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Objectives Azithromycin is commonly used to treat Neisseria gonorrhoeae. We compared its gastrointestinal side effects using 1 g single, 2 g single or 2 g split (i.e. 1 g plus 1 g 6–12 h later) dosing, representing our clinic’s changing guidelines over the study period. Methods We recruited consecutive sexual health clinic patients who received azithromycin (and 500 mg ceftriaxone) for uncomplicated gonorrhoea. Each patient received a text message 48 h after their attendance to complete a questionnaire. Results Patients received 1 g single (n = 271), 2 g single (218) or 2 g split (105) doses. Vomiting was less common for 1 g versus 2 g single dose [1.1% versus 3.7%; risk difference (RD): −2.6%; 95% CI: −0.2 to −5.4] and 2 g split versus 2 g single dose (0.9% versus 3.7%; RD: −2.8%; 95% CI: −0.3 to −5.8). Nausea was less common for 1 g versus 2 g single dose (13.7% versus 43.1%; RD: −29.5%; 95% CI: −21.7 to −37.2) and 2 g split versus 2 g single dose (16.4% versus 43.1%; RD: −26.8; 95% CI: −17.2 to −36.3). Diarrhoea was less common for 1 g versus 2 g single dose (25.5% versus 50.9%; RD: −25.5%; 95% CI: −17.0 to −33.9) and 2 g split versus 2 g single dose (30.9% versus 50.9%; RD: −20.0; 95% CI: −9.1 to −30.9). Almost all were willing to retake the same dosing for gonorrhoea in the future: 97% for 1 g single; 94% for 2 g single; and 97% for 2 g split dose. Conclusions Azithromycin 2 g split dose for gonorrhoea resulted in significantly less vomiting, nausea and diarrhoea than a 2 g single dose.

Background Acute otitis media (AOM) is a common cause for antibiotic prescription. Most guidelines endorse abstaining from immediate antibiotic treatment (‘watchful waiting’, WW) in mild–moderate episodes. We studied adherence rates to the latest AOM guidelines (2013), in terms of antibiotic type and prescription options. Methods In this population-based study, AOM episodes were identified in Clalit Health Services-insured children aged 0–10 years between 2011 and 2018, using a data-sharing platform. After identifying the index, prescription and issuing dates for antibiotics for each AOM episode, treatment was categorized as immediate (≤2 days after diagnosis) or WW (antibiotic not prescribed/issued; prescribed ≤2 days after diagnosis but issued on Days 2–7; or prescribed/issued on Days 2–7). Guideline adherence was measured according to age. Results Of the 491 106 episodes, 361 518 (73.6%) were treated with antibiotics. Following the 2013 guidelines, the ratio of episodes in children aged ≤6 months that were adherent (immediate treatment) was higher (OR = 1.22; 95% CI 1.15–1.29; P Conclusions Improved adherence to the 2013 guidelines was observed only in children aged ≤6 months and over-treatment with antibiotics was still high.

Background The prevalence of azithromycin resistance in Neisseria gonorrhoeae is increasing in numerous populations worldwide. Objectives To characterize the genetic pathways leading to high-level azithromycin resistance. Methods A customized morbidostat was used to subject two N. gonorrhoeae reference strains (WHO-F and WHO-X) to dynamically sustained azithromycin pressure. We tracked stepwise evolution of resistance by whole genome sequencing. Results Within 26 days, all cultures evolved high-level azithromycin resistance. Typically, the first step towards resistance was found in transitory mutations in genes rplD, rplV and rpmH (encoding the ribosomal proteins L4, L22 and L34 respectively), followed by mutations in the MtrCDE-encoded efflux pump and the 23S rRNA gene. Low- to high-level resistance was associated with mutations in the ribosomal proteins and MtrCDE efflux pump. However, high-level resistance was consistently associated with mutations in the 23S ribosomal RNA, mainly the well-known A2059G and C2611T mutations, but also at position A2058G. Conclusions This study enabled us to track previously reported mutations and identify novel mutations in ribosomal proteins (L4, L22 and L34) that may play a role in the genesis of azithromycin resistance in N. gonorrhoeae.

Background Neisseria gonorrhoeae has developed resistance to all antimicrobials used to treat gonorrhoea, with even ceftriaxone being undermined. It is therefore important to examine any potential to redeploy older antimicrobials routinely used for other infections to treat ceftriaxone-resistant gonococcal infections. Objectives We examined the susceptibility of N. gonorrhoeae to aztreonam, chloramphenicol, co-trimoxazole, fosfomycin, piperacillin/tazobactam and rifampicin. Methods N. gonorrhoeae isolates (n = 94) were selected to include a range of antimicrobial susceptibilities: 58 were collected in the Gonococcal Resistance to Antimicrobials Surveillance Programme; 17 were clinical isolates referred to the PHE reference laboratory; and 19 were control strains. MICs were determined by agar dilution for the six study antimicrobials and for ceftriaxone and azithromycin as comparators. Results There was correlation between piperacillin/tazobactam and ceftriaxone MICs, but all five isolates with high ceftriaxone MICs (>0.5 mg/L) were inhibited by piperacillin/tazobactam at 0.06–0.5 mg/L. Aztreonam MICs for ceftriaxone-resistant isolates exceeded those of ceftriaxone. Among non-β-lactams, fosfomycin and co-trimoxazole had low, tightly clustered MICs, suggesting widespread susceptibility, rifampicin split the collection into highly susceptible and highly resistant groups and chloramphenicol had a wide MIC distribution. Conclusions Although unsuitable for empirical use, piperacillin/tazobactam, fosfomycin, co-trimoxazole, rifampicin and, possibly, chloramphenicol could be considered for individual patients with ceftriaxone-resistant gonococcal infection once MICs are known. Wider surveillance of the susceptibility of N. gonorrhoeae to these agents is needed, along with clinical trials and the establishment of clinical breakpoints for N gonorrhoeae.

BackgroundNeisseria gonorrhoeae (NG) isolates with high-level azithromycin resistance (HL-AziR) have emerged worldwide in recent decades, threatening the sustainability of current dual-antimicrobial therapy.ObjectivesThis study aimed to characterize the first 16 NG isolates with HL-AziR in Barcelona between 2016 and 2018.MethodsWGS was used to identify the mechanisms of antimicrobial resistance, to establish the MLST ST, NG multiantigen sequence typing (NG-MAST) ST and NG sequence typing for antimicrobial resistance (NG-STAR) ST and to identify the clonal relatedness of the isolates with other closely related NG previously described in other countries based on a whole-genome SNP analysis approach. The sociodemographic characteristics of the patients included in the study were collected by comprehensive review of their medical records.ResultsTwelve out of 16 HL-AziR isolates belonged to the MLST ST7823/NG-MAST ST5309 genotype and 4 to MLST ST9363/NG-MAST ST3935. All presented the A2059G mutation in all four alleles of the 23S rRNA gene. MLST ST7823/NG-MAST ST5309 isolates were only identified in men who have sex with women and MLST ST9363/NG-MAST ST3935 were found in MSM. Phylogenomic analysis revealed the presence of three transmission clusters of three different NG strains independently associated with sexual behaviour.ConclusionsOur findings support the first appearance of three mild outbreaks of NG with HL-AziR in Spain. These results highlight the continuous capacity of NG to develop antimicrobial resistance and spread among sexual networks. The enhanced resolution of WGS provides valuable information for outbreak investigation, complementing the implementation of public health measures focused on the prevention and dissemination of MDR NG.

ObjectivesTo compare the effectiveness of doxycycline 100 mg twice daily for 7 days and azithromycin 1 g single dose for the treatment of rectal Chlamydia trachomatis infection among MSM in a real clinical setting.MethodsA prospective study was performed to compare the effectiveness of doxycycline and azithromycin for the treatment of rectal C. trachomatis among MSM in Tokyo, Japan. Subjects diagnosed with rectal C. trachomatis infection were treated and test-of-cure examination (TOC) was performed at least 3 weeks after the treatment. Treatment of rectal C. trachomatis infection was decided prospectively in a time-dependent manner; in the period between January 2017 and October 2018, azithromycin was administered to all subjects, whereas from October 2018 through March 2020, doxycycline was administered to all subjects. Effectiveness of these treatments was calculated by the number of rectal C. trachomatis-negative subjects at TOC divided by the number of subjects treated.ResultsTwo hundred and ninety-six MSM with rectal C. trachomatis infection were treated with azithromycin (80 patients) and doxycycline (216 patients) in a time-dependent manner. Of the 296 MSM, 274 (92.6%) were treated successfully [67 (83.7%, 95% CI = 79.6%–87.9%) in the azithromycin group versus 207 (95.8%, 95% CI = 94.5%–97.2%) in the doxycycline group, P ConclusionsThe treatment with doxycycline 100 mg twice daily for 7 days was superior to that with azithromycin 1 g single dose for rectal C. trachomatis among MSM in a real-world setting.

BackgroundChloroquine (CQ) and hydroxychloroquine (HCQ) show anti-SARS-CoV-2 activity in vitro; however, clinical studies have reported conflicting results. We sought to systematically evaluate the effect of CQ and HCQ with or without azithromycin (AZ) on outcomes of COVID-19 patients.MethodsWe searched Medline, Embase, EBM Reviews, Scopus, Web of Science, preprints and grey literature up to July 7, 2020. We included studies that assessed COVID-19 patients treated with CQ or HCQ, with or without AZ. We pooled only adjusted effect estimates of mortality using a random effect model. We summarized the effect of CQ or HCQ on viral clearance and ICU admission/ mechanical ventilation.ResultsOut of 1463 citations screened for eligibility, five RCTs and 14 cohort studies were included (20,263 hospitalized patients). Thirteen studies (1 RCT and 12 cohorts) with 15,938 patients examined the effect of HCQ on short term mortality. The pooled adjusted OR was 1.05 (95% CI 0.96-1.15, I2=0 %, p=0.647). Six cohort studies examined the effect of HCQ and AZ combination among 14,016 patients. The pooled adjusted OR was 0.93 (95% CI 0.79-1.11, I2=59.3%, p=0.003). Two cohort studies and three RCTs found no significant effect of HCQ on viral clearance. One RCT with 48 patients demonstrated improved viral clearance in patients treated with CQ and HCQ. Three cohort studies found that HCQ with or without AZ had no significant effect on mechanical ventilation/ ICU admission.ConclusionModerate certainty evidence suggests that HCQ, with or without AZ, lacks efficacy in reducing short-term mortality in patients hospitalized with COVID-19.SummaryThis systematic review and meta-analysis showed that in-hospital treatment of COVID-19 patients with antimalarials medications failed to reduce short-term mortality and morbidity with potential harm if used in combination with azithromycin.

Background Reducing Neisseria gonorrhoeae colonies in the oropharynx is a viable solution to minimize the transmission of this bacterium amongst individuals. Objectives A strategy involving the electrostatic interaction between a common antiseptic and a discontinued antibiotic (i.e. octenidine and carbenicillin) was evaluated as a potential treatment for gonorrhoea. Octenidine/carbenicillin is a novel group of uniform materials based on organic salts (GUMBOS) with inherent in vitro antibacterial activity that comes from its parent antiseptic and antibacterial ions, octenidine and carbenicillin, respectively. Methods Antibacterial activities for octenidine dihydrochloride, disodium carbenicillin, octenidine/carbenicillin and stoichiometrically equivalent 1:1 octenidine dihydrochloride to disodium carbenicillin were assessed using the Kirby–Bauer disc diffusion assay for N. gonorrhoeae (ATCC 49226) and three clinical isolates. Predictive permeability using the Parallel Artificial Membrane Permeability Assay and cytotoxicity against HeLa cells was also evaluated. Results Additive in vitro antibacterial activities against N. gonorrhoeae were observed in this study, which suggests octenidine/carbenicillin could be a useful agent in reducing N. gonorrhoeae transmission and minimizing gonorrhoea infections. Octenidine/carbenicillin also exhibited bioequivalence to azithromycin and doxycycline, two currently prescribed antibiotics. Likewise, octenidine/carbenicillin had improved predicted permeability compared with octenidine dihydrochloride. Conclusions Antimicrobial GUMBOS synthesized in this study could be used as an adjunctive treatment approach to current drug therapies for oropharyngeal gonorrhoea infection control and prevention.

Background The incidence of antimicrobial-resistant Neisseria gonorrhoeae (GC) is rising in Canada; however, antimicrobial resistance (AMR) surveillance data are unavailable for infections diagnosed directly from clinical specimens by nucleic acid amplification tests (NAATs), representing over 80% of diagnoses. We developed a set of 10 improved molecular assays for surveillance of GC-AMR and prediction of susceptibilities in NAAT specimens. Methods Multiplex real-time PCR (RT–PCR) assays were developed to detect SNPs associated with cephalosporin (ponA, porB, mtrR −35delA, penA A311V, penA A501, N513Y, G545S), ciprofloxacin (gyrA S91, parC D86/S87/S88) and azithromycin [23S (A2059G, C2611T), mtrR meningitidis-like promoter] resistance. The assays were validated on 127 gonococcal isolates, 51 non-gonococcal isolates and 50 NAATs with matched culture isolates. SNPs determined from the assay were compared with SNPs determined from in silico analysis of WGS data. MICs were determined for culture isolates using the agar dilution method. Results SNP analysis of the 50 NAAT specimens had 96% agreement with the matched culture RT–PCR analysis. When compared with MICs, presence of penA A311V or penA A501 and two or more other SNPs correlated with decreased susceptibility and presence of three or more other SNPs correlated with intermediate susceptibility to cephalosporins; presence of any associated SNP correlated with ciprofloxacin or azithromycin resistance. NAAT-AMR predictions correlated with matched-culture cephalosporin, ciprofloxacin and azithromycin MICs at 94%, 100% and 98%, respectively. Conclusions We expanded molecular tests for N. gonorrhoeae AMR prediction by adding new loci and multiplexing reactions to improve surveillance where culture isolates are unavailable.

Background Antibiotic-resistant gonorrhoea has been a chronic public health burden since the mid-1930s. Recent emergence of isolates resistant to the current recommended antibiotics for gonorrhoea further magnifies the threat of untreatable gonorrhoea. The lack of new, effective antibiotics highlights the need for better understanding of the population structure of Neisseria gonorrhoeae in order to provide greater insight on how to curtail the spread of antimicrobial-resistant N. gonorrhoeae. Objectives To explore a potential application of MALDI-TOF MS to differentiate N. gonorrhoeae displaying different levels of susceptibility to the antibiotic azithromycin. Methods We conducted MALDI-TOF MS using the Bruker Biotyper on 392 N. gonorrhoeae isolates collected through the Gonococcal Isolate Surveillance Project (GISP) and/or the Strengthening the United States Response to Resistant Gonorrhea (SURRG) project. The MALDI-TOF MS spectra were visually analysed to assess the presence of distinctive peak(s). Statistical analysis was performed to assess the relationship between gonococcal isolates with the distinct protein peak and antibiotic susceptibility. Results In this study, we were able to differentiate N. gonorrhoeae isolates into two distinct subpopulations using MALDI-TOF MS. Isolates were distinguished by the presence or absence of a spectral peak at 11 300 Da. Notably, these two groups exhibited different levels of susceptibility to azithromycin. Conclusions We have shown that in addition to its ability to identify N. gonorrhoeae, MALDI-TOF MS could also be used to differentiate gonococcal isolates with different levels of susceptibility to azithromycin.

Background Azithromycin resistance is emerging in typhoidal Salmonella. Confirmation of azithromycin MIC is the most frequent antibiotic susceptibility request made to the Gastrointestinal Bacteria Reference Unit (GBRU) laboratory in England by local diagnostic laboratories. Objectives (i) Determine concordance between local diagnostic and reference laboratory estimations of azithromycin MIC by gradient strip in Salmonella enterica serovars Typhi and Paratyphi. (ii) Consider causes of variation. Methods Isolates from patients with enteric fever attending a central London hospital between May 2011 and April 2019 were tested for azithromycin susceptibility using gradient strips, according to EUCAST methodology. Matched local diagnostic and reference laboratory estimations of azithromycin and ciprofloxacin (as a comparator) MICs were included; concordance in estimations was examined. Results Local diagnostic laboratory readings overestimated azithromycin MIC values compared with the reference laboratory, resulting in poor concordance in susceptibility/resistance attribution (concordant susceptibility interpretation in 8/19, κ = 0). In contrast, ciprofloxacin MIC estimation demonstrated superior concordance (concordant susceptibility interpretation in 16/17, κ = 0.85). None of the isolates was resistant to azithromycin at the reference laboratory and no known genes associated with azithromycin resistance were detected in any isolate using WGS. Conclusions Overestimation of azithromycin resistance is likely to be due to difficulty in interpreting the point of intersection of the ‘trailing edge’ with the gradient strip, used to determine MIC. We advise local diagnostic laboratories to review their experience and consider adopting a ‘second reader’ system to mitigate this.

Background Post-exposure prophylaxis (PEP) for close contacts of invasive meningococcal disease (IMD) cases is recommended in most countries to avoid secondary cases by eradicating supposed meningococcal colonization. Currently, rifampicin, ciprofloxacin and ceftriaxone are recommended in many countries including Germany. Azithromycin has been shown to eradicate meningococcal colonization. Objectives To assess the azithromycin susceptibility of invasive Neisseria meningitidis isolates. Methods A subset of German invasive meningococcal isolates from 2006–18 was selected for this study. Azithromycin MIC was determined using broth microdilution and agar gradient diffusion. Results Azithromycin MICs as determined by broth microdilution ranged from Conclusions Azithromycin is an eligible antibiotic for PEP of IMD close contacts. It is approved for adults as well as children and may even be used in pregnancy. Because of easier application and lower toxicity, it might be an alternative to rifampicin and ciprofloxacin, as we found no resistant isolates. Since a gonococcal gene associated with elevated azithromycin MICs has been reported in N. meningitidis, careful monitoring of the emergence of resistant strains is nevertheless necessary for meningococci. Lack of concordance of MICs between broth microdilution and agar gradient diffusion needs to be considered.

Background Oral azithromycin given during labour reduces carriage of bacteria responsible for neonatal sepsis, including Staphylococcus aureus. However, there is concern that this may promote drug resistance. Objectives Here, we combine genomic and epidemiological data on S. aureus isolated from mothers and babies in a randomized intra-partum azithromycin trial (PregnAnZI) to describe bacterial population dynamics and resistance mechanisms. Methods Participants from both arms of the trial, who carried S. aureus in day 3 and day 28 samples post-intervention, were included. Sixty-six S. aureus isolates (from 7 mothers and 10 babies) underwent comparative genome analyses and the data were then combined with epidemiological data. Trial registration (main trial): ClinicalTrials.gov Identifier NCT01800942. Results Seven S. aureus STs were identified, with ST5 dominant (n = 40, 61.0%), followed by ST15 (n = 11, 17.0%). ST5 predominated in the placebo arm (73.0% versus 49.0%, P = 0.039) and ST15 in the azithromycin arm (27.0% versus 6.0%, P = 0.022). In azithromycin-resistant isolates, msr(A) was the main macrolide resistance gene (n = 36, 80%). Ten study participants, from both trial arms, acquired azithromycin-resistant S. aureus after initially harbouring a susceptible isolate. In nine (90%) of these cases, the acquired clone was an msr(A)-containing ST5 S. aureus. Long-read sequencing demonstrated that in ST5, msr(A) was found on an MDR plasmid. Conclusions Our data reveal in this Gambian population the presence of a dominant clone of S. aureus harbouring plasmid-encoded azithromycin resistance, which was acquired by participants in both arms of the study. Understanding these resistance dynamics is crucial to defining the public health drug resistance impacts of azithromycin prophylaxis given during labour in Africa.

ObjectivesTo investigate the relationship between MIC and clinical outcome in a randomized controlled trial that compared gentamicin 240 mg plus azithromycin 1 g with ceftriaxone 500 mg plus azithromycin 1 g. MIC analysis was performed on Neisseria gonorrhoeae isolates from all participants who were culture positive before they received treatment.MethodsViable gonococcal cultures were available from 279 participants, of whom 145 received ceftriaxone/azithromycin and 134 received gentamicin/azithromycin. Four participants (6 isolates) and 14 participants (17 isolates) did not clear infection in the ceftriaxone/azithromycin and gentamicin/azithromycin arms, respectively. MICs were determined by Etest on GC agar base with 1% Vitox. The geometric mean MICs of azithromycin, ceftriaxone and gentamicin were compared using logistic and linear regression according to treatment received and N. gonorrhoeae clearance.ResultsAs the azithromycin MIC increased, gentamicin/azithromycin treatment was less effective than ceftriaxone/azithromycin at clearing N. gonorrhoeae. There was a higher geometric mean MIC of azithromycin for isolates from participants who had received gentamicin/azithromycin and did not clear infection compared with those who did clear infection [ratio 1.95 (95% CI 1.28–2.97)], but the use of categorical MIC breakpoints did not accurately predict the treatment response. The geometric mean MIC of azithromycin was higher in isolates from the pharynx compared with genital isolates.ConclusionsWe found that categorical resistance to azithromycin or ceftriaxone in vitro, and higher gentamicin MICs in the absence of breakpoints, were poorly predictive of treatment failure.

Objectives Gonorrhoea and antimicrobial resistance (AMR) in Neisseria gonorrhoeae are major public health concerns globally. Enhanced AMR surveillance for gonococci is essential worldwide; however, recent quality-assured gonococcal AMR surveillance in Latin America, including Brazil, has been limited. Our aims were to (i) establish the first nationwide gonococcal AMR surveillance, quality assured according to WHO standards, in Brazil, and (ii) describe the antimicrobial susceptibility of clinical gonococcal isolates collected from 2015 to 2016 in all five main regions (seven sentinel sites) of Brazil. Methods Gonococcal isolates from 550 men with urethral discharge were examined for susceptibility to ceftriaxone, cefixime, azithromycin, ciprofloxacin, benzylpenicillin and tetracycline using the agar dilution method, according to CLSI recommendations and quality assured according to WHO standards. Results The levels of resistance (intermediate susceptibility) to tetracycline, ciprofloxacin, benzylpenicillin and azithromycin were 61.6% (34.2%), 55.6% (0.5%), 37.1% (60.4%) and 6.9% (8.9%), respectively. All isolates were susceptible to ceftriaxone and cefixime using the US CLSI breakpoints. However, according to the European EUCAST cefixime breakpoints, 0.2% (n = 1) of isolates were cefixime resistant and 6.9% (n = 38) of isolates had a cefixime MIC bordering on resistance. Conclusions This study describes the first national surveillance of gonococcal AMR in Brazil, which was quality assured according to WHO standards. The high resistance to ciprofloxacin (which promptly informed a revision of the Brazilian sexually transmitted infection treatment guideline), emerging resistance to azithromycin and decreasing susceptibility to extended-spectrum cephalosporins necessitate continuous surveillance of gonococcal AMR and ideally treatment failures, and increased awareness when prescribing treatment in Brazil.

Background Mycoplasma genitalium is estimated to be the second most common cause of bacterial sexually transmitted infection in Europe. It is of increasing public health concern due to the rapid development of resistance to different antimicrobial classes, including the preferred first- and second-line treatments azithromycin and moxifloxacin. Thus, new antimicrobial agents are urgently needed, especially for the treatment of MDR strains. Methods The in vitro activity of the new spiropyrimidinetrione zoliflodacin against 47 M. genitalium strains was assessed by growing M. genitalium in Vero cell culture and measuring growth by quantitative PCR. The collection included 34 moxifloxacin-susceptible (MIC

ObjectivesAntimicrobial 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.MethodsTwenty-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.ResultsMICs 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.ConclusionsA 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.

The antimicrobial susceptibility of Neisseria gonorrhoeae isolates from Saskatchewan was determined retrospectively (2003-15) to ascertain temporal trends to both current and older antimicrobials used for treatment.The agar dilution method was used to test the antimicrobial susceptibilities of 685 isolates to seven antibiotics.Over the period, only three (0.4%) gonococcal isolates had reduced susceptibility to cefixime and/or ceftriaxone. All isolates were susceptible to spectinomycin. Over 95% of the isolates tested were susceptible to azithromycin except in 2010 and 2013 (27.6% and 7.2% resistant, respectively). One isolate was resistant to both azithromycin and cefixime. Ciprofloxacin resistance was seen in 5% of isolates prior to 2010, but in 5% thereafter. From 2006 to 2012, and in 2015, penicillin resistance was detected in 5% (0%-4.0%) of isolates, but in 5% for the rest of the study period. Tetracycline resistance remained5% (11.8%-89.1%) throughout the study. Plasmid-mediated resistance to tetracycline fluctuated between 0% and 17.5% of isolates tested. Four isolates were MDR and two isolates were XDR.N. gonorrhoeae isolates were largely susceptible (∼85%) to antibiotics no longer recommended for treatment, such as penicillin and ciprofloxacin. Gonorrhoea in Saskatchewan is primarily (95%) diagnosed by nucleic acid amplification testing, which does not permit antimicrobial susceptibility testing. The development of molecular testing, or point-of-care tests, to evaluate antimicrobial susceptibility, would enhance knowledge of true levels of resistance and allow discretion as to whether older but still effective antibiotics could be used in individual patient care.

Objectives To determine if ceftaroline and ceftazidime combined with avibactam are efficacious against pulmonary Mycobacterium avium complex (MAC) disease. Methods First, we performed a concentration-effect study of ceftaroline and ceftaroline/avibactam against extracellular MAC in test tubes. Given the difficulty of obtaining avibactam at the time of experimentation, we used a single concentration of commercial ceftazidime/avibactam, and two sets of non-treated controls, one with ceftazidime/avibactam and the other without. After finding antimicrobial activity with the ceftazidime/avibactam 'control', we performed ceftazidime/avibactam dose-effect studies in test tubes against extracellular MAC and in 24-well plates against intracellular MAC. We then performed a ceftazidime/avibactam exposure-effect and dose-fractionation studies in the hollow-fibre system model of intracellular pulmonary MAC (HFS-MAC). In each experiment, we repetitively sampled each HFS-MAC at specified times to validate ceftazidime/avibactam pharmacokinetics and to quantify bacterial burden. Results Ceftaroline killed extracellular MAC with maximal microbial kill (Emax) of 4.87 ± 0.26 log10 cfu/mL. However, the ceftazidime/avibactam 'control' also killed MAC compared with the non-treated control. Ceftazidime/avibactam Emax was 3.8 log10 cfu/mL against extracellular bacilli and 3.6 log10 cfu/mL against intracellular MAC. In the HFS-MAC, ceftazidime/avibactam achieved a half-life of 2.5-3.3 h and killed MAC 0.61-2.40 log10 cfu/mL below the starting bacterial burden. The ceftazidime/avibactam efficacy was linked to the proportion of the dosing interval for which the concentration persists above the MIC (fT>MIC), with optimal efficacy at free-drug fT>MIC of 52% (r2 = 0.95). Conclusions Ceftazidime/avibactam effectively kills MAC at exposures easily achieved in the lung by clinical doses. Efficacy was higher than with clinically achievable doses of azithromycin and ethambutol.

Objectives To investigate the performance of the two backbone drugs in the standard combination therapy regimen in the hollow-fibre system (HFS) model of pulmonary Mycobacterium avium complex (MAC) infection. Methods Six HFS were inoculated with human-derived monocytes infected with MAC, and treated with 15 mg/kg of ethambutol and 500 mg of azithromycin daily for 28 days to recapitulate the concentration-time profiles seen in the lungs of humans treated with these drugs and doses. The concentration-time profiles achieved were validated by sampling the central compartment at seven timepoints over 24 h. The total MAC burden, as well as the subpopulation resistant to 3 × MIC of each drug, was identified based on sampling the peripheral compartment of each system on days 0, 3, 7, 14, 21 and 28 of therapy. The experiment was performed twice. Results In non-treated control HFS, MAC grew from 5.0 to 8.53 log10 cfu/mL in 28 days. The dual therapy killed a maximum of 1.52 ± 0.43 log10 cfu/mL during the first 7 days, after which it failed. By day 28 there was no difference in MAC burden between the combination-therapy-treated and non-treated systems. Failure arose in parallel with the emergence of acquired ethambutol resistance. By day 28, 100% of the bacterial population was ethambutol resistant in the combination-therapy-treated HFS replicates. Conclusions The backbone combination of macrolide and ethambutol has poor MAC kill rates and is ineffective. Microbial kill is rapidly abrogated by acquired drug resistance. This backbone should be replaced.

Objectives: To investigate the phenotypic effect of expression of selected acquired macrolide resistance genes (AMRGs) in non-typeable Haemophilus influenzae (NTHi). Methods: The AMRGs erm(A), erm(B) and erm(C) were cloned into Escherichia coli JM109 using the shuttle vector pLS88; constructed plasmids extracted from suitable clones were used to transform H. influenzae Rd by electroporation. Erythromycin and azithromycin MICs for suitable transformants were determined by broth microdilution. AMRG expression was determined using quantitative PCR on transformant cDNA with locked nucleic acid dual-labelled hydrolysis probes. Results: Expression of all AMRGs was observed in the transformants. Some variation in expression between the AMRGs was apparent, but expression of all genes was associated with a notable increase in erythromycin and azithromycin MICs compared with untransformed H. influenzae Rd. Conclusions: While the establishment of erm genes within WT populations of NTHi remains contentious, H. influenzae is capable of expression of erm. Expression may be associated with a subsequent decreased susceptibility to macrolides in isolates and future monitoring of these genes in NTHi isolates is warranted.

Objectives To develop a method to enable the large-scale antimicrobial susceptibility screening of Orientia tsutsugamushi clinical isolates, using one timepoint and one concentration of antibiotics to considerably speed up the time to result. Methods Growth, harvesting, multiplicity of infection (moi) and the day to determine the MICs were optimized using five O. tsutsugamushi reference strains [susceptible (Karp, Kato and Gilliam) and putatively resistant (AFC-3 and AFSC-4)], one clinical isolate (UT76) and one rodent isolate (TA763). Subsequently, the MICs of azithromycin, chloramphenicol and doxycycline for these strains and 51 clinical isolates including AFSC-7 were determined. An optimal concentration was calculated using the epidemiological cut-off value. Results The conditions for O. tsutsugamushi infection, growth and harvesting were determined to be an moi of 100:1 and trypsinization with the peak growth on day 10. The resulting MICs were in line with previously published susceptibility data for all reference strains, except for Karp and AFSC-4, which showed azithromycin MICs of 0.0156 and 0.0313 mg/L, compared with 0.0078 and 0.0156 mg/L, respectively, in previous reports. The MIC of doxycycline for AFC-3 was 0.125 mg/L compared with >4 mg/L in earlier reports. The final single screening concentrations were identified as: azithromycin, 0.125 mg/L; chloramphenicol, 8 mg/L; and doxycycline, 1 mg/L. Conclusions This simplified procedure facilitates the simultaneous screening of 48 isolates for actively monitoring potential resistance of this important fever pathogen, with an 8-fold throughput improvement over early methods. The data do not support the existence of doxycycline- and chloramphenicol-resistant scrub typhus.

OBJECTIVES: To elucidate the genome-based epidemiology and phylogenomics of azithromycin-resistant (MIC >2 mg/L) Neisseria gonorrhoeae strains collected in 2009-14 in Europe and clarify the azithromycin resistance mechanisms. METHODS: Seventy-five azithromycin-resistant (MIC 4 to >256 mg/L) N. gonorrhoeae isolates collected in 17 European countries during 2009-14 were examined using antimicrobial susceptibility testing and WGS. RESULTS: Thirty-six N. gonorrhoeae multi-antigen sequence typing STs and five phylogenomic clades, including 4-22 isolates from several countries per clade, were identified. The azithromycin target mutation A2059G (Escherichia coli numbering) was found in all four alleles of the 23S rRNA gene in all isolates with high-level azithromycin resistance (n = 4; MIC ≥256 mg/L). The C2611T mutation was identified in two to four alleles of the 23S rRNA gene in the remaining 71 isolates. Mutations in mtrR and its promoter were identified in 43 isolates, comprising isolates within the whole azithromycin MIC range. No mutations associated with azithromycin resistance were found in the rplD gene or the rplV gene and none of the macrolide resistance-associated genes [mef(A/E), ere(A), ere(B), erm(A), erm(B), erm(C) and erm(F)] were identified in any isolate. CONCLUSIONS: Clonal spread of relatively few N. gonorrhoeae strains accounts for the majority of the azithromycin resistance (MIC >2 mg/L) in Europe. The four isolates with high-level resistance to azithromycin (MIC ≥256 mg/L) were widely separated in the phylogenomic tree and did not belong to any of the main clades. The main azithromycin resistance mechanisms were the A2059G mutation (high-level resistance) and the C2611T mutation (low- and moderate-level resistance) in the 23S rRNA gene. The present work was supported by grants from theO¨ rebro County Council Research Committee and the Foundation for Medical Research at O¨ rebro University Hospital, Sweden info:eu-repo/semantics/publishedVersion

Objectives The Gram-negative bacillus Stenotrophomonas maltophilia (SM) is an emerging MDR opportunistic pathogen. Recent studies identify a potentially relevant activity of azithromycin against Gram-negative bacteria overlooked in standard bacteriological testing. We investigated azithromycin activity against SM in testing conditions incorporating mammalian tissue culture medium and host defence factors. Methods MIC testing, chequerboard assays, time-kill assays and fluorescence microscopy were performed for azithromycin, the cationic peptide antibiotic colistin and the human defence peptide cathelicidin LL-37 alone or in combination in cation-adjusted Mueller-Hinton broth or mammalian tissue culture media. Azithromycin sensitization of SM to host immune clearance was tested in a human neutrophil killing assay and a murine pneumonia model. Results We observed potent bactericidal activity of azithromycin against SM in mammalian tissue culture medium absent in bacteriological medium. Colistin and LL-37 strongly potentiated azithromycin killing of SM by increasing drug entry. Additionally, azithromycin sensitized SM to neutrophil killing and increased SM clearance in the murine pneumonia model. Conclusions Despite lack of activity in standard MIC testing, azithromycin synergizes with cationic peptide antibiotics to kill SM in medium mimicking tissue fluid conditions. Azithromycin, alone or in combination with colistin, merits further exploration in therapy of drug-resistant SM infections.

The objectives of this study were to evaluate the time to a Mycoplasma genitalium-negative test after start of treatment and to monitor if and when antibiotic resistance developed.Sexually transmitted disease (STD) clinic attendees with suspected or verified M. genitalium infection were treated with azithromycin (5 days, 1.5 g; n = 85) or moxifloxacin (n = 5). Subjects with symptomatic urethritis or cervicitis of unknown aetiology were randomized to either doxycycline (n = 49) or 1 g of azithromycin as a single dose (n = 51). Women collected vaginal specimens and men collected first-catch urine 12 times during 4 weeks. Specimens were tested for M. genitalium with a quantitative MgPa PCR and for macrolide resistance-mediating mutations with a PCR targeting 23S rRNA.NCT01661985.Ninety M. genitalium cases were enrolled. Of 56 patients with macrolide-susceptible strains before treatment with azithromycin (1.5 g, n = 46; 1 g single oral dose, n = 10), 54 (96%) had a negative PCR test within 8 days. In four patients, M. genitalium converted from macrolide susceptible to resistant after a 10 day lag time with negative tests (azithromycin 1.5 g, n = 3; 1 g single oral dose, n = 1). Moxifloxacin-treated subjects (n = 4) were PCR negative within 1 week. Six of eight (75%) remained positive despite doxycycline treatment.PCR for M. genitalium rapidly became negative after azithromycin treatment. Macrolide-resistant strains were detected after initially negative tests. Test of cure should be recommended no earlier than 3-4 weeks.

Objectives: Mycoplasma genitalium is a sexually transmitted pathogen, and infection with it is usually treated with macrolides. Unfortunately, emerging resistance to the macrolides has been associated with mutations in region V of the 23S rRNA gene. The aim of this retrospective study was to describe the incidence of macrolide resistance-associated mutations in M. genitalium from patients in the Netherlands. Methods: All urogenital samples obtained from patients visiting a general practitioner or hospital in the east of the Netherlands that tested positive using the routine M. genitalium real-time PCR (February 2012-November 2014) were included. Following a PCR targeting the 23S rRNA gene, sequencing of the PCR fragments was performed to identify possible macrolide resistance-associated mutations. Results: Forty-eight of the 153 samples (31.4%) included in this study contained a mutation in the 23S rRNA gene. This was reduced to 44/146 (30.1%) if only samples from unique patients were included. The predominant mutations identified were A2058G (16/44; 36.3%), A2059G (14/44; 31.8%) and a unique high proportion of A2058T (12/44; 27.3%). Treatment failure was observed in four patients initially infected with M. genitalium containing macrolide resistance-associated mutations, while in one of these patients subsequent treatment with moxifloxacin resulted in a microbiological cure. Conclusions: This study shows that macrolide resistance-associated mutations in M. genitalium occur with a high frequency. In contrast to studies from other regions, Dutch M. genitalium isolates carry the A2058T mutation at high frequency. Our data confirm the need for prospective detection of macrolide resistance-associated mutations prior to treating patients.

Objectives Antimicrobial resistance in Neisseria gonorrhoeae is a major public health concern globally. We report the first verified treatment failure of gonorrhoea with 2 g of azithromycin (extended-release formulation) in Japan and characteristics of the corresponding N. gonorrhoeae isolates. Methods Pre- and post-treatment isolates (n = 4) were investigated by Etest for antimicrobial susceptibility. The isolates were examined for molecular epidemiology by multilocus sequence typing (MLST), N. gonorrhoeae multi-antigen sequence typing (NG-MAST) and multiple-locus variable-number tandem repeat analysis (MLVA), and for the presence of azithromycin resistance determinants (23S rRNA gene mutations, erm genes and mtrR mutations). Results All isolates were resistant to azithromycin (MIC 4 mg/L) and ciprofloxacin, but remained susceptible to cefixime, ceftriaxone and spectinomycin. All isolates were assigned to MLST ST1901 and NG-MAST ST1407 and three of four isolates possessed MLVA profile 8-3-21-16-1. All isolates contained the previously described C2599T mutation (N. gonorrhoeae numbering) in all four 23S rRNA alleles and the previously described single-nucleotide (A) deletion in the mtrR promoter region. Conclusions This verified treatment failure occurred in a patient infected with an MLST ST1901/NG-MAST ST1407 strain of N. gonorrhoeae. While this international strain commonly shows resistance or decreased susceptibility to multiple antimicrobials, including extended-spectrum cephalosporins, the strain reported here remained fully susceptible to the latter antimicrobials. Hence, two subtypes of azithromycin-resistant gonococcal MLST ST1901/NG-MAST ST1407 appear to have evolved and to be circulating in Japan. Azithromycin should not be recommended as a single antimicrobial for first-line empirical treatment of gonorrhoea.

Background Gastroenteritis (GE) is a frequent reason for consultating a general practitioner. Yet little is known about antibiotic prescribing in primary care patients with GE. In this study, we quantified empirical and targeted antibiotic treatment of GE, compliance with recommendations from primary care clinical practice guidelines (CPGs) and the degree of antimicrobial resistance in patients receiving diagnostic faeces testing (DFT). Methods We performed a cohort study using routine care data of 160 general practitioners, including electronic patient records from 2013 to 2014. GE episodes were extracted and linked to microbiological laboratory records to retrieve results of DFT. For each episode, data on patient characteristics, DFT results including antimicrobial resistance testing, and antibiotic prescriptions were collected. Results We identified 13217 GE episodes. Antibiotic treatment was prescribed in 1163 (8.8%) episodes, most frequently with metronidazole (n = 646, 4.9%), azithromycin (n = 254, 1.9%) or ciprofloxacin (n = 184, 1.4%). Treatment was empirical for 641 (5%) GE episodes, of which 30% (n = 191) followed the CPG-recommended antibiotic choice. Targeted treatment following DFT results was prescribed for 537 GE episodes (4%), of which 99% (n = 529) followed CPG recommendations. Non-susceptibility to first- or second-choice antibiotics was demonstrated in three Salmonella isolates (9%-13% of all isolates) and one Campylobacter isolate (1%). Conclusions Antibiotic treatment of GE in primary care is relatively infrequent, with 1 in 11 episodes treated. Empirical treatment was more frequent compared with targeted treatment and mostly with non-CPG-recommended antibiotics. However, treatment based upon DFT results followed CPG recommendations.

OBJECTIVES Unlike most of the world, penicillin resistance in Neisseria gonorrhoeae from remote regions of Western Australia (WA) with high gonorrhoea notification rates has not increased despite many years of empirical oral therapy. With the advent of non-culture molecular diagnosis of gonorrhoea and the consequent decline in culture-based susceptibility, it is imperative to ensure the ongoing reliability of combination oral azithromycin, amoxicillin and probenecid for uncomplicated gonorrhoea in this setting. PCR-based non-culture N. gonorrhoeae antimicrobial resistance surveillance for penicillinase production was therefore employed. METHODS Genital and non-genital specimens that were PCR-positive for N. gonorrhoeae were assessed for penicillinase production by detection of the N. gonorrhoeae TEM-1 plasmid using specific real-time PCR. RESULTS In remote regions of WA where gonorrhoea is highly endemic

Of the non-tuberculous mycobacteria, Mycobacterium abscessus is particularly refractory to antimicrobial therapy and new agents with activity against these pathogens are urgently needed. The screening of candidate antimicrobial agents against M. abscessus requires a relevant and reproducible animal model of chronic infection. Granulocyte-macrophage colony-stimulating factor knockout (GM-CSF KO) mice were used to develop a new animal model of chronic pulmonary M. abscessus infection that can be used for preclinical efficacy testing of antimicrobial drugs.GM-CSF KO mice were infected with a clinical isolate of M. abscessus via intrapulmonary aerosol delivery using a microsprayer device. The clinical condition, histology and cfu of M. abscessus-infected GM-CSF KO mice were evaluated over a period of 4 months. Mice were treated with azithromycin (100 mg/kg) by oral gavage and the clinical condition, histology and bacterial burden was determined after 2 weeks of treatment.We show that pulmonary infection of GM-CSF KO mice with M. abscessus results in a chronic pulmonary infection that lends itself to preclinical testing of new antimicrobial drugs against this bacterium. Azithromycin treatment of M. abscessus-infected GM-CSF KO mice resulted in a lower bacterial burden in the lungs and spleen, weight gain and significant improvement in lung pathology.Intrapulmonary aerosol infection of GM-CSF KO mice with M. abscessus is a useful animal model for studying pathogenesis as well as pre-clinical testing of new compounds against M. abscessus in acute or chronic phases of infection.

Detailed characterization of the first high-level azithromycin-resistant Neisseria gonorrhoeae cases in Ireland

Objectives The objective of this study was to determine the prevalence of specific acquired macrolide resistance genes previously reported as present in clinical isolates of Haemophilus influenzae. Methods A collection of 172 clinical respiratory isolates of H. influenzae, including 59 isolates from cystic fibrosis patients and 27 from non-cystic fibrosis bronchiectasis patients with significant prior macrolide use, was established. This collection was tested for azithromycin susceptibility using Etest and screened for the presence of erm(A), erm(B), erm(C), erm(F), mef(A) and mef(E) using locked nucleic acid dual-labelled hydrolysis probes. Results The azithromycin MICs ranged from 0.09 to >256 mg/L, with 2 (1.2%) isolates susceptible, 163 (94.8%) intermediate and 7 (4%) resistant according to EUCAST breakpoints (susceptible, ≤0.12 mg/L; resistant, >4 mg/L). None of the acquired macrolide resistance genes erm(A), erm(B), erm(C), erm(F), mef(A) or mef(E) was detected in any of the isolates. Conclusions The specific acquired macrolide resistance genes are not widespread in H. influenzae and the high prevalence of these genes previously reported might be unique to the specific circumstances of that study.

Previous work showed a higher prevalence of macrolide/azalide resistance in provinces of Canada where azithromycin was the major treatment for Streptococcus pneumoniae as compared with regions where clarithromycin was the dominant treatment. These data provided a way to test the mutant selection window hypothesis, which predicts that the serum drug concentration (AUC(24)) relative to the mutant prevention concentration (MPC) would be higher for clarithromycin than for azithromycin.The MIC and MPC were determined for 191 penicillin/macrolide-susceptible clinical isolates of S. pneumoniae with azithromycin, clarithromycin and erythromycin using agar plate assays.The MIC(50/90) (mg/L) and MPC(50/90) (mg/L), respectively, were as follows: azithromycin 0.13/0.25 and 1/4; clarithromycin 0.031/0.063 and 0.13/0.5; erythromycin 0.063/0.13 and 0.25/2. We calculated from published pharmacokinetic values that the AUC(24)/MPC(90) for azithromycin was 0.85; for clarithromycin it was 96, and for erythromycin base and estolate it was 4 and 10, respectively. Thus the AUC(24)/MPC(90) was about 50 times higher for clarithromycin than for azithromycin.The elevated prevalence of azithromycin resistance may derive in part from a low value of AUC(24)/MPC(90) and/or time above MPC, since previous work indicates that the number of prescriptions per person was similar in the geographical regions examined.

Mycoplasma genitalium is a sexually transmitted organism associated with non-gonococcal urethritis in men and several inflammatory reproductive tract syndromes in women. Resistance to macrolides has been recently associated with point mutations in the 23S rRNA gene. The aim of this study was to detect these mutations using a large French collection of M. genitalium-positive specimens. We evaluated whether these mutations were related to azithromycin treatment failure and whether macrolide-resistant M. genitalium may be spreading.A retrospective study conducted in France between 2003 and 2010 included 156 urogenital clinical specimens from 136 patients that were positive for M. genitalium. Mutations in domain V of M. genitalium 23S rRNA were detected using amplification and sequencing. The mutated strains were genotyped by studying single nucleotide polymorphisms in the mgpB gene.We have detected macrolide resistance-associated mutations in M. genitalium since 2006 at a rate of 13.2%, ranging from 10% to 15.4% of patients per year. Nine mutations at position 2059 as well as two A2058G substitutions, one A2062T substitution and one C2038T substitution (Escherichia coli numbering) were identified in M. genitalium. These patients had treatment failure with azithromycin in 75% (6/8) of cases. For one patient, genotyping showed selection for the mutation during treatment with azithromycin.For the first time, we describe macrolide resistance for M. genitalium in France and demonstrate that its detection has increased since 2006. Epidemiological surveillance of M. genitalium is necessary to adapt treatments to M. genitalium infections.

To undertake laboratory and clinical surveillance of gonococcal antimicrobial susceptibility to various therapeutic agents in Hong Kong, so as to monitor for emerging resistance and to inform on appropriate choice of empirical therapy.Trends in susceptibility of gonococci to ceftriaxone, spectinomycin, ceftibuten and azithromycin were monitored over time. Isolates with reduced susceptibility to oral extended-spectrum cephalosporins were further characterized by detection of the mosaic penA gene and typing by Neisseria gonorrhoeae multi-antigen sequence typing (NG-MAST). Correlation with clinical and epidemiological findings was undertaken on isolates positive for the mosaic penA gene.Trends in susceptibility of gonococci to ceftriaxone, spectinomycin and ceftibuten remained stable between 2005 and 2010. In 2010, 30.3% of tested strains were not susceptible to azithromycin. The percentage of gonococcal strains harbouring the mosaic penA gene increased from 1.0% during April to December 2010 to 8.2% during January to September 2011 (P 0.0001). Review of available clinical records showed that, out of 35 patients infected by strains positive for the mosaic penA gene, 30 had laboratory-documented treatment failure.This study showed that ceftriaxone and spectinomycin remained effective against gonorrhoea in Hong Kong. There was an alarming increase in strains with reduced susceptibility to oral extended-spectrum cephalosporins associated with clinical treatment failure. One-third of gonococcal isolates were non-susceptible to azithromycin. The need to switch to agents other than oral extended-spectrum cephalosporins for empirical treatment is imminent. Continued surveillance with strain characterization is essential to monitor the effectiveness of currently recommended therapy.

Objectives We treated gonococcal urethritis in men with a single 2 g dose of azithromycin extended-release formulation (azithromycin-SR) to determine its microbiological outcomes and tolerability. Patients and methods We enrolled 189 Japanese men with gonococcal urethritis between April 2009 and December 2013. The patients were given a single 2 g dose of azithromycin-SR. Microbiological efficacy was evaluated by the results of the post-treatment molecular testing of Neisseria gonorrhoeae. MIC testing was performed only for pretreatment isolates of N. gonorrhoeae collected from the patients. Results We evaluated 130 patients for microbiological outcomes. Of these patients, 122 (93.8%) were judged to be microbiologically cured on the basis of negative test results. All isolates for which the azithromycin MICs were ≤0.25 mg/L were eradicated, whereas 5 of 12 isolates for which the MICs were 1 mg/L persisted after the treatment. Forty-six adverse events occurred in 41 patients. However, all adverse events were classified as mild. Conclusions The eradication rate of N. gonorrhoeae was 93.8% in men with gonococcal urethritis treated with a single 2 g dose of azithromycin-SR. The breakpoint MIC of a 2 g dose of azithromycin-SR for gonococcal urethritis associated with clinical treatment failures appeared to be 1 mg/L. With regard to side effects of higher doses of azithromycin, the 2 g dose of azithromycin-SR appeared to improve tolerability. However, the widespread use of a high-dose regimen of azithromycin might lead to the development of further resistance to azithromycin.

it is known that the efficacy of azithromycin, in animal infection models, is best correlated with AUC/MIC. The pharmacokinetic-pharmacodynamic (PK-PD) relationship for azithromycin, however, has not been previously confirmed with clinical data. The objectives of this PK-PD analysis were to characterize exposure-response relationships for the efficacy and safety of azithromycin extended release (ER) in Japanese patients, and to evaluate the effects of potential covariates on the prediction of response.sparse serum azithromycin concentration, MIC, efficacy and safety data were collected from three Japanese Phase 3 studies of a 2 g single dose of azithromycin-ER for respiratory tract infections. These sparse concentration data were combined with data from eight Phase 1 PK studies in Japanese and Western populations, to develop a robust population PK model using a non-linear mixed effects approach. The exposure-response relationships for efficacy and safety were evaluated using logistic regression.a two-compartment model with first-order absorption and first-order elimination with a lag time adequately described the PK of azithromycin-ER, without any significant ethnic differences in AUC. The percentage of bacteriological and clinical success in patients with AUC/MIC 5 (95.8% and 100%, respectively) was much higher than in those with AUC/MIC ≤ 5 (60.0% and 83.3%, respectively).as expected, the probabilities of success in the clinical and bacteriological responses were positively associated with AUC/MIC, but not with AUC. For the exposure-safety relationship, the incidence of treatment-related diarrhoea was inversely associated with azithromycin exposure.

Objectives: The aim of this study was to characterize a putative novel macrolide efflux gene located in the vicinity of sul3 in porcine Escherichia coli. Methods: Five sul3-encoding E. coli isolates of porcine origin were investigated by plasmid characterization and random amplification of polymorphic DNA (RAPD) PCR. Unknown DNA adjacent to the sul3 genes was amplified using a PCR approach, followed by sequencing of the fragments. The putative macrolide efflux gene was cloned into pK18. The cloned gene was characterized by susceptibility testing by Etest in the presence and absence of efflux inhibitors. Results: Five sul3-encoding isolates, demonstrated to be unrelated by RAPD PCR, were characterized. The immediate genetic context of sul3 in five isolates was identical to that in plasmid pVP440, and in all cases, sul3 was associated with class 1 integrons. In three isolates, an open reading frame (orf2) encoding a putative protein with 38% amino acid identity to Mef(A) was found, while the two remaining isolates contained a fragment of orf2 truncated by IS26 insertion. In three of the isolates, this DNA region was demonstrated to be located on non-conjugative plasmids. When the complete orf2 was cloned, it conferred high-level resistance to erythromycin and azithromycin, and the resistance property could be partially inhibited using the efflux inhibitor Phe-Arg b-naphthylamide dihydrochloride. The gene was named mef(B). Conclusions: A new macrolide efflux protein, Mef(B), with 38% amino acid identity to Mef(A), has been characterized and represents the second member of the mef family of genes.

Objectives: Mucus hypersecretion is a prominent feature in patients with chronic respiratory tract infections such as cystic fibrosis and diffuse panbronchiolitis, and the clinical effectiveness of macrolide antibiotics has been reported in these patients. Because human neutrophil peptide-1 (HNP-1), an antimicrobial peptide in neutrophils, exists in high concentrations in the airway fluid of these patients, we examined the direct effect of HNP-1 on MUC5AC mucin production using NCI-H292 cells. The effects of macrolide antibiotics on the response were also examined. Methods: MUC5AC synthesis was assayed using RT-PCR and ELISA. Phosphorylation of ERK1/2 was determined by western blotting. Results: Stimulation with HNP-1 or lipopolysaccharide (LPS) derived from Pseudomonas aeruginosa increases the production of MUC5AC mRNA and protein, and an additive effect was found upon co-stimulation with both HNP-1 and LPS. Azithromycin and clarithromycin had inhibitory effects on overproduction of MUC5AC induced by HNP-1 or LPS stimulation. Telithromycin also had an inhibitory effect on MUC5AC production induced by LPS, but not on production by HNP-1. Phosphorylation of ERK1/2 was induced by HNP-1 or LPS stimulation, and azithromycin, clarithromycin and telithromycin had inhibitory effects on ERK1/2 phosphorylation induced by LPS, but not by HNP-1. Conclusions: These findings suggest that neutrophil-derived defensins as bacterial components contribute to excessive mucus production in patients with respiratory tract infections, and that macrolide and ketolide antibiotics directly inhibit these actions by interfering with intracellular signal transduction. However, the mechanism of telithromycin inhibition of MUC5AC synthesis may differ from the response induced by azithromycin and clarithromycin.

The use of macrolide antibiotics in food animals has the potential to select for macrolide-resistant strains of resident bacterial flora. This may include the animal pathogens that are the intended targets of macrolide antibiotic intervention and Campylobacter, common inhabitants of the intestinal tract of food animals that are zoonotic pathogens in man. Such Campylobacter strains are not only resistant to the macrolide antibiotics used in food animals, e.g. tylosin, tilmicosin and tulathromycin, but to the macrolide antibiotics used in human medicine, e.g. erythromycin, azithromycin and clarithromycin, as well. Retail meat is a possible source of Campylobacter and persons consuming the meat derived from macrolide-treated food animals could acquire infections due to macrolide-resistant strains of this organism. Erythromycin is sometimes used to treat human cases of campylobacteriosis and those infected with animal-derived macrolide-resistant Campylobacter may not respond to treatment. The actual risk to human health from the use of macrolide antibiotics in food animals has been difficult to determine because of a lack of information about the macrolide-resistant Campylobacter found on the farm and in the clinic. Recently, however, a plethora of new information has become available on this topic. This review discusses what is currently known about the selection of macrolide-resistant Campylobacter in food animals, the prevalence of macrolide-resistant Campylobacter on retail meat, the prevalence of animal-derived macrolide-resistant Campylobacter in the clinic and the human health consequences associated with macrolide-resistant Campylobacter infection. This work will emphasize the comprehensive body of data generated in Denmark and the US as part of government-sponsored research studies over the last 10 years. These scientific findings may allow informed decisions to be made in the future about how macrolide antibiotics should be used in food animals while still safeguarding human health.

Objectives: Patients with cystic fibrosis (CF) are frequently colonized by macrolide-resistant Staphylococcus aureus, a result of maintenance macrolide therapy. As transmission of S. aureus between household contacts is common, we examined the prevalence of macrolide-resistant S. aureus colonization in CF patients on maintenance azithromycin therapy and their household contacts and compared this with the S. aureus macrolide resistance prevalence in the community. Patients and methods: Sixty-five CF patients on maintenance macrolide therapy and 194 household contacts were screened for S. aureus colonization by culturing sputa, cough swabs and nasal swabs. Resistance to macrolide, lincosamide and methicillin was determined by disc diffusion tests. The prevalence of macrolide-resistant S. aureus colonization in both groups was compared with figures from a nationwide study into S. aureus carriership and resistance. To assess possible transmission, genotyping of S. aureus was performed using the spa-typing method. Results: Macrolide resistance among CF patients with S. aureus colonization was 69.6%; 75% of these isolates displayed lincosamide resistance too. Among household contacts, macrolide resistance prevalence did not differ significantly from resistance prevalence in the community (9.6% versus 6.3%; P 5 0.358). No methicillin resistance was observed. No identical (macrolide-resistant and -susceptible) S. aureus genotypes were observed between CF patients and their household contacts except for one household, suggesting a probable transmission. Conclusions: No significant increase in macrolide-resistant S. aureus colonization was observed among household contacts of CF patients on long-term azithromycin therapy. Transmission of macrolide-resistant S. aureus could not be proved by genotyping in the majority of households.

Objectives Azithromycin is not associated with significant adverse effects or restricted usage in certain populations unlike standard antileptospirosis agents. In this study, the utility of short courses of azithromycin in treating or preventing leptospirosis was investigated in a lethal hamster model. Methods All hamsters were infected intraperitoneally with 10(5) leptospires. In experiment one, animals received 5 mg/kg of doxycycline or 10 mg/kg of azithromycin via intraperitoneal injection beginning on the second day after infection and continuing once daily for 1, 2, 3 or 5 days. In experiment two, animals received 1 or 2 day courses of azithromycin initiated 2 or 4 days following infection, or 4 days prior to infection. Results All untreated control animals died between the sixth and ninth day following infection. In experiment one, survival rates in the doxycycline groups were 0, 50, 80 and 100% for those animals treated for 1, 2, 3 and 5 days, respectively. Except for the 1 day treatment group (which had an 80% survival), there was 100% survival in all azithromycin-treated groups. In experiment two, all animals treated after infection survived until study completion. No animals survived with 1 day of therapy started 4 days prior to infection while only 20% survived if they received 2 days. Conclusions These results suggest short-course therapy with azithromycin, even started well after infection, is efficacious in preventing mortality from acute leptospirosis.

Objectives: To investigate antimicrobial effects on a mixed culture of five Streptococcus pneumoniae serotypes (S) as an approach to ecology of population dynamics. Methods: A computerized pharmacodynamic model simulating concentrations obtained after levofloxacin, ciprofloxacin and azithromycin doses was used. Resistance patterns were S12, susceptible to study drugs; S31, low-level macrolide-resistant (efflux phenotype); S11, high-level macrolide-resistant (erm genotype); S9V, low-level quinolone-resistant; and S3, high-level quinolone-resistant. Initial mixed inocula (time 0) included similar percentages of each serotype. Results: At 24 h of control drug-free experiments, dominant strains were S9V (57.4%) and S12 (41.8%) with marginal populations of S31, S3 and S11. Azithromycin selected to a much higher extent the strain with low-level resistance to macrolides (S31) rather than the strain with high-level resistance (S11) (accounting for 99.9% versus 0.1% of the total population at 24 h). Ciprofloxacin selected to a higher extent low-level (S9V) rather than high-level (S3) quinolone resistance (72.4% versus 27.6%). Levofloxacin decreased the proportion of the predominant S9V in controls to 22.2% (an intermediate-resistant strain with MIC = 4 mg/L) and unmasked the high-level resistant strain (MIC = 32 mg/L) up to 77.8%. Conclusions: Strain distribution in an antibiotic-free environment depends on bacterial fitness in mono-and multi-strain niches. Selective pressure of antimicrobial regimens eradicate some populations and unmask minor populations, thus redistributing the whole population. Selective potential only for resistance phenotypes with very low prevalence (such as high-level quinolone resistance) in the community should be preferred to that selecting more prevalent resistance phenotypes.

To investigate the azithromycin pharmacodynamic parameters predicting bacterial killing in epithelial lining fluid (ELF) versus serum against macrolide-susceptible and -resistant Streptococcus pneumoniae isolates (with different resistance genotypes), through the simulation of concentrations achieved after a 500 mg intravenous (iv) once a day regimen.An in vitro computer-controlled pharmacodynamic simulation of human azithromycin concentrations in serum and ELF was carried out, and colony counts were determined over 24 h. Four strains with MIC values (mg/L) of 0.5 [mef(A) and erm(B) negative], 2 [mef(A) positive and erm(B) negative], 8 [mef(A) positive and erm(B) negative] and 256 [mef(A) negative and erm(B) positive] were used.Significant (P0.05) azithromycin antibacterial activity versus antibiotic-free controls was found in serum and ELF against the susceptible and mef(A) positive strains, but not against the erm(B) positive strain. AUC(0-24)/MIC values around or higher than 25 were needed to achieve (time to 99.9% reduction of initial inocula of around 6 h) and maintain (24 h inocula reductionor =3 log(10)cfu/mL) bactericidal activity without regrowth. This was achieved only with the susceptible strain in serum, but also with the mef(A) positive strain exhibiting an MIC of 2 mg/L in ELF.The results of this study support that the suggested breakpoint for susceptibility (or =2 mg/L) may be adequate to predict S. pneumoniae eradication with ELF but not with serum concentrations obtained after a 500 mg iv once a day regimen.

Objectives Azithromycin is used to modulate exuberant inflammatory response in patients with cystic fibrosis (CF). The purpose of this study was to determine the association between long-term use of azithromycin in CF patients and change over time in macrolide susceptibility of Staphylococcus aureus and Haemophilus spp. Methods The study was performed at the Erasmus MC-Sophia Children's Hospital. CF patients' sputum cultures were obtained at routine visits and at pulmonary exacerbations. All cultures between January 1999 and March 2004 were included. Antibiotic susceptibility of S. aureus and Haemophilus spp. was tested routinely. Susceptibility was compared with isolates from sputum of non-CF patients. Logistic regression was used to analyse the association between azithromycin use and resistance, adjusting for age, Pseudomonas carriage and time-trends. Results In March 2004 one-third of CF patients were on azithromycin maintenance treatment. S. aureus (715 isolates) and/or Haemophilus (537 isolates) were cultured in 141 of the 155 patients on one or more occasions. The study period was divided into octiles. Erythromycin resistance in S. aureus increased from 6.9 to 53.8% and clarithromycin resistance in Haemophilus spp. from 3.7 to 37.5%. Resistance but also isolation rates were strongly related to azithromycin use. Resistance of 3217 S. aureus control isolates remained stable and resistance of 3257 Haemophilus controls increased, although at a slower rate than CF isolates. Conclusions Over a 4 year period, azithromycin maintenance therapy in our CF population was associated with an increase in macrolide resistance in S. aureus and Haemophilus spp.

Objectives: A homologue of the MacA–MacB ABC transporter of Escherichia coli, which recognizes and exports macrolides, was identified in Neisseria gonorrhoeae. This study was undertaken to determine whether gonococci could use the MacA–MacB homologue to express decreased susceptibility to macrolides. Methods:Techniques of DNA sequencing, gene cloning and expression of recombinant proteinsinE. coli, gene mutation construction, transcriptional analysis and antimicrobial susceptibility testing were used in the study. Results: Although the gonococcal MacA–MacB efflux pump enhanced bacterial resistance to macrolides when overexpressed in an E. coli background, its loss in a gonococcal clinical isolate only slightly decreasedbacterialresistancetoazithromycinanderythromycin.However,amutationinthe –10sequence ofthepromoterusedinmacABexpressionenhancedthemacrolideresistanceofgonococcithatproduced a defective MtrC–MtrD–MtrE pump, which also recognizes macrolides. Conclusions: The results from this study indicate that gonococci can employ both the MacA–MacB and MtrC–MtrD–MtrEeffluxpumpstodevelop resistancetomacrolides, particularlyifmutations developinthe promoter that drives transcription of macAB.