Determination of four aminoglycoside antibiotics and spectinomycin in feed at cross-contamination level: Development and in-house validation of a LC-MS/MS method for enforcing EU Regulation.

Combating antimicrobial resistance is a top priority worldwide involving a concerted action by several high-level institutions and organisations in the health sector. To ensure that a meaningful progress is achieved, several campaigns and political initiatives have been launched targeting the health professionals, the industry, the farmers, and the general public. The Regulation (EU) 2019/4 on medicated feed contains provisions for the limitation and control of the contamination of non-target compound feed with 24 antimicrobials. The purpose of this work was to develop a reliable and effective method for the determination of four aminoglycoside antibiotics (apramycin, paromomycin, tobramycin and neomycin) and spectinomycin in feed at cross-contamination level, where an absolute lack of suitable methods was identified. Four candidate methods described in the literature failed to provide adequate recoveries of all analytes. Therefore, an in-depth investigation was carried out to identify the bottleneck variable. The optimised method was then in-house validated and showed performance features appropriate for the intended purpose. The selected compounds could be analysed by LC-MS/MS in five animal feeds with LOQs between 2.6 and 9.2 µg kg-1 for the AGs and between 28 and 86 µg kg-1 for spectinomycin. Using isotopically labelled internal standards, the recovery rates varied from 63 % to 103 % and the intermediate precision (RSDip) varied from 1.1 % to 14 %. This work represents a step forward in the reliable determination of antibiotics in compound feed as the developed method has shown to be precise and sensitive. It is expected that this method gains wide acceptance and can supplement the legislation with effective control tools for antibiotic residues.

Detection of antimicrobial resistance in <5 h in Neisseria gonorrhoeae isolates using flow cytometry-proof of concept for seven clinically relevant antimicrobials.

INTRODUCTION: Antimicrobial resistance in Neisseria gonorrhoeae compromises gonorrhoea treatment and rapid antimicrobial susceptibility testing (AST) would be valuable. We have developed a rapid and accurate flow cytometry method (FCM) for AST of gonococci. METHODS: The 2016 WHO gonococcal reference strains, and WHO Q, R and S (n = 17) were tested against seven clinically relevant antibiotics (ceftriaxone, cefixime, azithromycin, spectinomycin, ciprofloxacin, tetracycline and gentamicin). After 4.5 h incubation of inoculated broth, the fluorescent dye Syto™ 9 was added, followed by FCM analysis. After gating, the relative remaining population of gonococci, compared with unexposed growth control samples, was plotted against antimicrobial concentration, followed by non-linear curve regression analysis. Furthermore, the response at one single concentration/tested antibiotic was evaluated with the intention to use as a screening test for detection of resistant gonococci. RESULTS: A dose-dependent response was seen in susceptible isolates for all tested antimicrobials. There was a clear separation between susceptible/WT and resistant/non-WT isolates for ceftriaxone, cefixime, spectinomycin, ciprofloxacin and tetracycline. In contrast, for azithromycin, only high-level-resistant isolates were distinguished, while resistant isolates with MICs of 4 mg/L were indistinguishable from WT (MIC ≤ 1 mg/L) isolates. For gentamicin, all tested 17 isolates were WT and FCM analysis resulted in uniform dose-response curves. Using a single antibiotic concentration and a 50% remaining cell population cut-off, the overall sensitivity and specificity for resistance detection were 93% and 99%, respectively. CONCLUSIONS: By providing results in <5 h for gonococcal isolates, FCM-based AST can become a rapid screening method for antimicrobial resistance or antimicrobial susceptibility in gonococci.

[Establishment and Evaluation of a Resazurin-Based Microdilution Assay for Microbial Sensitivity Test of Neisseria gonorrhoeae]. / æ·‹ç— å¥ˆç‘ŸèŒè¯æ•è¯•éªŒåˆƒå¤©é’å¾®é‡ç¨€é‡Šæ³•çš„å»ºç«‹ä¸Žè¯„ä»·.

Objective: To establish and evaluate a microbial sensitivity test method for Neisseria gonorrhoeae based on resazurin coloration. Methods: Based on the broth microdilution method, resazurin was added as a live bacteria indicator. WHO G, a WHO gonococcal reference strain, was used to optimize the incubation time for resazurin-stained bacteria and the color change was visually observed to obtain the results. Agar dilution method (the gold standard) and resazurin-based microdilution assay were used to determine the minimum inhibitory concentration (MIC) of azithromycin, ceftriaxone, and spectinomycin for 3 reference strains and 32 isolates of Neisseria gonorrhoeae. The results were analyzed based on essential agreement (EA), which reflected the consistency of the MIC values, category agreement (CA), which reflected the consistency in the determination of drug resistance, intermediary, and sensitivity, very major error (VME), which reflected false sensitivity, and major error (ME), which reflected pseudo drug resistance, to evaluate the accuracy of resazurin-based microdilution assay as a microbial sensitivity test of of Neisseria gonorrhoeae. CA and EA rates≥90% and VME and ME rates≤3% were found to be the acceptable performance rates. Results: The results obtained 6 hours after resazurin was added were consistent with those of the agar dilution method and the resazurin-based microdilution assay was established accordingly based on this parameter. The EA of resazurin-based microdilution assay for measuring the MIC results of azithromycin, ceftriaxone, and spectinomycin was 97.1%, 91.5%, and 94.3%, respectively, and the CA was 88.6%, 94.3%, and 94.3%, respectively. The VME was 0% for all three antibiotics, while the ME was 11.4%, 5.7%, and 5.7%, respectively. Conclusion: The resazurin-based microdilution assay established in this study showed good agreement with agar dilution method for measuring the MIC of antibiotics against Neisseria gonorrhoeae. Moreover, the sensitivity results of this method were highly reliable and could be easily obtained through naked eye observation. Nonetheless, the results of drug resistance should be treated with caution and the optimization of parameters should be continued.

Development of 2nd generation aminomethyl spectinomycins that overcome native efflux in Mycobacterium abscessus.

Mycobacterium abscessus (Mab), a nontuberculous mycobacterial (NTM) species, is an emerging pathogen with high intrinsic drug resistance. Current standard-of-care therapy results in poor outcomes, demonstrating the urgent need to develop effective antimycobacterial regimens. Through synthetic modification of spectinomycin (SPC), we have identified a distinct structural subclass of N-ethylene linked aminomethyl SPCs (eAmSPCs) that are up to 64-fold more potent against Mab over the parent SPC. Mechanism of action and crystallography studies demonstrate that the eAmSPCs display a mode of ribosomal inhibition consistent with SPC. However, they exert their increased antimicrobial activity through enhanced accumulation, largely by circumventing efflux mechanisms. The N-ethylene linkage within this series plays a critical role in avoiding TetV-mediated efflux, as lead eAmSPC 2593 displays a mere fourfold susceptibility improvement against Mab ΔtetV, in contrast to the 64-fold increase for SPC. Even a minor shortening of the linkage by a single carbon, akin to 1st generation AmSPC 1950, results in a substantial increase in MICs and a 16-fold rise in susceptibility against Mab ΔtetV. These shifts suggest that longer linkages might modify the kinetics of drug expulsion by TetV, ultimately shifting the equilibrium towards heightened intracellular concentrations and enhanced antimicrobial efficacy. Furthermore, lead eAmSPCs were also shown to synergize with various classes of anti-Mab antibiotics and retain activity against clinical isolates and other mycobacterial strains. Encouraging pharmacokinetic profiles coupled with robust efficacy in Mab murine infection models suggest that eAmSPCs hold the potential to be developed into treatments for Mab and other NTM infections.

Antimicrobial susceptibility profile of Neisseria gonorrhoeae from patients attending a medical laboratory, Institut Pasteur de Madagascar between 2014 and 2020: phenotypical and genomic characterisation in a subset of Neisseria gonorrhoeae isolates.

OBJECTIVES: Antimicrobial-resistant Neisseria gonorrhoeae (NG) is a concern. Little is known about antimicrobial susceptibility profiles and associated genetic resistance mechanisms of NG in Madagascar. We report susceptibility data of NG isolates obtained by the medical laboratory (CBC) of the Institut Pasteur de Madagascar, Antananarivo, Madagascar, during 2014-2020. We present antimicrobial resistance mechanisms data and phenotype profiles of a subset of isolates. METHODS: We retrieved retrospective data (N=395) from patients with NG isolated during 2014-2020 by the CBC. We retested 46 viable isolates including 6 found ceftriaxone and 2 azithromycin resistant, as well as 33 isolated from 2020. We determined minimal inhibitory concentrations for ceftriaxone, ciprofloxacin, azithromycin, penicillin, tetracycline and spectinomycin using Etest. We obtained whole-genome sequences and identified the gene determinants associated with antimicrobial resistance and the sequence types (STs). RESULTS: Over the study period, ceftriaxone-resistant isolates exceeded the threshold of 5% in 2017 (7.4% (4 of 54)) and 2020 (7.1% (3 of 42)). All retested isolates were found susceptible to ceftriaxone, azithromycin and spectinomycin, and resistant to ciprofloxacin. The majority were resistant to penicillin (83% (38 of 46)) and tetracycline (87% (40 of 46)). We detected chromosomal mutations associated with antibiotic resistance in gyrA, parC, penA, ponA, porB and mtrR genes. None of the retested isolates carried the mosaic penA gene. The high rate of resistance to penicillin and tetracycline is explained by the presence of bla TEM (94.7% (36 of 38)) and tetM (97.5% (39 of 40)). We found a high number of circulating multilocus STs. Almost half of them were new types, and one new type was among the four most predominant. CONCLUSIONS: Our report provides a detailed dataset obtained through phenotypical and genotypical methods which will serve as a baseline for future surveillance of NG. We could not confirm the occurrence of ceftriaxone-resistant isolates. Our results highlight the importance of implementing quality-assured gonococcal antimicrobial resistance surveillance in Madagascar.

Engineering of insecticidal hybrid gene into potato chloroplast genome exhibits promising control of Colorado potato beetle, Leptinotarsa decemlineata (Coleoptera: Chrysomelidae).

The potato chloroplast was transformed with codon optimized synthetic hybrid cry gene (SN19) to mitigate crop losses by Colorado potato beetle (CPB). The bombarded explants (leaves and internode) were cultured on MS medium supplemented with BAP (2.0 mg/l), NAA (0.2 mg/l), TDZ (2.0 mg/l) and GA3 (0.1 mg/l); spectinomycin 50 mg/l was used as a selection agent in the medium. Leaf explants of cultivar Kuroda induced highest percentage (92%) of callus where cultivar Santae produced the highest percentage (85.7%) of transplastomic shoots. Sante and Challenger showed 9.6% shoot regeneration efficiency followed by cultivar Simply Red (8.8%). PCR amplification yielded 16 postive transplastomic plantlets out of 21 spectinomycin resistant ones. Target gene integration was confirmed by PCR and Southern blot, whereas RT-qPCR was used to assess the expression level of transgene. The localization of visual marker gene gfp was tracked by laser scanning confocal microscopy which confirmed its expression in chloroplasts of leaf cells. The transplastomic plants ensured high mortality to both larvae and adult CPB. Foliage consumption and weight gain of CPB fed on transplastomic leaves were lower compared to the control plants. Sucessful implementation of current research findings can lead to a viable solution to CPB mediated potato losses globally.

The third nationwide surveillance of antimicrobial susceptibility against Neisseria gonorrhoeae from male urethritis in Japan, 2016-2017.

Neisseria gonorrhoeae is one of the important pathogens of sexually transmitted infections. N. gonorrhoeae is rapidly becoming antimicrobial resistant, and there are few drugs that are effective in the initial treatment of gonorrhea. To understand the trends of antimicrobial susceptibility of N. gonorrhoeae, the Surveillance Committee of the Japanese Society of Infectious Diseases, the Japanese Society for Chemotherapy, and the Japanese Society of Clinical Microbiology conducted the third nationwide antimicrobial susceptibility surveillance of N. gonorrhoeae isolated from male urethritis. The specimens were collected from male patients with urethritis at 30 facilities from May 2016 to July 2017. From the 159 specimens collected, 87 N. gonorrhoeae strains were isolated, and 85 were tested for susceptibility to 21 antimicrobial agents. All strains were non-susceptible to penicillin G. Seven strains (8.2%) were ß-lactamase-producing strains. The rates of susceptibility to cefixime and cefpodoxime were 96.5% and 52.9%, respectively. Three strains were non-susceptible with a minimum inhibitory concentration (MIC) of 0.5 mg/L for cefixime. None of the strains were resistant to ceftriaxone or spectinomycin. The susceptibility rate for ciprofloxacin was 23.5% (20 strains), and no strains showed intermediate susceptibility. The susceptibility rate against azithromycin was 81.2%, with one strain isolated with a MIC of 8 mg/L against azithromycin. The results of this surveillance indicate that ceftriaxone and spectinomycin, which are currently recommended for gonococcal infections in Japan, appear to be effective. It will be necessary to further expand the scale of the next surveillance to understand the current status of drug-resistant N. gonorrhoeae in Japan.

Sanguinarine, similar to the MICs of spectinomycin, exhibits good anti-Neisseria gonorrhoeae activity in vitro.

The increasing antibiotic resistance of Neisseria gonorrhoeae (NG) is an urgent need to explore new and effective drugs. The antibacterial activities of spectinomycin and sanguinarine against 117 clinical NG isolates and time-kill curve of sanguinarine were evaluated. Almost all isolates were resistant to penicillin (91.5%) and ciprofloxacin (96.5%), 8.5% showed resistance to azithromycin, 10.3% and 10.3% had decreased susceptibility/resistance to ceftriaxone and cefixime, respectively, whereas 100% were susceptible to spectinomycin. The minimum inhibitory concentration (MIC) ranges, MIC50, MIC90 and MICmean values of sanguinarine were 2-64 µg/ml, 16 µg/ml, 32 µg/ml and 16.9 µg/ml, respectively, and time-kill curve showed killing of bacteria in a dose-dependent manner during the assay time of 6h, very similar to spectinomycin. Sanguinarine has great potential as an effective and novel anti-NG agent.

ANT(9)-Ic, a Novel Chromosomally Encoded Aminoglycoside Nucleotidyltransferase from Brucella intermedia.

Aminoglycoside-modifying enzymes are among the most important mechanisms of resistance to aminoglycoside antibiotics, typically conferring high-level resistance by enzymatic drug inactivation. Previously, we isolated a multidrug-resistant Brucella intermedia strain ZJ499 from a cancer patient, and whole-genome sequencing revealed several putative novel aminoglycoside-modifying enzyme genes in this strain. Here, we report the characterization of one of them that encodes an intrinsic, chromosomal aminoglycoside nucleotidyltransferase designated ANT(9)-Ic, which shares only 33.05% to 47.44% amino acid identity with the most closely related ANT(9)-I enzymes. When expressed in Escherichia coli, ANT(9)-Ic conferred resistance only to spectinomycin and not to any other aminoglycosides tested, indicating a substrate profile typical of ANT(9)-I enzymes. Consistent with this, deletion of ant(9)-Ic in ZJ499 resulted in a specific and significant decrease in MIC of spectinomycin. Furthermore, the purified ANT(9)-Ic protein showed stringent substrate specificity for spectinomycin with a Km value of 44.83 µM and a kcat/Km of 2.8 × 104 M-1 s-1, echoing the above observations of susceptibility testing. In addition, comparative genomic analysis revealed that the genetic context of ant(9)-Ic was conserved in Brucella, with no mobile genetic elements found within its 20-kb surrounding region. Overall, our results demonstrate that ANT(9)-Ic is a novel member of the ANT(9)-I lineage, contributing to the intrinsic spectinomycin resistance of ZJ499. IMPORTANCE The emergence, evolution, and worldwide spread of antibiotic resistance present a significant global public health crisis. For aminoglycoside antibiotics, enzymatic drug modification is the most common mechanism of resistance. We identify a novel chromosomal aminoglycoside nucleotidyltransferase from B. intermedia, called ANT(9)-Ic, which shares the highest identity (47.44%) with the previously known ANT(9)-Ia and plays an important role in spectinomycin resistance of the host strain. Analysis of the genetic environment and origin of ant(9)-Ic shows that the gene and its surrounding region are widely conserved in Brucella, and no mobile elements are detected, indicating that ANT(9)-Ic may be broadly important in the natural resistance to spectinomycin of Brucella species.

Characterization of spectinamide 1599 efficacy against different mycobacterial phenotypes.

Spectinamides are a novel series of spectinomycin analogs being developed for the treatment of tuberculosis. The preclinical lead spectinamide 1599 is an antituberculosis drug that possesses robust in vivo efficacy, good pharmacokinetic properties, and excellent safety profiles in rodents. In individuals infected with Mycobacterium tuberculosis or Mycobacterium bovis, causative agents of tuberculosis, the host immune system is capable of restraining these mycobacteria within granulomatous lesions. The harsh microenvironmental conditions of these granuloma lead to phenotypic transformation of mycobacteria. Phenotypically transformed bacteria display suboptimal growth, or complete growth arrest and are frequently associated with drug tolerance. Here we quantified the effect of spectinamide 1599 on log-phase and phenotypically tolerant isoforms of Mycobacterium bovis BCG using various in vitro approaches as a first indicator of spectinamide 1599 activity against various mycobacterial isoforms. We also used the hollow fiber infection model to establish time-kill curves and deployed pharmacokinetic/pharmacodynamic modeling to characterize the activity differences of spectinamide 1599 towards the different phenotypic subpopulations. Our results indicate that spectinamide 1599 is more efficacious against log phase bacteria when compared to its activity against other phenotypically tolerant forms such as acid phase bacteria and hypoxic phase bacteria, a behavior similar to the established antituberculosis drug isoniazid.

Mab2780c, a TetV-like efflux pump, confers high-level spectinomycin resistance in mycobacterium abscessus.

Mycobacterium abscessus is highly resistant to spectinomycin (SPC) thereby making it unavailable for therapeutic use. Sublethal exposure to SPC strongly induces whiB7 and its regulon, and a ΔMab_whiB7 strain is SPC sensitive suggesting that the determinants of SPC resistance are included within its regulon. In the present study we have determined the transcriptomic changes that occur in M. abscessus upon SPC exposure and have evaluated the involvement of 11 genes, that are both strongly SPC induced and whiB7 dependent, in SPC resistance. Of these we show that MAB_2780c can complement SPC sensitivity of ΔMab_whiB7 and that a ΔMab_2780c strain is ∼150 fold more SPC sensitive than wildtype bacteria, but not to tetracycline (TET) or other aminoglycosides. This is in contrast to its homologues, TetV from M. smegmatis and Tap from M. tuberculosis, that confer low-level resistance to TET, SPC and other aminoglycosides. We also show that the addition of the efflux pump inhibitor (EPI), verapamil results in >100-fold decrease in MIC of SPC in bacteria expressing Mab2780c to the levels observed for ΔMab_2780c; moreover a deletion of MAB_2780c results in a decreased efflux of the drug into the cell supernatant. Together our data suggest that Mab2780c is an SPC antiporter. Finally, molecular docking of SPC and TET on models of TetVMs and Mab2780c confirmed our antibacterial susceptibility findings that the Mab2780c pump preferentially effluxes SPC over TET. To our knowledge, this is the first report of an efflux pump that confers high-level drug resistance in M. abscessus. The identification of Mab2780c in SPC resistance opens up prospects for repurposing this relatively well-tolerated antibiotic as a combination therapy with verapamil or its analogs against M. abscessus infections.

Base-catalyzed hydrolysis of spectinomycin in aqueous solutions: Kinetics and mechanisms.

Hydrolysis plays an imperative role in the abiotic transformation process of antibiotics in aqueous solutions. However, little information is available on the hydrolysis process of spectinomycin (an aminocyclitol antibiotic). This study systematically investigated the spectinomycin hydrolysis kinetics and mechanisms under different pH via experiments and density functional theory (DFT) computation. Hydrolysis was first conducted in a pure water system under pH of 4.0-9.0 and temperature of 25 °C, 50 °C and 70 °C, respectively. Results showed that hydrolysis was highly dependent on pH and temperature. When pH > 6.0, spectinomycin hydrolysis was accelerated by the catalysis of OH-. Meanwhile, the hydrolysis rate increased with the elevation of temperature. Then, for the reference of the practical environment, the general base-catalyzed hydrolysis and mechanisms were studied under environmental pH 6.0-8.0 and 25 °C. DFT calculation demonstrated that base-catalyzed hydrolysis of spectinomycin could be more thermodynamically and kinetically favorable based on the lower Gibbs free energies of reaction and Gibbs free energies of activation. Further, instead of specific base catalysis (OH-), the general base catalysis (e.g., phosphate buffer) was also found to promote hydrolysis efficiency. The antibacterial activity and ecotoxicities of the hydrolysis product were analyzed to be lower than the precursor, thereby decreasing the environmental impact of spectinomycin.

Penicillin and amikacin mixture has bactericidal activity equivalent to gentamicin, tylosin, lincomycin and spectinomycin mixture in equine frozen semen.

Neat stallion semen can contain a variety of microorganisms, some of which may impair sperm quality and/or cause infection of the mares' reproductive tract. For this reason, antibiotics are commonly added to semen extenders. A combination of gentamicin, tylosin, lincomycin and spectinomycin (GTLS) has been recommended for use, but there are no reports on the use of this mixture in equine semen extender. Penicillin and amikacin (PA) are safe for preserving sperm quality while effectively controlling bacterial growth in equine cooled stored semen, but data on frozen semen are scarce. Therefore, a bioequivalence study was performed to assess the bactericidal activity of GTLS and PA in equine frozen semen. Nine mature, healthy stallions were used in the study. Split ejaculates were processed using media without antibiotics (Control) or with different antibiotics. For the GTLS group, centrifugation medium and freezing extender were prepared with gentamicin 250 µg/ml, tylosin 50 µg/ml, lincomycin 150 µg/ml and spectinomycin 300 µg/ml. For the PA group, the centrifugation medium was prepared with potassium penicillin G (PPG) 1200 units/ml and the freezing extender was prepared with PPG 1200 units/ml and amikacin 500 µg/ml. Semen processed in extenders without antibiotics had higher (p < .005) bacterial loads throughout all cryopreservation processing steps than semen samples processed using antibiotics. There were no differences in semen bacterial load after centrifugation, 15 and 30 min after final extension, and after thawing between GTLS and PA groups, but PA had faster (p < .05) kill-time kinetics than GTLS. Only minor differences in sperm kinetic parameters were observed among groups. In conclusion, this study demonstrated bioequivalence between GTLS and PA in mitigating end-point bacterial loads. Prudent concentrations of the antibiotic mixtures evaluated in this study can be considered both effective and sperm-safe for equine frozen semen.

Invasive Streptococcus suis isolated in Spain contain a highly promiscuous and dynamic resistome.

Introduction: Streptococcus suis is a major pathogen for swine and human. Here we aimed to know the rates of antimicrobial resistance (AMR) in invasive S. suis isolates recovered along Spain between 2016 - 2021 and elucidate their genetic origin. Methods: Antibiotic susceptibility testing was performed for 116 isolates of different genetic backgrounds and geographic origins against 18 antibiotics of 9 families. The association between AMR and genotypes and the origin of the isolates were statistically analyzed using Pearson´s chi-square test and the likelihood ratio. The antimicrobial resistant genes were identified by whole genome sequencing analysis and PCR screenings. Results: High AMR rates (>80%) were detected for tetracyclines, spectinomycin, lincosamides, and marbofloxacin, medium (20-40%) for sulphonamides/trimethoprim, tiamulin, penicillin G, and enrofloxacin, and low (< 20%) for florfenicol, and four additional ß-lactams. The occurrence of multidrug resistance was observed in 90% of isolates. For certain antibiotics (penicillin G, enrofloxacin, marbofloxacin, tilmicosin, and erythromycin), AMR was significantly associated with particular sequence types (STs), geographic regions, age of pigs, and time course. Whole genome sequencing comparisons and PCR screenings identified 23 AMR genes, of which 19 were previously reported in S. suis (aph(3')-IIIa, sat4, aadE, spw, aac(6')-Ie-aph(2'')-Ia, fexA, optrA, erm(B), mef(A/E), mrs(D), mph(C), lnu(B), lsa(E), vga(F), tet(M), tet(O), tet(O/W/32/O), tet(W)), and 4 were novel (aph(2'')-IIIa, apmA, erm(47), tet(T)). These AMR genes explained the AMR to spectinomycin, macrolides, lincosamides, tiamulin, and tetracyclines. Several genes were located on mobile genetic elements which showed a variable organization and composition. As AMR gene homologs were identified in many human and animal pathogens, the resistome of S. suis has a different phylogenetic origin. Moreover, AMR to penicillin G, fluoroquinolones, and trimethoprim related to mutations in genes coding for target enzymes (pbp1a, pbp2b, pbp2x, mraY, gyrA, parC, and dhfr). Bioinformatic analysis estimated traits of recombination on target genes, also indicative of gene transfer events. Conclusions: Our work evidences that S. suis is a major contributor to AMR dissemination across veterinary and human pathogens. Therefore, control of AMR in S. suis should be considered from a One Health approach in regions with high pig production to properly tackle the issue of antimicrobial drug resistance.

A molecular dynamics study on the diffusion and imprint ability of spectinomycin under different sizes of aniline oligomers.

Molecularly imprinted polymers (MIP) are the polymers created by molecular imprinting techniques that leave cavities for the specific interactions with a template molecule and have been applied in molecular selectivity tasks. In this study, the molecular dynamics (MD) simulation technique was used to demonstrate that aniline oligomer could be developed as a potential MIP for detection and separation of the spectinomycin drug molecule for gonorrhea treatment. MD simulations were performed for the systems of a spectinomycin within aniline oligomers of different sizes. The mean square displacement (MSD) and the diffusivity calculated from MD simulations showed that the diffusion coefficient was significantly dropped when the length of aniline oligomer was greater than two. The diffusion coefficient of spectinomycin became the lowest within aniline trimers, corresponded to the highest atomic distribution of MIP around the template. Then, the specific cavity in MIP systems with and without spectinomycin was calculated to assess the stability of the cavity created by the template. The volume of a cavity created within the trimer system was closest to the spectinomycin volume and therefore became the optimal oligomer size for further development of MIP.

"Upcycling" known molecules and targets for drug-resistant TB.

Despite reinvigorated efforts in Tuberculosis (TB) drug discovery over the past 20 years, relatively few new drugs and candidates have emerged with clear utility against drug resistant TB. Over the same period, significant technological advances and learnings around target value have taken place. This has offered opportunities to re-assess the potential for optimization of previously discovered chemical matter against Mycobacterium tuberculosis (M.tb) and for reconsideration of clinically validated targets encumbered by drug resistance. A re-assessment of discarded compounds and programs from the "golden age of antibiotics" has yielded new scaffolds and targets against TB and uncovered classes, for example beta-lactams, with previously unappreciated utility for TB. Leveraging validated classes and targets has also met with success: booster technologies and efforts to thwart efflux have improved the potential of ethionamide and spectinomycin classes. Multiple programs to rescue high value targets while avoiding cross-resistance are making progress. These attempts to make the most of known classes, drugs and targets complement efforts to discover new chemical matter against novel targets, enhancing the chances of success of discovering effective novel regimens against drug-resistant TB.

Prevention of COVID-19 superspreading events in buses boarded by presymptomatic cases.

INTRODUCTION: COVID-19 superspreader events have occurred when symptomatic individuals without wearing face masks boarded buses. OBJECTIVE: To report the risk of superspreader events when presymptomatic individuals boarded buses to-gether with unvaccinated passengers, but with non-pharmacological preventive interventions being maintained. METHODS: Prospec-tive study of health personnel transported in buses to a COVID-19 vaccination center for two weeks. Open windows, correct use of face masks and exclusion of symptomatic individuals were mandatory. Prospective surveillance identified workers with COVID-19 within 14 days after vaccination. Each asymptomatic passenger of buses where cases were identified was monitored for a similar time period. Voluntary screening results were available for workers who were tested in the month before or after vaccination. RESULTS: 1,879 workers boarded 65 buses. On-board time ranged from three to eight hours. Twenty-nine cases of COVID-19 and four asymptomatic cases were identified among 613 passengers of 21 buses. Median time between vaccina-tion and COVID-19 symptoms onset was six days. One case of suspected transmission on a bus was identi-fied. CONCLUSIONS: Strict nonpharmacological preventive interventions substantially reduced the risk of COVID-19 super-spreader events in buses boarded by presymptomatic individuals.

ANTECEDENTES: Ha ocurrido superpropagación de COVID-19 cuando individuos sintomáticos sin uso de cubrebocas abordaron autobuses. OBJETIVO: Reportar el riesgo de superpropagación cuando individuos presintomáticos abordaron autobuses junto con pasajeros no vacunados pero se mantuvieron intervenciones preventivas no farmacológicas. MATERIAL Y MÉTODOS: Estudio prospec­tivo de personal de salud transportado durante dos semanas en autobuses a un centro de vacunación contra COVID-19. Fue obligatorio llevar ventanas abiertas, uso correcto de cubrebocas y exclusión de personas con síntomas. La vigilancia prospectiva identificó a trabajadores con COVID-19 los 14 días siguientes a la vacunación. Cada pasajero asintomático de autobuses donde se detectaron casos fue vigilado durante un periodo de tiempo similar. Los resultados de tamizaje voluntario estuvieron disponibles para los trabajadores que se realizaron prueba el mes previo o el siguiente a la vacunación. RESULTADOS: 1879 trabajadores abordaron 65 autobuses. El tiempo a bordo varió de tres a ocho horas. Veintinueve casos de COVID-19 y 4 casos asintomáticos fueron identificados entre 613 pasajeros de 21 autobuses. La mediana de tiempo entre la vacunación y el inicio de síntomas en casos de COVID-19 fue de seis días. Fue identificado un caso de transmisión sospechada en autobús. CONCLUSIONES: Las intervenciones preventivas no farmacológicas estrictas redujeron sustancialmente el riesgo de superpropagación de COVID-19 en autobuses ocupados por individuos presintomáticos.

A One Health Genomic Investigation of Gentamicin Resistance in Escherichia coli from Human and Chicken Sources in Canada, 2014 to 2017.

We investigated whether gentamicin resistance (Genr) in Escherichia coli isolates from human infections was related to Genr E. coli in chicken and whether resistance may be due to coselection from use of lincomycin-spectinomycin in chickens on farms. Whole-genome sequencing was performed on 483 Genr E. coli isolates isolated between 2014 and 2017. These included 205 human-source isolates collected by the Canadian Ward (CANWARD) program and 278 chicken-source isolates: 167 from live/recently slaughtered chickens (animals) and 111 from retail chicken meat collected by the Canadian Integrated Program for Antimicrobial Resistance Surveillance (CIPARS). The predominant Genr gene was different in human and chicken sources; however, both sources carried aac(3)-IId, aac(3)-VIa, and aac(3)-IVa. Forty-one percent of human clinical isolates of Genr E. coli contained a blaCTX-M extended-spectrum beta-lactamase (ESBL) gene (84/205), and 53% of these were sequence type 131 (ST131). Phylogenomic analysis revealed a high diversity of Genr isolates; however, there were three small clusters of closely related isolates from human and chicken sources. Genr and spectinomycin resistance (Specr) genes were colocated in 148/167 (89%) chicken animal isolates, 94/111 (85%) chicken retail meat isolates, and 137/205 (67%) human-source isolates. Long-read sequencing of 23 isolates showed linkage of the Genr and Specr genes on the same plasmid in 14/15 (93%) isolates from chicken(s) and 6/8 (75%) isolates from humans. The use of lincomycin-spectinomycin on farms may be coselecting for gentamicin-resistant plasmids in E. coli in broiler chickens; however, Genr isolates and plasmids were mostly different in chickens and humans.

Associations of antimicrobial use with antimicrobial susceptibility at the calf level in bacteria isolated from the respiratory and digestive tracts of veal calves before slaughter.

OBJECTIVES: Antimicrobial drugs are frequently administered in veal calves, but investigations on associations with antimicrobial susceptibility of bacteria are scarce and convey partly contradictory findings. The aim of this study was to investigate associations of antimicrobial use (AMU) during the fattening period with antimicrobial susceptibility shortly before slaughter. METHODS: Detailed treatment data of 1905 veal calves from 38 farms were collected prospectively during monthly farm visits for 1 year (n = 1864 treatments, n = 535 visits); 1582 Escherichia coli, 1059 Pasteurella multocida and 315 Mannheimia haemolytica were isolated from rectal and nasopharyngeal swabs collected before slaughter and subjected to antimicrobial susceptibility testing by microdilution. Associations of antimicrobial treatments with resistant isolates were investigated at the calf level. RESULTS: Associations of AMU with antimicrobial resistance were observed using generalized linear models. For E. coli, the odds of being resistant were increased with increased AMU (OR 1.36 when number of treatments >1, P = 0.066). Use of tetracyclines was associated with resistance to tetracycline (OR 1.86, P < 0.001) and use of penicillins was associated with resistance to ampicillin (OR 1.66, P = 0.014). No significant associations were observed for P. multocida (use of aminoglycosides: OR 3.66 for resistance to spectinomycin, P = 0.074). For M. haemolytica, the odds of being resistant were increased with increased AMU (OR 4.63, P < 0.001), and use of tetracyclines was associated with resistance to tetracycline (OR 6.49, P < 0.001). CONCLUSIONS: Occurrence of resistant bacteria shortly before slaughter was associated with AMU in veal calves. Prudent and appropriate use may contribute to limit the selection of resistant bacteria on veal farms.

Antibiotic resistance genes and bacterial diversity: A comparative molecular study of treated sewage from different origins and their impact on irrigated soils.

Present study aims to investigate how is soil affected following irrigation with treated effluents of different origins by analysing the bacterial diversity, metabolic diversity and antibiotic resistance genes (ARGs). Comparative analysis with previously reported ARGs in effluents was performed to understand the mobility of ARGs from treated wastewater to the irrigated soil with respect to the control soil regimen. Acinetobacter, Burkholderia and Pseudomonas were observed as the most abundant genera in all the samples. The metabolic gene abundance of all the samples suggests a prominent contribution to natural mineral recycling. Most abundant ARGs observed encode resistance for clindamycin, kanamycin A, macrolides, paromomycin, spectinomycin and tetracycline. Treated effluent reuse did not appear to enhance the ARG levels in soils in most cases except for institutional treatment site (M), where the ARGs for aminoglycosides, ß-lactams and sulfonamides were found to be abundantly present in both treated effluent and the irrigated soil. This study finds the importance of wastewater treatment from different origins and the impact of treated wastewater reuse in irrigation. This study also emphasises on the better understanding of ARGs mobility from water to soil.