Augmented surveillance of antimicrobial resistance with high-throughput robotics detects transnational flow of fluoroquinolone-resistant Escherichia coli strain into poultry.

BACKGROUND: Food animal AMR surveillance programs assess only small numbers of Escherichia coli (from 100 to 600 per animal class) nationally each year, severely limiting the evaluation of public health risk(s). Here we demonstrate an affordable approach for early detection of emerging resistance on a broad scale that can also accurately characterize spatial and temporal changes in resistance. METHODS: Caecal samples (n = 295) obtained from 10 meat poultry were screened using high-throughput robotics. Initial screening via agar dilution (5310 plates) quantified AMR carriage (cfu/g) for each sample. Ciprofloxacin-resistant isolates (n = 91) proceeded to downstream broth microdilution susceptibility testing. A subset of 28 ciprofloxacin-resistant isolates underwent WGS and phylogenetic analysis. RESULTS: Intra- and inter-flock carriage of resistance varied with drug class. Ampicillin and tetracycline resistance was ubiquitous to most birds in all flocks with an average carriage rate of 5.8 log10 cfu/g. Gentamicin and ciprofloxacin-resistant E. coli colonized fewer birds, and had an average carriage rate of 1.2 log10 cfu/g and 1.0 log10 cfu/g of faeces, respectively. Resistance to extended-spectrum cephalosporins was absent. ST354 was the dominant ST among the WGS isolates, but they demonstrated markedly lower resistance gene carriage than their international counterparts. CONCLUSIONS: These data amply demonstrate the ineffectiveness of commonly relied-on approaches to AMR surveillance for achieving early detection of emergence, or for measuring spatial and temporal resistance trends. Genetic analysis suggested there has been transnational flow of a ciprofloxacin-resistant strain into Australian poultry flocks, explaining their detection in a nation that prohibits fluoroquinolone use in poultry.

Thieno[3,2-b]pyrrole 5-carboxamides as potent and selective inhibitors of Giardia duodenalis.

Giardia duodenalis is the causative agent of the neglected diarrhoeal disease giardiasis. While often self-limiting, giardiasis is ubiquitous and impacts hundreds of millions of people annually. It is also a common gastro-intestinal disease of domestic pets, wildlife, and livestock animals. However, despite this impact, there is no vaccine for Giardia currently available. In addition, treatment relies on chemotherapies that are associated with increasing failure rates. To identify new treatment options for giardiasis we recently screened the Compounds Australia Scaffold Library for new chemotypes with selective anti-Giardia activity, identifying three compounds with sub-µM activity and promising selectivity. Here we extended these studies by examining the anti-Giardia activity of series CL9569 compounds. This compound series was of interest given the promising activity (IC50 1.2 µM) and selectivity demonstrated by representative compound, SN00798525 (1). Data from this work has identified an additional three thieno [3,2-b]pyrrole 5-carboxamides with anti-Giardia activity, including 2 which displayed potent cytocidal (IC50 ≤ 10 nM) and selective activity against multiple Giardia strains, including representatives from both human-infecting assemblages and metronidazole resistant parasites. Preclinical studies in mice also demonstrated that 2 is well-tolerated, does not impact the normal gut microbiota and can reduce Giardia parasite burden in these animals.

Cardiac tissue engineering for the treatment of hypoplastic left heart syndrome (HLHS).

Hypoplastic left heart syndrome (HLHS) is a deadly congenital heart disease that arises when the left ventricle and outflow tract fail to develop appropriately, inhibiting the adequate perfusion of the rest of the body. Historically, this disease has been treated via a series of surgeries that allows the heart to use a single ventricle. These surgeries are often a palliative measure, and heart transplantation is the only definitive therapy that exists for this condition. It has been hypothesized that stem cell-based regenerative therapies could have a role in promoting cardiac tissue regeneration in HLHS patients who are undergoing palliative surgery. Several clinical trials have demonstrated that introducing pluripotent cells into the heart is safe, feasible, and capable of improving right ventricular ejection fraction (RVEF). However, while these approaches show great promise, there is still room for development. There is a substantial body of pre-clinical work that is focused on generating increasingly large and complex pieces of cardiac tissue in the form of cardiac patches, with the idea that these could be used to rebuild and strengthen the heart in a robust and long-lasting manner. In total, stem cell-based therapies have much to offer when it comes to improving the treatment of HLHS.

A national study confirms that Escherichia coli from Australian commercial layer hens remain susceptible to critically important antimicrobials.

Controlling the use of the most critically important antimicrobials (CIAs) in food animals has been identified as one of the key measures required to curb the transmission of antimicrobial resistant bacteria from animals to humans. Expanding the evidence demonstrating the effectiveness of restricting CIA usage for preventing the emergence of resistance to key drugs amongst commensal organisms in animal production would do much to strengthen international efforts to control antimicrobial resistance (AMR). As Australia has strict controls on antimicrobial use in layer hens, and internationally comparatively low levels of poultry disease due to strict national biosecurity measures, we investigated whether these circumstances have resulted in curtailing development of critical forms of AMR. The work comprised a cross-sectional national survey of 62 commercial layer farms with each assessed for AMR in Escherichia coli isolates recovered from faeces. Minimum inhibitory concentration analysis using a panel of 13 antimicrobials was performed on 296 isolates, with those exhibiting phenotypic resistance to fluoroquinolones (a CIA) or multi-class drug resistance (MCR) subjected to whole genome sequencing. Overall, 53.0% of isolates were susceptible to all antimicrobials tested, and all isolates were susceptible to cefoxitin, ceftiofur, ceftriaxone, chloramphenicol and colistin. Resistance was observed for amoxicillin-clavulanate (9.1%), ampicillin (16.2%), ciprofloxacin (2.7%), florfenicol (2.4%), gentamicin (1.0%), streptomycin (4.7%), tetracycline (37.8%) and trimethoprim/sulfamethoxazole (9.5%). MCR was observed in 21 isolates (7.0%), with two isolates exhibiting resistance to four antimicrobial classes. Whole genome sequencing revealed that ciprofloxacin-resistant (fluoroquinolone) isolates were devoid of both known chromosomal mutations in the quinolone resistance determinant regions and plasmid-mediated quinolone resistance genes (qnr)-other than in one isolate (ST155) which carried the qnrS gene. Two MCR E. coli isolates with ciprofloxacin-resistance were found to be carrying known resistance genes including aadA1, dfrA1, strA, strB, sul1, sul2, tet(A), blaTEM-1B, qnrS1 and tet(A). Overall, this study found that E. coli from layer hens in Australia have low rates of AMR, likely due to strict control on antimicrobial usage achieved by the sum of regulation and voluntary measures.

Proximity to human settlement is directly related to carriage of critically important antimicrobial-resistant Escherichia coli and Klebsiella pneumoniae in Silver Gulls.

Human population and activities play an important role in dissemination of antimicrobial resistant bacteria. This study investigated the relationship between carriage rates of critically important antimicrobial-resistant (CIA-R) Escherichia coli and Klebsiella pneumoniae by Silver Gulls and their proximity to human populations. Faecal swabs (n = 229) were collected from Silver Gulls across 10 southern coastline locations in Western Australia (WA) traversing 650 kms. The sampling locations included main town centres and remote areas. Fluoroquinolone and extended-spectrum cephalosporin-resistant E. coli and K. pneumoniae were isolated and tested for antimicrobial sensitivity. Genome sequencing was performed on n = 40 subset out of 98 E. coli and n = 14 subset out of 27 K. pneumoniae isolates to validate phenotypic resistance profiles and determine the molecular characteristics of strains. CIA-R E. coli and K. pneumoniae were detected in 69 (30.1 %) and 20 (8.73 %) of the faecal swabs respectively. Two large urban locations tested positive for CIA-R E. coli (frequency ranging from 34.3 % to 84.3 %), and/or for CIA-R K. pneumoniae (frequency ranging from 12.5 % to 50.0 %). A small number of CIA-R E. coli (3/31, 9.7 %) were identified at a small tourist town, but no CIA-R bacteria were recovered from gulls at remote sites. Commonly detected E. coli sequence types (STs) included ST131 (12.5 %) and ST1193 (10.0 %). Five K. pneumoniae STs were detected which included ST4568, ST6, ST485, ST967 and ST307. Resistance genes including blaCTX-M-3, blaCTX-M-15 and blaCTX-M-27 were identified in both bacterial species. High-level colonisation of CIA-R E. coli and K. pneumoniae in Silver Gulls in and around urban areas compared to remote locations substantiates that anthropogenic activities are strongly associated with acquisition of resistant bacteria by gulls.

Antimicrobial Resistance of and Genomic Insights into Pasteurella multocida Strains Isolated from Australian Pigs.

Infection with Pasteurella multocida represents a significant economic threat to Australian pig producers, yet our knowledge of its antimicrobial susceptibilities is lagging, and genomic characterization of P. multocida strains associated with porcine lower respiratory disease is internationally scarce. This study utilized high-throughput robotics to phenotypically and genetically characterize an industry-wide collection of 252 clinical P. multocida isolates that were recovered between 2014 and 2019. Overall, antimicrobial resistance was found to be low, with clinical resistance below 1% for all tested antimicrobials except those from the tetracycline class. Five dominant sequence types, representing 64.8% of all isolates, were identified; they were disseminated across farms and had previously been detected in various animal hosts and countries. P. multocida in Australian farms remain controllable via current antimicrobial therapeutic protocols. The identification of highly dominant, interspecies-infecting strains provides insight into the epidemiology of the opportunistic pathogen, and it highlights a biosecurity threat to the Australian livestock industry. IMPORTANCE Pasteurellosis is rated by the World Animal Health Organisation (OIE) as a high-impact disease in livestock. Although it is well understood in many host-disease contexts, our understanding of the organism in porcine respiratory disease is limited. Given its high frequency of involvement in porcine respiratory disease complex (PRDC), it is important that we are aware of its antimicrobial susceptibilities so that we can respond quickly and appropriately with antimicrobial therapy. Genetic insights about the organism can help us to better understand its epidemiology and inform our biosecurity practices and prophylactic management.

Antigiardial Activity of Novel Guanidine Compounds.

From four focused compound libraries based on the known anticoccidial agent robenidine, 44 compounds total were synthesised and screened for antigiardial activity. All active compounds were counter-screened for antibiotic and cytotoxic action. Of the analogues examined, 21 displayed IC50 <5 µM, seven with IC50 <1.0 µM. Most active were 2,2'-bis{[4-(trifluoromethoxy)phenyl]methylene}carbonimidic dihydrazide hydrochloride (30), 2,2'-bis{[4-(trifluoromethylsulfanyl)phenyl]methylene}carbonimidic dihydrazide hydrochloride (32), and 2,2'-bis[(2-bromo-4,5-dimethoxyphenyl)methylene]carbonimidic dihydrazide hydrochloride (41) with IC50 =0.2 µM. The maximal observed activity was a 5 h IC50 value of 0.2 µM for 41. The clinically used metronidazole was inactive at this timepoint at a concentration of 25 µM. Robenidine off-target effects at bacteria and cell line toxicity were removed. Analogue 41 was well tolerated in mice treated orally (100 mg/kg). Following 5 h treatment with 41, no Giardia regrowth was noted after 48 h.

GS-2: A Novel Broad-Spectrum Agent for Environmental Microbial Control.

The environmental control of microbial pathogens currently relies on compounds that do not exert long-lasting activity on surfaces, are impaired by soil, and contribute to the growing problem of antimicrobial resistance. This study presents the scientific development and characterization of GS-2, a novel, water-soluble ammonium carboxylate salt of capric acid and L-arginine that demonstrates activity against a range of bacteria (particularly Gram-negative bacteria), fungi, and viruses. In real-world surface testing, GS-2 was more effective than a benzalkonium chloride disinfectant at reducing the bacterial load on common touch-point surfaces in a high-traffic building (average 1.6 vs. 32.6 CFUs recovered from surfaces 90 min after application, respectively). Toxicology testing in rats confirmed GS-2 ingredients were rapidly cleared and posed no toxicities to humans or animals. To enhance the time-kill against Gram-positive bacteria, GS-2 was compounded at a specific ratio with a naturally occurring monoterpenoid, thymol, to produce a water-based antimicrobial solution. This GS-2 with thymol formulation could generate a bactericidal effect after five minutes of exposure and a viricidal effect after 10 min of exposure. Further testing of the GS-2 and thymol combination on glass slides demonstrated that the compound retained bactericidal activity for up to 60 days. Based on these results, GS-2 and GS-2 with thymol represent a novel antimicrobial solution that may have significant utility in the long-term reduction of environmental microbial pathogens in a variety of settings.

Specific in situ inflammatory states associate with progression to renal failure in lupus nephritis.

BACKGROUNDIn human lupus nephritis (LN), tubulointerstitial inflammation (TII) on biopsy predicts progression to end-stage renal disease (ESRD). However, only about half of patients with moderate-to-severe TII develop ESRD. We hypothesized that this heterogeneity in outcome reflects different underlying inflammatory states. Therefore, we interrogated renal biopsies from LN longitudinal and cross-sectional cohorts.METHODSData were acquired using conventional and highly multiplexed confocal microscopy. To accurately segment cells across whole biopsies, and to understand their spatial relationships, we developed computational pipelines by training and implementing several deep-learning models and other computer vision techniques.RESULTSHigh B cell densities were associated with protection from ESRD. In contrast, high densities of CD8+, γδ, and other CD4-CD8- T cells were associated with both acute renal failure and progression to ESRD. B cells were often organized into large periglomerular neighborhoods with Tfh cells, while CD4- T cells formed small neighborhoods in the tubulointerstitium, with frequency that predicted progression to ESRD.CONCLUSIONThese data reveal that specific in situ inflammatory states are associated with refractory and progressive renal disease.FUNDINGThis study was funded by the NIH Autoimmunity Centers of Excellence (AI082724), Department of Defense (LRI180083), Alliance for Lupus Research, and NIH awards (S10-OD025081, S10-RR021039, and P30-CA14599).

Antimicrobial resistance and genomic relationships of Salmonella enterica from Australian cattle.

The aim of this study was to evaluate phenotypic and genotypic AMR characteristics of Salmonella enterica isolates from Australian cattle collected through a structured national survey utilizing 1001 faecal samples collected from healthy cattle at slaughter. A total of 184 Salmonella isolates were subsequently derived and subjected to microbroth dilution to 16 drugs from 11 classes with interpretation of minimum inhibitory concentrations (MICs) using epidemiological cut off (ECOFF) values to distinguish between wild-type and non-wild-type populations. Most isolates were susceptible (wild type) to all antimicrobials tested, with no resistance (non-wild type) detected for colistin, nalidixic acid, meropenem, gentamicin, florfenicol or chloramphenicol. Low rates of resistance were detected for ampicillin (2.2%), cefoxitin (2.2%), ceftiofur (2.2%), ceftriaxone (2.2%), ciprofloxacin (0.5%), streptomycin (3.3%) and tetracycline (0.5%). Isolates resistant to ceftriaxone (a critically important antimicrobial, CIA) carried the extended spectrum cephalosporin gene blaCMY-2 while no known mutation in the QRDR region or qnrS genes were detected for the CIA ciprofloxacin-resistant isolate. Thirty-six serovars were detected among the 184 Salmonella isolates using whole genome sequencing, dominated by Typhimurium (n = 36), Saintpaul (n = 22) and Anatum (n = 16). Genomic analysis clustered the cattle isolates based on serovar, with the majority of serovars containing a single sequence type (ST). Further analysis of the bovine Typhimurium isolates (ST19) and comparison with publicly available data from human Typhimurium isolates from Australia, revealed the majority of cattle isolates were unrelated to human isolates. In conclusion, this study demonstrates the continued low prevalence of AMR among Salmonella within the beef, dairy and veal industries in Australia. Salmonella Typhimurium from cattle were genetically distinct from isolates sourced from human infections. Further investigations are warranted to expand on the potential clinical and public health relevance of these findings to inform risk-management of this key pathogen.

In Vitro Demonstration of Targeted Phage Therapy and Competitive Exclusion as a Novel Strategy for Decolonization of Extended-Spectrum-Cephalosporin-Resistant Escherichia coli.

Extended-spectrum cephalosporin-resistant (ESC-R) Escherichia coli have disseminated in food-producing animals globally, attributed to horizontal transmission of blaCTX-M variants, as seen in the InCI1-blaCTX-M-1 plasmid. This ease of transmission, coupled with its demonstrated long-term persistence, presents a significant One Health antimicrobial resistance (AMR) risk. Bacteriophage (phage) therapy is a potential strategy in eliminating ESC-R E. coli in food-producing animals; however, it is hindered by the development of phage-resistant bacteria and phage biosafety concerns. Another alternative to antimicrobials is probiotics, with this study demonstrating that AMR-free commensal E. coli, termed competitive exclusion clones (CECs), can be used to competitively exclude ESC-R E. coli. This study isolated and characterized phages that lysed E. coli clones harboring the InCI1-blaCTX-M-1 plasmid, before investigation of the effect and synergy of phage therapy and competitive exclusion as a novel strategy for decolonizing ESC-resistant E. coli. In vitro testing demonstrated superiority in the combined therapy, reducing and possibly eliminating ESC-R E. coli through phage-mediated lysis coupled with simultaneous prevention of regrowth of phage-resistant mutants due to competitive exclusion with the CEC. Further investigation into this combined therapy in vivo is warranted, with on-farm application possibly reducing ESC-R prevalence, while constricting newly emergent ESC-R E. coli outbreaks prior to their dissemination throughout food-producing animals or humans. IMPORTANCE The emergence and global dissemination of resistance toward critically important antimicrobials, including extended-spectrum cephalosporins in the livestock sector, deepens the One Health threat of antimicrobial resistance. This resistance has the potential to disseminate to humans, directly or indirectly, nullifying these last lines of defense in life-threatening human infections. This study explores a novel strategy, the coadministration of bacteriophages (phages) and a competitive exclusion clone (antimicrobial-susceptible commensal E. coli), to revert an antimicrobial-resistant population to a susceptible population. While phage therapy is vulnerable to the emergence of phage-resistant bacteria, no phage-resistant bacteria emerged when a competitive exclusion clone was used in combination with the phage. Novel strategies that reduce the prevalence and slow the dissemination of extended-spectrum cephalosporin-resistant E. coli in food-producing animals have the potential to extend the time frame in which antimicrobials remain available for effective use in animal and human health.

Phenotypic and Genotypic Assessment of Antimicrobial Resistance in Escherichia coli from Australian Cattle Populations at Slaughter.

ABSTRACT: Australia relies on periodic antimicrobial resistance (AMR) surveys to determine trends and changes in AMR in animal production systems. This study is a follow-up to a survey of Escherichia coli from healthy cattle at slaughter conducted in 2013, which provided baseline data on AMR prevalence across cattle groups and production practices. In this study, 591 beef cattle, 194 dairy cattle, and 216 veal calf fecal samples were collected from 25 beef and veal processing establishments in Australia, representing approximately 77% of total export volume. A total of 969 matrix-assisted laser desorption-ionization results confirmed commensal E. coli isolates from 574 beef cattle, 186 dairy cattle, and 209 veal calves were recovered, and antimicrobial susceptibility testing was carried out by microbroth dilution to 16 drugs from 10 classes interpreted against epidemiological cutoff breakpoints. Overall, a high proportion of E. coli isolates (83.8%) were wild type for all antimicrobials assessed. In addition, isolates that were non-wild type (NWT) for three or more classes of antimicrobial did not exceed 4% for any of the cattle groups. The prevalence of E. coli that were NWT for antimicrobials that are critical or of high importance to human health was very low, with 1.4% of all isolates tested determined to be NWT for fluoroquinolones, third-generation cephalosporins, or polymyxins. Genomic analysis of NWT isolates identified one beef cattle isolate (ST-10) harboring blaCMY-2 and a dairy isolate (ST-58) and two veal calf isolates (ST-58 and ST-394) that had qnrS1, which confer resistance to extended-spectrum cephalosporins and fluoroquinolones, respectively. The low levels of AMR reported in this study confirm previous Australian studies, which indicated that there is minimal evidence that specific production practices lead to widespread disproportionate development of NWT isolates.

Diversity detected in commensals at host and farm level reveals implications for national antimicrobial resistance surveillance programmes.

BACKGROUND: A key component to control of antimicrobial resistance (AMR) is the surveillance of food animals. Currently, national programmes test only limited isolates per animal species per year, an approach tacitly assuming that heterogeneity of AMR across animal populations is negligible. If the latter assumption is incorrect then the risk to humans from AMR in the food chain is underestimated. OBJECTIVES: To demonstrate the extent of phenotypic and genetic heterogeneity of Escherichia coli in swine to assess the need for improved protocols for AMR surveillance in food animals. METHODS: Eight E. coli isolates were obtained from each of 10 pigs on each of 10 farms. For these 800 isolates, AMR profiles (MIC estimates for six drugs) and PCR-based fingerprinting analysis were performed and used to select a subset (n = 151) for WGS. RESULTS: Heterogeneity in the phenotypic AMR traits of E. coli was observed in 89% of pigs, with 58% of pigs harbouring three or more distinct phenotypes. Similarly, 94% of pigs harboured two or more distinct PCR-fingerprinting profiles. Farm-level heterogeneity was detected, with ciprofloxacin resistance detected in only 60% of pigs from a single farm. Furthermore, 58 STs were identified, with the dominant STs being ST10, ST101, ST542 and ST641. CONCLUSIONS: Phenotypic and genotypic heterogeneity of AMR traits in bacteria from animal populations are real phenomena posing a barrier to correct interpretation of data from AMR surveillance. Evolution towards a more in-depth sampling model is needed to account for heterogeneity and increase the reliability of inferences.

Validation of Selective Agars for Detection and Quantification of Escherichia coli Strains Resistant to Critically Important Antimicrobials.

Success in the global fight against antimicrobial resistance (AMR) is likely to improve if surveillance can be performed on an epidemiological scale. An approach based on agars with incorporated antimicrobials has enormous potential to achieve this. However, there is a need to identify the combinations of selective agars and key antimicrobials yielding the most accurate counts of susceptible and resistant organisms. A series of experiments involving 1,202 plates identified the best candidate combinations from six commercially available agars and five antimicrobials, using 18 Escherichia coli strains as either pure cultures or inocula-spiked feces. The effects of various design factors on colony counts were analyzed in generalized linear models. Without antimicrobials, Brilliance E. coli and CHROMagar ECC agars yielded 28.9% and 23.5% more colonies, respectively, than MacConkey agar. The order of superiority of agars remained unchanged when fecal samples with or without spiking of resistant E. coli strains were inoculated onto agars with or without specific antimicrobials. When antimicrobials were incorporated at various concentrations, it was revealed that ampicillin, tetracycline, and ciprofloxacin were suitable for incorporation into Brilliance and CHROMagar agars at all defined concentrations. Gentamicin was suitable for incorporation only at 8 and 16 µg/ml, while ceftiofur was suitable only at 1 µg/ml. CHROMagar extended-spectrum ß-lactamase (ESBL) agar supported growth of a wider diversity of extended-spectrum-cephalosporin-resistant E. coli strains. The findings demonstrate the potential for agars with incorporated antimicrobials to be combined with laboratory-based robotics to deliver AMR surveillance on a vast scale with greater sensitivity of detection and strategic relevance. IMPORTANCE Established models of surveillance for AMR in livestock typically have a low sampling intensity, which creates a tremendous barrier to understanding the variation of resistance among animal and food enterprises. However, developments in laboratory robotics now make it possible to rapidly and affordably process large volumes of samples. Combined with modern selective agars incorporating antimicrobials, this forms the basis of a novel surveillance process for identifying resistant bacteria by chromogenic reactions, including accurately detecting and quantifying the presence of bacteria even when they are present at low concentrations. Because Escherichia coli is a widely preferred indicator bacterium for AMR surveillance, this study identifies the optimal selective agar for quantifying resistant E. coli strains by assessing the growth performance on agars with antimicrobials. The findings are the first step toward exploiting laboratory robotics in an up-scaled approach to AMR surveillance in livestock, with wider adaptations in food, clinical microbiology, and public health.

Artificial Intelligence and Cellular Segmentation in Tissue Microscopy Images.

With applications in object detection, image feature extraction, image classification, and image segmentation, artificial intelligence is facilitating high-throughput analysis of image data in a variety of biomedical imaging disciplines, ranging from radiology and pathology to cancer biology and immunology. Specifically, a growth in research on deep learning has led to the widespread application of computer-visualization techniques for analyzing and mining data from biomedical images. The availability of open-source software packages and the development of novel, trainable deep neural network architectures has led to increased accuracy in cell detection and segmentation algorithms. By automating cell segmentation, it is now possible to mine quantifiable cellular and spatio-cellular features from microscopy images, providing insight into the organization of cells in various pathologies. This mini-review provides an overview of the current state of the art in deep learning- and artificial intelligence-based methods of segmentation and data mining of cells in microscopy images of tissue.

Robotic Antimicrobial Susceptibility Platform (RASP): a next-generation approach to One Health surveillance of antimicrobial resistance.

BACKGROUND: Surveillance of antimicrobial resistance (AMR) is critical to reducing its wide-reaching impact. Its reliance on sample size invites solutions to longstanding constraints regarding scalability. A robotic platform (RASP) was developed for high-throughput AMR surveillance in accordance with internationally recognized standards (CLSI and ISO 20776-1:2019) and validated through a series of experiments. METHODS: Experiment A compared RASP's ability to achieve consistent MICs with that of a human technician across eight replicates for four Escherichia coli isolates. Experiment B assessed RASP's agreement with human-performed MICs across 91 E. coli isolates with a diverse range of AMR profiles. Additionally, to demonstrate its real-world applicability, the RASP workflow was then applied to five faecal samples where a minimum of 47 E. coli per animal (239 total) were evaluated using an AMR indexing framework. RESULTS: For each drug-rater-isolate combination in Experiment A, there was a clear consensus of the MIC and deviation from the consensus remained within one doubling dilution (the exception being gentamicin at two dilutions). Experiment B revealed a concordance correlation coefficient of 0.9670 (95% CI: 0.9670-0.9670) between the robot- and human-performed MICs. RASP's application to the five faecal samples highlighted the intra-animal diversity of gut commensal E. coli, identifying between five and nine unique isolate AMR phenotypes per sample. CONCLUSIONS: While adhering to internationally accepted guidelines, RASP was superior in throughput, cost and data resolution when compared with an experienced human technician. Integration of robotics platforms in the microbiology laboratory is a necessary advancement for future One Health AMR endeavours.

Antimicrobial Resistance in Porcine Enterococci in Australia and the Ramifications for Human Health.

Enterococci are ubiquitous opportunistic pathogens that have become a major public health issue globally. The increasing prevalence of antimicrobial resistance in hospital-adapted enterococci had been thought to originate from livestock. However, this association between livestock and hospital-adapted enterococci is currently unclear. This study investigates the antimicrobial susceptibilities of enterococci isolated from pig cecal samples and compares the genomic characteristics of Enterococcus faecium from pigs to those of isolates from meat chickens and from human sepsis cases. From 200 cecal samples, antimicrobial susceptibility testing was performed for E. faecium (n = 84), E. hirae (n = 36), and E. faecalis (n = 17). Whole-genome sequencing was performed for all E. faecium isolates, and the sequences were compared to those of previously studied isolates from meat chickens and human sepsis cases through bioinformatics analysis. Resistance (non-wild type) to erythromycin, gentamicin, tetracycline, ampicillin, daptomycin, virginiamycin, and quinupristin-dalfopristin was identified. More importantly, except for a single isolate harboring the vanC operon, no resistance was observed in the three species to vancomycin, teicoplanin, and linezolid, which are critically important antimicrobials used to treat enterococcal infections in humans. The E. faecium isolates from chickens were genetically distinct from human and pig isolates, which were more closely related. Human strains that were closely related to pig strains were not typical "hospital-adapted strains" as previously identified. The results of this study show that enterococci from Australian finisher pigs are not a source of resistance to critically important antimicrobials and that E. faecium from pigs is not part of the current human hospital-adapted population.IMPORTANCE Resistance to the critically important antimicrobials vancomycin, teicoplanin, and linezolid is not found in enterococci collected from Australian finisher pigs. However, some antimicrobial resistance was observed. In particular, resistance to quinupristin-dalfopristin, a combination of two streptogramin class antimicrobials, was identified despite the absence of streptogramin use Australia-wide since 2005. Other observed resistance among enterococci from pigs include chloramphenicol, erythromycin, and tetracycline resistance. Genomic comparison of E. faecium from Australian pigs to isolates collected from previous studies on chickens and humans indicate that E. faecium from pigs are genetically more similar to those of humans than those from chickens. Despite the increased genetic similarities, E. faecium strains from pigs are phylogenetically distinct and did not belong to the dominant sequence types found in hospital-adapted strains causing sepsis in humans. Therefore, the results indicate that Australian finisher pigs are not a source of hospital-adapted E. faecium in Australia.

Quantifying the effects of biopsy fixation and staining panel design on automatic instance segmentation of immune cells in human lupus nephritis.

SIGNIFICANCE: Lupus nephritis (LuN) is a chronic inflammatory kidney disease. The cellular mechanisms by which LuN progresses to kidney failure are poorly characterized. Automated instance segmentation of immune cells in immunofluorescence images of LuN can probe these cellular interactions. AIM: Our specific goal is to quantify how sample fixation and staining panel design impact automated instance segmentation and characterization of immune cells. APPROACH: Convolutional neural networks (CNNs) were trained to segment immune cells in fluorescence confocal images of LuN biopsies. Three datasets were used to probe the effects of fixation methods on cell features and the effects of one-marker versus two-marker per cell staining panels on CNN performance. RESULTS: Networks trained for multi-class instance segmentation on fresh-frozen and formalin-fixed, paraffin-embedded (FFPE) samples stained with a two-marker panel had sensitivities of 0.87 and 0.91 and specificities of 0.82 and 0.88, respectively. Training on samples with a one-marker panel reduced sensitivity (0.72). Cell size and intercellular distances were significantly smaller in FFPE samples compared to fresh frozen (Kolmogorov-Smirnov, p ≪ 0.0001). CONCLUSIONS: Fixation method significantly reduces cell size and intercellular distances in LuN biopsies. The use of two markers to identify cell subsets showed improved CNN sensitivity relative to using a single marker.

Machine Learning to Quantify In Situ Humoral Selection in Human Lupus Tubulointerstitial Inflammation.

In human lupus nephritis, tubulointerstitial inflammation (TII) is associated with in situ expansion of B cells expressing anti-vimentin antibodies (AVAs). The mechanism by which AVAs are selected is unclear. Herein, we demonstrate that AVA somatic hypermutation (SHM) and selection increase affinity for vimentin. Indeed, germline reversion of several antibodies demonstrated that higher affinity AVAs can be selected from both low affinity B cell germline clones and even those that are strongly reactive with other autoantigens. While we demonstrated affinity maturation, enzyme-linked immunosorbent assays (ELISAs) suggested that affinity maturation might be a consequence of increasing polyreactivity or even non-specific binding. Therefore, it was unclear if there was also selection for increased specificity. Subsequent multi-color confocal microscopy studies indicated that while TII AVAs often appeared polyreactive by ELISA, they bound selectively to vimentin fibrils in whole cells or inflamed renal tissue. Using a novel machine learning pipeline (CytoSkaler) to quantify the cellular distribution of antibody staining, we demonstrated that TII AVAs were selected for both enhanced binding and specificity in situ. Furthermore, reversion of single predicted amino acids in antibody variable regions indicated that we could use CytoSkaler to capture both negative and positive selection events. More broadly, our data suggest a new approach to assess and define antibody polyreactivity based on quantifying the distribution of binding to native and contextually relevant antigens.

Antimicrobial resistance and genomic insights into bovine mastitis-associated Staphylococcus aureus in Australia.

The aim of this study was to investigate antimicrobial resistance and population structure of bovine mastitis-associated Staphylococcus aureus isolates, and compare them to human isolates obtained from Western Australian hospitals and overseas strains to determine relatedness to human isolates from a zoonotic or reverse zoonotic aspect. Antimicrobial susceptibility testing was performed on 202 S. aureus isolates of which 166 isolates underwent whole genome sequencing. Only resistance to penicillin (12.4%) and erythromycin (0.5%) was identified and of note, no resistance was demonstrated to oxacillin. Genomic characterisation identified 14 multilocus sequence types (STs), with most isolates belonging to clonal complexes 97, 705, and 1. Four distinct clades based on virulence gene composition were identified. The four clades were predominantly ST based, consisting of ST352, ST97, ST81/ST1, and ST705. Core genome comparison of the bovine and human S. aureus isolates demonstrated defined clustering by ST, with the Australian bovine S. aureus isolates clustering together according to their ST separately from human isolates. In addition, a bovine specific cluster comprising Australian ST151 and ST705 isolates, and ST151 isolates from Irish dairy cattle was clearly delineated. Examination of a detailed ST352 phylogeny provided evidence for geographical clustering of Australian strains into a distinct grouping separate from international strains. This study has identified Australian S. aureus isolates have limited genetic diversity and are genetically distinct from human and international bovine S. aureus isolates. Current first line therapies for bovine mastitis in Australian dairy cattle remain appropriate.