Explainable deep learning approach for multilabel classification of antimicrobial resistance with missing labels

Predicting Antimicrobial Resistance (AMR) from genomic sequence data has become a significant component of overcoming the AMR challenge, especially given its potential for facilitating more rapid diagnostics and personalised antibiotic treatments. With the recent advances in sequencing technologies and computing power, deep learning models for genomic sequence data have been widely adopted to predict AMR more reliably and error-free. There are many different types of AMR; therefore, any practical AMR prediction system must be able to identify multiple AMRs present in a genomic sequence. Unfortunately, most genomic sequence datasets do not have all the labels marked, thereby making a deep learning modelling approach challenging owing to its reliance on labels for reliability and accuracy. This paper addresses this issue by presenting an effective deep learning solution, Mask-Loss 1D convolution neural network (ML-ConvNet), for AMR prediction on datasets with many missing labels. The core component of ML- ConvNet utilises a masked loss function that overcomes the effect of missing labels in predicting AMR. The proposed ML-ConvNet is demonstrated to outperform state-of-the-art methods in the literature by 10.5%, according to the F1 score. The proposed model’s performance is evaluated using different degrees of the missing label and is found to outperform the conventional approach by 76% in the F1 score when 86.68% of labels are missing. Furthermore, the ML-ConvNet was established with an explainable artificial intelligence (XAI) pipeline, thereby making it ideally suited for hospital and healthcare settings, where model interpretability is an essential requirement

Metabonomics-based analysis of Brachyspira pilosicoli's response to tiamulin reveals metabolic activity despite significant growth inhibition

Pathogenic anaerobes Brachyspira spp. are responsible for an increasing number of Intestinal Spirochaetosis (IS) cases in livestock against which few approved treatments are available. Tiamulin is used to treat swine dysentery caused by Brachyspira spp. and recently has been used to handle avian intestinal spirochaetosis (AIS). The therapeutic dose used in chickens requires further evaluation since cases of bacterial resistance to tiamulin have been reported. In this study, we evaluated the impact of tiamulin at varying concentrations on the metabolism of B. pilosicoli using a 1H-NMR-based metabonomics approach allowing the capture of the overall bacterial metabolic response to antibiotic treatment. Based on growth curve studies, tiamulin impacted bacterial growth even at very low concentration (0.008 μg/mL) although its metabolic activity was barely affected 72 h post exposure to antibiotic treatment. Only the highest dose of tiamulin tested (0.250 μg/mL) caused a major metabolic shift. Results showed that below this concentration, bacteria could maintain a normal metabolic trajectory despite significant growth inhibition by the antibiotic, which may contribute to disease reemergence post antibiotic treatment. Indeed, we confirmed that B. pilosicoli remained viable even after exposition to the highest antibiotic dose. This paper stresses the need to ensure new evaluation of bacterial viability post bacteriostatic exposure such as tiamulin to guarantee treatment efficacy and decrease antibiotic resistance development

Curing vector for IncI1 plasmids and its use to provide evidence for a metabolic burden of IncI1 CTX-M-1 plasmid pIFM3791 on Klebsiella pneumoniae

Using a sequence based approach we previously identified an IncI1 CTX-M-1 plasmid,pIFM3791, on a single pig farm in the UK that was harboured by K. pneumoniae,Escherichia coli and Salmonella enterica serotype 4,5,12,i:-. To test the hypothesis that the plasmid had spread rapidly into these differing host bacteria we wished to assess whether the plasmid conferred a fitness advantage. To do this an IncI1 curing vector was constructed and used to displace the IncI1 CTX-M-1 plasmids from K. pneumoniae strain B3791 and several other unrelated IncI1 harbouring strains indicating the potential wider application of the curing plasmid. The IncI1 CTX-M-1 plasmid was re-introduced by conjugation into the cured K. pneumoniae strain and also a naturally IncI1 plasmid free S. enterica serotype 4,5,12,i:-, S348/1. Original, cured and complemented strains were tested for metabolic competence using BiologTM technology and in competitive growth, association to mammalian cells and biofilm formation experiments. The plasmid-cured K. pneumoniae strain grew more rapidly than either the original plasmid-carrying strain or plasmid-complemented strains in competition experiments. Additionally, the plasmid-cured strain was significantly better at respiring with L-sorbose as a carbon source and putrescine, γ-amino-n-butyric acid,L-alanine, L-proline as a nitrogen sources. By contrast, no differences in phenotype were found when comparing plasmid harbouring and plasmid free S. enterica S348/11. In conclusion, the IncI1 curing vector successfully displaced multiple IncI plasmids. The IncI1 CTX-M1 plasmid conferred a growth disadvantage upon K. pneumoniae, possibly by imposing a metabolic burden the mechanism of which remains to be determined

Craniometric Analysis of the Hindbrain and Craniocervical Junction of Chihuahua, Affenpinscher and Cavalier King Charles Spaniel Dogs With and Without Syringomyelia Secondary to Chiari-Like Malformation

Objectives To characterize and compare the phenotypic variables of the hindbrain and craniocervical junction associated with syringomyelia (SM) in the Chihuahua, Affenpinscher and Cavalier King Charles Spaniel (CKCS). Method Analysis of 273 T1-weighted mid-sagittal DICOM sequences of the hindbrain and craniocer-vical junction from 99 Chihuahuas, 42 Affenpinschers and 132 CKCSs. The study compared 22 morphometric features (11 lines, eight angles and three ratios) of dogs with and without SM using refined techniques based on previous studies of the Griffon Bruxellois (GB) using Discriminant Function Analysis and ANOVA with post-hoc corrections. Results The analysis identified 14/22 significant traits for SM in the three dog breeds, five of which were identical to those reported for the GB and suggest inclusion of a common aetiology. One ratio, caudal fossa height to the length of the skull base extended to an imaginary point of alignment between the atlas and supraoccipital bones, was common to all three breeds (p values 0.029 to <0.001). Associated with SM were a reduced occipital crest and two acute changes in angulation i) 'sphenoid flexure' at the spheno-occipital synchondrosis ii) 'cervical flexure' at the foramen magnum allied with medulla oblongata elevation. Comparing dogs with and without SM, each breed had a unique trait: Chihuahua had a smaller angle between the dens, atlas and basioccipital bone (p value <0.001); Affenpinschers had a smaller dis-tance from atlas to dens (p value 0.009); CKCS had a shorter distance between the spheno-occipital synchondrosis and atlas (p value 0.007). Conclusion The selected morphometries successfully characterised conformational changes in the brain and craniocervical junction that might form the basis of a diagnostic tool for all breeds. The severity of SM involved a spectrum of abnormalities, incurred by changes in both angulation and size that could alter neural parenchyma compliance and/or impede cerebrospinal fluid channels.Peer reviewe

Biofilm regulation in <i>Clostridioides difficile</i>: Novel systems linked to hypervirulence

Clostridiodes difficile (C. difficile) was ranked an “urgent threat” by the Centers for Disease Control and Prevention (CDC) in 2019. C. difficile infection (CDI) is the most common healthcare-associated infection (HAI) in the United States of America as well as the leading cause of antibiotic-associated gastrointestinal disease. C. difficile is a gram-positive, rod-shaped, spore-forming, anaerobic bacterium that causes infection of the epithelial lining of the gut. CDI occurs most commonly after disruption of the human gut microflora following the prolonged use of broad-spectrum antibiotics. However, the recurrent nature of this disease has led to the hypothesis that biofilm formation may play a role in its pathogenesis. Biofilms are sessile communities of bacteria protected from extracellular stresses by a matrix of self-produced proteins, polysaccharides, and extracellular DNA. Biofilm regulation in C. difficile is still incompletely understood, and its role in disease recurrence has yet to be fully elucidated. However, many factors have been found to influence biofilm formation in C. difficile, including motility, adhesion, and hydrophobicity of the bacterial cells. Small changes in one of these systems can greatly influence biofilm formation. Therefore, the biofilm regulatory system would need to coordinate all these systems to create optimal biofilm-forming physiology under appropriate environmental conditions. The coordination of these systems is complex and multifactorial, and any analysis must take into consideration the influences of the stress response, quorum sensing (QS), and gene regulation by second messenger molecule cyclic diguanosine monophosphate (c-di-GMP). However, the differences in biofilm-forming ability between C. difficile strains such as 630 and the “hypervirulent” strain, R20291, make it difficult to assign a “one size fits all” mechanism to biofilm regulation in C. difficile. This review seeks to consolidate published data regarding the regulation of C. difficile biofilms in order to identify gaps in knowledge and propose directions for future study

Fecal Enterobacteriales enrichment is associated with increased in vivo intestinal permeability in humans

Type 2 diabetes (T2D) has been linked with increased intestinal permeability, but the clinical significance of this phenomenon remains unknown. The objective of this study was to investigate the potential link between glucose control, intestinal permeability, diet and intestinal microbiota in patients with T2D. Thirty‐two males with well‐controlled T2D and 30 age‐matched male controls without diabetes were enrolled in a case–control study. Metabolic parameters, inflammatory markers, endotoxemia, and intestinal microbiota in individuals subdivided into high (HP) and normal (LP) colonic permeability groups, were the main outcomes. In T2D, the HP group had significantly higher fasting glucose (P = 0.034) and plasma nonesterified fatty acid levels (P = 0.049) compared with the LP group. Increased colonic permeability was also linked with altered abundances of selected microbial taxa. The microbiota of both T2D and control HP groups was enriched with Enterobacteriales. In conclusion, high intestinal permeability was associated with poorer fasting glucose control in T2D patients and changes in some microbial taxa in both T2D patients and nondiabetic controls. Therefore, enrichment in the gram‐negative order Enterobacteriales may characterize impaired colonic permeability prior to/independently from a disruption in glucose tolerance

In Vitro Antibacterial Activity of Curcumin-Polymyxin B Combinations against Multidrug-Resistant Bacteria Associated with Traumatic Wound Infections

Bacterial infections resulting from nonsurgical traumatic wounds can be life threatening, especially those caused by multidrug-resistant (MDR) bacteria with limited therapeutic options. The antimicrobial activity of polymyxin B (1) and curcumin (2) alone and in combination was determined versus MDR bacterial isolates associated with traumatic wound infections. Cytotoxicity assays for 1 and 2 were undertaken in keratinocyte cell lines. Minimum inhibitory concentrations of 1 were significantly reduced in the presence of 2 (3- to 10-fold reduction), with synergy observed. Time− kill assays showed the combinations produced bactericidal activity. Cytotoxicity assays indicate the toxicity of 2 was reduced in the presence of 1

Low pathogenic avian influenza virus infection retards colon microbiota diversification in two different chicken lines

Background: A commensal microbiota regulates and is in turn regulated by viruses during host infection which can influence virus infectivity. In this study, analysis of colon microbiota population changes following a low pathogenicity avian influenza virus (AIV) of the H9N2 subtype infection of two different chicken breeds was conducted. Methods: Colon samples were taken from control and infected groups at various timepoints post infection. 16S rRNA sequencing on an Illumina MiSeq platform was performed on the samples and the data mapped to operational taxonomic units of bacterial using a QIIME based pipeline. Microbial community structure was then analysed in each sample by number of observed species and phylogenetic diversity of the population. Results: We found reduced microbiota alpha diversity in the acute period of AIV infection (day 2–3) in both Rhode Island Red and VALO chicken lines. From day 4 post infection a gradual increase in diversity of the colon microbiota was observed, but the diversity did not reach the same level as in uninfected chickens by day 10 post infection, suggesting that AIV infection retards the natural accumulation of colon microbiota diversity, which may further influence chicken health following recovery from infection. Beta diversity analysis indicated a bacterial species diversity difference between the chicken lines during and following acute influenza infection but at phylum and bacterial order level the colon microbiota dysbiosis was similar in the two different chicken breeds. Conclusion: Our data suggest that H9N2 influenza A virus impacts the chicken colon microbiota in a predictable way that could be targeted via intervention to protect or mitigate disease

Intermittent Escherichia coli O157:H7 colonisation at the terminal rectum mucosa of conventionally-reared lambs§

In cattle, the lymphoid rich regions of the rectal-anal mucosa at the terminal rectum are the preferred site for Escherichia coli O157:H7 colonisation. All cattle infected by rectal swab administration demonstrate long-term E. coli O157:H7 colonisation, whereas orally challenged cattle do not demonstrate long-term E. coli O157:H7 colonisation in all animals. Oral, but not rectal challenge of sheep with E. coli O157:H7 has been reported, but an exact site for colonisation in sheep is unknown. To determine if E. coli O157:H7 can effectively colonise the ovine terminal rectum, in vitro organ culture (IVOC) was initiated. Albeit sparsely, large, densely packed E. coli O157:H7 micro-colonies were observed on the mucosa of ovine and control bovine terminal rectum explants. After necropsy of orally inoculated lambs, bacterial enumeration of the proximal and distal gastrointestinal tract did suggest a preference for E. coli O157:H7 colonisation at the ovine terminal rectum, albeit for both lymphoid rich and non-lymphoid sites. As reported for cattle, rectal inoculation studies were then conducted to determine if all lambs would demonstrate persistent colonisation at the terminal rectum. After necropsy of E. coli O157:H7 rectally inoculated lambs, most animals were not colonised at gastrointestinal sites proximal to the rectum, however, large densely packed micro-colonies of E. coli O157:H7 were observed on the ovine terminal rectum mucosa. Nevertheless, at the end point of the study (day 14), only one lamb had E. coli O157:H7 micro-colonies associated with the terminal rectum mucosa. A comparison of E. coli O157:H7 shedding yielded a similar pattern of persistence between rectally and orally inoculated lambs. The inability of E. coli O157:H7 to effectively colonise the terminal rectum mucosa of all rectally inoculated sheep in the long term, suggests that E. coli O157:H7 may colonise this site, but less effectively than reported previously for cattle

A multi-country One Health foodborne outbreak simulation exercise: cross-sectoral cooperation, data sharing and communication

IntroductionThe awareness of scientists and policy makers regarding the requirement for an integrated One Health (OH) approach in responding to zoonoses has increased in recent years. However, there remains an overall inertia in relation to the implementation of practical cross-sector collaborations. Foodborne outbreaks of zoonotic diseases continue to affect the European population despite stringent regulations, evidencing the requirement for better ‘prevent, detect and response’ strategies. Response exercises play an essential role in the improvement of crisis management plans, providing the opportunity to test practical intervention methodologies in a controlled environment.MethodsThe One Health European Joint Programme simulation exercise (OHEJP SimEx) aimed at practicing the OH capacity and interoperability across public health, animal health and food safety sectors in a challenging outbreak scenario. The OHEJP SimEx was delivered through a sequence of scripts covering the different stages of a Salmonella outbreak investigation at a national level, involving both the human food chain and the raw pet feed industry.ResultsA total of 255 participants from 11 European countries (Belgium, Denmark, Estonia, Finland, France, Italy, Norway, Poland, Portugal, Sweden, the Netherlands) took part in national level two-day exercises during 2022. National evaluations identified common recommendations to countries aiming to improve their OH structure to establish formal communication channels between sectors, implement a common data sharing platform, harmonize laboratory procedures, and reinforce inter-laboratory networks within countries. The large proportion of participants (94%) indicated significant interest in pursuing a OH approach and desire to work more closely with other sectors.DiscussionThe OHEJP SimEx outcomes will assist policy makers in implementing a harmonized approach to cross-sector health-related topics, by highlighting the benefits of cooperation, identifying gaps in the current strategies and suggesting actions required to better address foodborne outbreaks. Furthermore, we summarize recommendations for future OH simulation exercises, which are essential to continually test, challenge and improve national OH strategies