Conjugation frequencies were calculated as the number of transconjugants obtained per input recipient cell exposed to donor cells. The plasmid profiles of the control strains, the ST1407 isolates and the transconjugants were established by extracting the plasmids using the QIAprep Spin Miniprep Kit (Qiagen, Manchester, UK) using the manufacturer’s standard protocol and growth from two culture plates. Extracted plasmids were run on 0.8% agarose gels incorporating GelRed dye (Biotium, Hayward, USA) for 5 h at 100 V. Plasmids were sized by comparison with those from isolates defined previously. Transconjugants were archived at #80 C and the stability of the b-lactamase plasmids was assessed after retrieval from#80 C and sub-culture without penicillin selection. The seven ST1407 recipients and strain WHO-K were all confirmed to be highly resistant to ciprofloxacin (MICs .32 mg/L) and, despite resistance to penicillin (MICs 1–2 mg/L), were b-lactamase negative (Table 1). Optimum selection of transconjugants was achieved using 4 mg/L for both penicillin and ciprofloxacin (Table 1). The presence of b-lactamase and the plasmid profiles of the transconjugants confirmed that the 3.2 MDa African b-lactamase plasmid was successfully transferred to all seven ST1407 isolates. The ST1407 transconjugants still harboured the b-lactamase plasmid after retrieval from #80 C, as indicated via the Nitrocefin test, but lost it after one sub-culture without penicillin selection. By contrast, WHO-K-derived transconjugants stably maintained the b-lactamase plasmid in the absence of penicillin selection for at least seven sub-cultures. N. gonorrhoeae isolates of a particular auxotype [one that requires proline, arginine and uracil and does not utilize ornithine (PAOU requiring)] do not acquire b-lactamase plasmids for reasons that are not fully elucidated, although the lack of the 2.6 MDa cryptic plasmid in these isolates may be a factor. However the ST1407 isolates studied here all contained the cryptic plasmid (by comparisons using agarose gel electrophoresis) and readily acquired an African b-lactamase plasmid via conjugation, so seem not to have mechanisms that prevent plasmid acquisition. The apparent absence of clinical ST1407 penicillinase-producing N. gonorrhoeae (PPNG) isolates may be due to an inability of the ST to maintain the plasmids stably in the absence of selection or due to a lack of exposure to other gonococci that harbour b-lactamase plasmids. For example, in England the ST1407 clone has been associated primarily with MSM, whereas contemporaneous PPNG were significantly associated with heterosexuals. A lack of bridging between heterosexual and MSM networks might provide an epidemiological explanation for why PPNG has never been detected in ST1407, rather than there being any underlying microbiological explanation. Nevertheless, in Europe ST1407 isolates were initially detected in both MSM and heterosexuals and an association between heterosexuals and PPNG has been established over some years (2011– 14) so there may have been more opportunity for ST1407 and PPNG isolates to infect the same host. The reason why the ST1407 isolates in this study could not maintain the African b-lactamase plasmid in the absence of selection may be elucidated by whole-genome sequence analysis and