Spatio-Temporal Distribution of Brucellosis in European Terrestrial and Marine Wildlife Species and Its Regional Implications.

Brucellosis is an important bacterial zoonosis of domestic and wildlife species. This disease has a significant public health concern and is characterized by reproductive failure resulting in economic losses in the livestock industry. Among thirteen known species, B. abortus, B. melitensis, B. suis, and B. canis are human pathogens. Brucellosis has been extensively investigated in humans and domestic animals. However, the situation in wildlife is still not completely reported and studied. Therefore, a systematic literature search and screening were done to clarify the situation of brucellosis in wildlife in Europe. Sixty-five articles from a total of 13,424 reports published between 1991 and 2021 were selected, applying defined inclusion criteria. Wild boars and brown hares were the most often studied terrestrial wildlife species, whereas seals and porpoises were the most often investigated marine wildlife. Poland, Croatia, and Belgium showed the highest seroprevalences of wild boars caused by B. suis biovar 2. In marine wildlife, brucellosis was mainly caused by B. ceti and B. pinnipedialis. Most samples were from carcasses. Thus, sera could not be collected. It is worrisome that B.abortus and B. melitensis were reported from both terrestrial and marine wild animals, posing a zoonotic threat to people exposed to wild animals. Currently, there is no approved vaccine available for wild animals. The main challenges are the development of specific diagnostics and their validation for use in wildlife.

Perspectives on the Ethics of Antibiotic Overuse and on the Implementation of (New) Antibiotics.

The continuing rise in global antimicrobial resistance is seen by many governments and international organizations as a major threat to worldwide health. This means that many publications have already described the problems concerning the overuse of currently available antibiotics and potential solutions to this crisis, including the development of new alternatives to antibiotics. However, in this manuscript, the authors approach the subject of increasing global antimicrobial resistance from two perspectives not normally covered by previous publications, namely the ethical use of antibiotics and potential issues relating to the implementation of new antibiotics.

Antibiotic Discovery and Resistance: The Chase and the Race.

The history of antimicrobial resistance (AMR) evolution and the diversity of the environmental resistome indicate that AMR is an ancient natural phenomenon. Acquired resistance is a public health concern influenced by the anthropogenic use of antibiotics, leading to the selection of resistant genes. Data show that AMR is spreading globally at different rates, outpacing all efforts to mitigate this crisis. The search for new antibiotic classes is one of the key strategies in the fight against AMR. Since the 1980s, newly marketed antibiotics were either modifications or improvements of known molecules. The World Health Organization (WHO) describes the current pipeline as bleak, and warns about the scarcity of new leads. A quantitative and qualitative analysis of the pre-clinical and clinical pipeline indicates that few antibiotics may reach the market in a few years, predominantly not those that fit the innovative requirements to tackle the challenging spread of AMR. Diversity and innovation are the mainstays to cope with the rapid evolution of AMR. The discovery and development of antibiotics must address resistance to old and novel antibiotics. Here, we review the history and challenges of antibiotics discovery and describe different innovative new leads mechanisms expected to replenish the pipeline, while maintaining a promising possibility to shift the chase and the race between the spread of AMR, preserving antibiotic effectiveness, and meeting innovative leads requirements.

Progress in Alternative Strategies to Combat Antimicrobial Resistance: Focus on Antibiotics.

Antibiotic resistance, and, in a broader perspective, antimicrobial resistance (AMR), continues to evolve and spread beyond all boundaries. As a result, infectious diseases have become more challenging or even impossible to treat, leading to an increase in morbidity and mortality. Despite the failure of conventional, traditional antimicrobial therapy, in the past two decades, no novel class of antibiotics has been introduced. Consequently, several novel alternative strategies to combat these (multi-) drug-resistant infectious microorganisms have been identified. The purpose of this review is to gather and consider the strategies that are being applied or proposed as potential alternatives to traditional antibiotics. These strategies include combination therapy, techniques that target the enzymes or proteins responsible for antimicrobial resistance, resistant bacteria, drug delivery systems, physicochemical methods, and unconventional techniques, including the CRISPR-Cas system. These alternative strategies may have the potential to change the treatment of multi-drug-resistant pathogens in human clinical settings.

Identification of Promising Genotypes Through Systematic Evaluation for Arsenic Tolerance and Exclusion in Rice (Oryza sativa L.).

Rice remains a major staple food source for the rapidly growing world population. However, regular occurrences of carcinogenic arsenic (As) minerals in waterlogged paddy topsoil pose a great threat to rice production and consumers across the globe. Although As contamination in rice has been well recognized over the past two decades, no suitable rice germplasm had been identified to exploit in adaptive breeding programs. Therefore, this current study identified suitable rice germplasm for As tolerance and exclusion based on a variety of traits and investigated the interlinkages of favorable traits during different growth stages. Fifty-three different genotypes were systematically evaluated for As tolerance and accumulation. A germination screening assay was carried out to identify the ability of individual germplasm to germinate under varying As stress. Seedling-stage screening was conducted in hydroponics under varying As stress to identify tolerant and excluder genotypes, and a field experiment was carried out to identify genotypes accumulating less As in grain. Irrespective of the rice genotypes, plant health declined significantly with increasing As in the treatment. However, genotype-dependent variation in germination, tolerance, and As accumulation was observed among the genotypes. Some genotypes (WTR1-BRRI dhan69, NPT-IR68552-55-3-2, OM997, and GSR IR1-5-Y4-S1-Y1) showed high tolerance by excluding As in the shoot system. Arsenic content in grain ranged from 0.12 mg kg-1 in Huang-Hua-Zhan (indica) from China to 0.48 mg kg-1 in IRAT 109 (japonica) from Brazil. This current study provides novel insights into the performance of rice genotypes under varying As stress during different growth stages for further use in ongoing breeding programs for the development of As-excluding rice varieties for As-polluted environments.

The potential impact of the COVID-19 pandemic on global antimicrobial and biocide resistance: an AMR Insights global perspective.

The COVID-19 pandemic presents a serious public health challenge in all countries. However, repercussions of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections on future global health are still being investigated, including the pandemic's potential effect on the emergence and spread of global antimicrobial resistance (AMR). Critically ill COVID-19 patients may develop severe complications, which may predispose patients to infection with nosocomial bacterial and/or fungal pathogens, requiring the extensive use of antibiotics. However, antibiotics may also be inappropriately used in milder cases of COVID-19 infection. Further, concerns such as increased biocide use, antimicrobial stewardship/infection control, AMR awareness, the need for diagnostics (including rapid and point-of-care diagnostics) and the usefulness of vaccination could all be components shaping the influence of the COVID-19 pandemic. In this publication, the authors present a brief overview of the COVID-19 pandemic and associated issues that could influence the pandemic's effect on global AMR.

Selection for Resistance to a Glyphosate-Containing Herbicide in Salmonella enterica Does Not Result in a Sustained Activation of the Tolerance Response or Increased Cross-Tolerance and Cross-Resistance to Clinically Important Antibiotics.

Evolution of bacterial tolerance to antimicrobials precedes evolution of resistance and may result in cross-tolerance, cross-resistance, or collateral sensitivity to other antibiotics. Transient exposure of gut bacteria to glyphosate, the world's most widely used herbicide, has been linked to the activation of the stress response and changes in susceptibility to antibiotics. In this study, we investigated whether chronic exposure to a glyphosate-based herbicide (GBH) results in resistance, a constitutive activation of the tolerance and stress responses, and cross-tolerance or cross-resistance to antibiotics. Of the 10 farm animal-derived clinical isolates of Salmonella enterica subjected to experimental evolution in increasing concentrations of GBH, three isolates showed stable resistance with mutations associated with the glyphosate target gene aroA and no fitness costs. Global quantitative proteomics analysis demonstrated activation of the cellular tolerance and stress response during the transient exposure to GBH but not constitutively in the resistant mutants. Resistant mutants displayed no cross-resistance or cross-tolerance to antibiotics. These results suggest that while transient exposure to GBH triggers cellular tolerance response in Salmonella enterica, this response does not become genetically fixed after selection for resistance to GBH and does not result in increased cross-tolerance or cross-resistance to clinically important antibiotics under our experimental conditions.IMPORTANCE Glyphosate-based herbicides (GBH) are among the world's most popular, with traces commonly found in food, feed, and the environment. Such high ubiquity means that the herbicide may come into contact with various microorganisms, on which it acts as an antimicrobial, and it may select for resistance and cross-resistance to clinically important antibiotics. It is therefore important to estimate whether the widespread use of pesticides may be an underappreciated source of antibiotic-resistant microorganisms that may compromise efficiency of antibiotic treatments in humans and animals.

Combined Methylome, Transcriptome and Proteome Analyses Document Rapid Acclimatization of a Bacterium to Environmental Changes.

Polynucleobacter asymbioticus strain QLW-P1DMWA-1T represents a group of highly successful heterotrophic ultramicrobacteria that is frequently very abundant (up to 70% of total bacterioplankton) in freshwater habitats across all seven continents. This strain was originally isolated from a shallow Alpine pond characterized by rapid changes in water temperature and elevated UV radiation due to its location at an altitude of 1300 m. To elucidate the strain's adjustment to fluctuating environmental conditions, we recorded changes occurring in its transcriptomic and proteomic profiles under contrasting experimental conditions by simulating thermal conditions in winter and summer as well as high UV irradiation. To analyze the potential connection between gene expression and regulation via methyl group modification of the genome, we also analyzed its methylome. The methylation pattern differed between the three treatments, pointing to its potential role in differential gene expression. An adaptive process due to evolutionary pressure in the genus was deduced by calculating the ratios of non-synonymous to synonymous substitution rates for 20 Polynucleobacter spp. genomes obtained from geographically diverse isolates. The results indicate purifying selection.

Pan-Proteomic Analysis and Elucidation of Protein Abundance among the Closely Related Brucella Species, Brucella abortus and Brucella melitensis.

Brucellosis is a zoonotic infection caused by bacteria of the genus Brucella. The species, B. abortus and B. melitensis, major causative agents of human brucellosis, share remarkably similar genomes, but they differ in their natural hosts, phenotype, antigenic, immunogenic, proteomic and metabolomic properties. In the present study, label-free quantitative proteomic analysis was applied to investigate protein expression level differences. Type strains and field strains were each cultured six times, cells were harvested at a midlogarithmic growth phase and proteins were extracted. Following trypsin digestion, the peptides were desalted, separated by reverse-phase nanoLC, ionized using electrospray ionization and transferred into an linear trap quadrapole (LTQ) Orbitrap Velos mass spectrometer to record full scan MS spectra (m/z 300-1700) and tandem mass spectrometry (MS/MS) spectra of the 20 most intense ions. Database matching with the reference proteomes resulted in the identification of 826 proteins. The Cluster of Gene Ontologies of the identified proteins revealed differences in bimolecular transport and protein synthesis mechanisms between these two strains. Among several other proteins, antifreeze proteins, Omp10, superoxide dismutase and 30S ribosomal protein S14 were predicted as potential virulence factors among the proteins differentially expressed. All mass spectrometry data are available via ProteomeXchange with identifier PXD006348.

Proteomics of Brucella: Technologies and Their Applications for Basic Research and Medical Microbiology.

Brucellosis is a global zoonosis caused by Gram-negative, facultative intracellular bacteria of the genus Brucella (B.). Proteomics has been used to investigate a few B. melitensis and B. abortus strains, but data for other species and biovars are limited. Hence, a comprehensive analysis of proteomes will significantly contribute to understanding the enigmatic biology of brucellae. For direct identification and typing of Brucella, matrix-assisted laser desorption ionization - time of flight mass spectrometry (MALDI - TOF MS) has become a reliable tool for routine diagnosis due to its ease of handling, price and sensitivity highlighting the potential of proteome-based techniques. Proteome analysis will also help to overcome the historic but still notorious Brucella obstacles of infection medicine, the lack of safe and protective vaccines and sensitive serologic diagnostic tools by identifying the most efficient protein antigens. This perspective summarizes past and recent developments in Brucella proteomics with a focus on species identification and serodiagnosis. Future applications of proteomics in these fields are discussed.

Bacteriophage amplification - A comparison of selected methods.

The bactericidal properties of bacteriophages have been used almost since the moment of the discovery of bacterial viruses. In the light of the rapidly growing number of antibiotic-resistant bacteria, phage therapy is considered one of the most promising alternatives to classical treatment. Phage amplification is one of the most common procedures of working with phages, and high-titer preparations are beneficial at the experimental stage of studies as well as in practice. The objective of this study was to compare five commonly applied methods of phage amplification: (i) pooled plaques method, (ii) the plate wash method, (iii) the agar culture method, (iv) the two-stage culture method, and (v) in liquid culture. All methods were tested for fifteen different phages. The results described herein indicate that there is no optimal, universal method for phage amplification, and the most effective method has to be established individually for each phage.

Treatment of Yersinia similis with the cationic lipid DOTAP enhances adhesion to and invasion into intestinal epithelial cells - A proof-of-principle study.

The monocationic quaternary surfactant DOTAP has been used for the delivery of nucleic acids and peptides into mammalian cells. This study tested the applicability of DOTAP for the enhancement of adhesion and invasion frequencies of Yersinia (Y.) similis to enable the analysis of the effects of low-pathogenic bacteria on intestinal epithelial cells. Incubation of Y. similis with DOTAP ahead of infection of C2BBe1 intestinal epithelial cells increased invasion and adhesion frequency four- and five-fold, respectively, in plating assays. Proteomic approaches confirmed the increased bacterial load on infected cells: analysis of protein extracts by two-dimensional difference gel electrophoresis (2D-DIGE) revealed higher amounts of bacterial proteins present in the cells infected with DOTAP-treated bacteria. MALDI-TOF mass spectrometry of selected spots from gel-separated protein extracts confirmed the presence of both bacterial and human cell proteins in the samples. Label-free quantitative proteomics analysis identified 1170 human cell proteins and 699 bacterial proteins. Three times more bacterial proteins (279 vs. 93) were detected in C2BBe1 cells infected with DOTAP-treated bacteria compared to infections with untreated bacteria. Infections with DOTAP-treated Y. similis led to a significant upregulation of the stress-inducible ubiquitin-conjugating enzyme UBE2M in C2BBe1 cells. This points towards a stronger impact of the stress and infection responsive transcription factor AP-1 by enhanced bacterial load. DOTAP-treatment of uninfected C2BBe1 cells led to a significant downregulation of the transmembrane trafficking protein TMED10. The application of DOTAP could be helpful for investigating the impact of otherwise low adherent or invasive bacteria on cultivated mammalian cells without utilisation of genetic modifications.

Identification of Chitinase-3-Like Protein 1 as a Novel Neutrophil Antigenic Target in Crohn's Disease.

BACKGROUND AND AIMS: There is an increasing incidence of inflammatory bowel disease [IBD]. Autoimmune responses are involved in the pathophysiology of IBD, but their underlying pathways and target antigens have not yet been fully elucidated. METHODS: Autoantigenic targets in IBD were identified after separation of whole cell proteins isolated from neutrophils using two-dimensional electrophoresis and matrix assisted laser desorption ionization - time of flight mass spectrometry-based protein identification of the spots that displayed Western blotting signals with anti-neutrophil cytoplasmic antibody-positive sera. The prevalence of IgG, IgA and secretory IgA [sIgA] to chitinase 3-like protein 1 [CHI3L1] was analysed by enzyme-linked immunosorbent assays using recombinant CHI3L1 in 110 patients with Crohn's disease [CD], 95 with ulcerative colitis [UC], 126 with coeliac disease [CeD] and 86 healthy controls [HCs]. RESULTS: The 18-glycosylhydrolase family member CHI3L1 was identified as a neutrophil autoantigenic target. CD patients displayed significantly higher levels of IgG to CHI3L1 than patients with UC and CeD (p < 0.0001, respectively). IgA and sIgA to CHI3L1 was significantly higher in CD than in UC, CeD and HCs [p < 0.0001, respectively]. IgA and sIgA to CHI3L1 demonstrated the highest prevalence in CD [25.5%, 28/110; and 41.8%%, 46/110] compared to HCs [2.3%, 2/86; and 4.7%%, 4/86; p = 0.0015 and p < 0.0001] and are associated with a more complicated progression of CD. CONCLUSION: CHI3L1 is a novel neutrophil autoantigenic target in CD. IgA and sIgA to CHI3L1 may serve as novel markers for CD and may facilitate the serological diagnosis of IBD.

Mass spectrometry data from identification of host-defense related proteins using label-free quantitative proteomic analysis of milk whey from cows with Staphylococcus aureus subclinical mastitis.

This dataset is associated with our research article 'Identification of host-defence related proteins using label-free quantitative proteomic analysis of milk whey from cows with Staphylococcus aureus Subclinical mastitis' published in International Journal of Molecular Sciences. Milk samples were collected from cows suffering from S. aureus-associated subclinical mastitis and the proteins abundance were identified in comparison with samples collected from the control cows using liquid chromatography-mass spectrometry (LC-MS)-based label free proteomics analysis. Following the MS measurements, the raw spectra were processed using MaxQuant-Andromeda software and the protein identification was carried out through a search against Uniprot FASTA files of the Bos taurus reference proteome. Perseus software analysis was applied for computation of protein abundance. The raw file presented in this DiB was deposition at PRIDE repository in the PRIDE repository with the dataset identifier PXD007516.

Concurrent Proteomic Fingerprinting and Molecular Analysis of Cyathostomins.

Rapid, cost-effective, efficient, and reliable helminth species identification is of considerable importance to understand host-parasite interactions, clinical disease, and drug resistance. Cyathostomins (Nematoda: Strongylidae) are considered to be the most important equine parasites, yet research on this group is hampered by the large number of 50 morphologically differentiated species, their occurrence in mixed infections with often more than 10 species and the difficulties associated with conventional identification methods. Here, MALDI-TOF MS, previously successfully applied to identify numerous organisms, is evaluated and compared with conventional and molecular genetic approaches. A simple and robust protocol for protein extraction and subsequent DNA isolation allowing molecular confirmation of proteomic findings is developed, showing that MALDI-TOF MS can discriminate adult stages of the two closely related cyathostomin species Cylicostephanus longibursatus and Cylicostephanus minutus. Intraspecific variability of proteomic profiles within morphospecies demonstrated an identification of morphospecies with an accuracy of close to 100%. In contrast, three genospecies within C. minutus and sex-specific profiles within both morphospecies could not be reliably discriminated using MALDI-TOF MS. In conclusion, MALDI-TOF MS complemented by the molecular protocol is a reliable and efficient approach for cyathostomin species identification.

Isolate Specific Cold Response of Yersinia enterocolitica in Transcriptional, Proteomic, and Membrane Physiological Changes.

Yersinia enterocolitica, a zoonotic foodborne pathogen, is able to withstand low temperatures. This psychrotrophic ability allows it to multiply in food stored in refrigerators. However, little is known about the Y. enterocolitica cold response. In this study, isolate-specific behavior at 4°C was demonstrated and the cold response was investigated by examining changes in phenotype, gene expression, and the proteome. Altered expression of cold-responsive genes showed that the ability to survive at low temperature depends on the capacity to acclimate and adapt to cold stress. This cold acclimation at the transcriptional level involves the transient induction and effective repression of cold-shock protein (Csp) genes. Moreover, the resumption of expression of genes encoding other non-Csp is essential during prolonged adaptation. Based on proteomic analyses, the predominant functional categories of cold-responsive proteins are associated with protein synthesis, cell membrane structure, and cell motility. In addition, changes in membrane fluidity and motility were shown to be important in the cold response of Y. enterocolitica. Isolate-specific differences in the transcription of membrane fluidity- and motility-related genes provided evidence to classify strains within a spectrum of cold response. The combination of different approaches has permitted the systematic description of the Y. enterocolitica cold response and gives a better understanding of the physiological processes underlying this phenomenon.

Methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-resistant Staphylococcus pseudintermedius (MRSP) among employees and in the environment of a small animal hospital.

The aim of the study was to investigate methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-resistant Staphylococcus pseudintermedius (MRSP) among employees of a small animal hospital and the hospital environment. In total, 96 swabs from employees and 73 swabs from the clinic environment were investigated. Cation-adjusted-Mueller-Hinton broth (CAMHB) + 6.5% NaCl was used for enrichment before plating on Mueller-Hinton (MH) agar with 2% NaCl and 0.25 mg/L oxacillin. The staphylococcal species was determined using MALDI-TOF MS. The isolates were subjected to mecA-PCR, macrorestriction analysis, and antimicrobial susceptibility testing. MRSA were present in five nasal swabs of the 55 employees tested and in six environmental samples, MRSP in two employees (nasal and hand swabs, each) and in three environmental samples. All isolates harboured mecA. Susceptibility testing revealed that all but one of the isolates were multiresistant. All isolates were resistant to ß-lactams and fluoroquinolones. All but one of the isolates were resistant to macrolides and lincosamides. A single MRSA was resistant to gentamicin. All MRSP were resistant to trimethoprim/sulfamethoxazole and non-susceptible to gentamicin. One isolate was also resistant to tetracycline. Macrorestriction analysis revealed three main SmaI patterns for MRSA and two main SmaI patterns for MRSP. All environmental isolates were found in areas of high people and animal traffic, such as dog ward areas, waiting and triage rooms. The finding of indistinguishable MRSA or MRSP among employees and in the environment of the small animal hospital suggests the possibility of transfer of these bacteria between humans, animals, and the hospital environment.

Medical phycology 2017.

In 2014, ISHAM formed a new working group: "Medical Phycology: Protothecosis and Chlorellosis." The purpose of this working group is to help facilitate collaboration and communication among people interested in the pathogenic algae, to share ideas and work together. Here we present reports on recent work we have done in five areas. 1. The history of medical phycology as a branch of science. 2. Aspects of the genetics of Prototheca. 3. Aspects of the proteins of Prototheca. 4. Human infections caused by Prototheca. 5. Dairy cow mastitis caused by Prototheca.

Trueperella pyogenes and Brucella abortus Coinfection in a Dog and a Cat on a Dairy Farm in Egypt with Recurrent Cases of Mastitis and Abortion.

Trueperella pyogenes was isolated from a dog and a cat with a mixed infection with Brucella abortus. Both lived on a dairy cattle farm with a history of regular cases of abortion and mastitis. Identification of the bacteria was done by means of MALDI-TOF MS, loop-mediated isothermal amplification (LAMP) based on cpn60, partial 16S rRNA sequencing, and growth on Loeffler Serum Medium. Isolation of Trueperella pyogenes on the dairy farm highlights its neglected role in reproduction failure and draws attention to its effects in the dairy industry in Egypt. Diagnosis and control of abortion in Egypt should include Trueperella pyogenes as one of possible causes of abortion.

MALDI Spectra Database for Rapid Discrimination and Subtyping of Mycobacterium kansasii.

Mycobacterium kansasii is an emerging non-tuberculous mycobacterial (NTM) pathogen capable of causing severe lung disease. Of the seven currently recognized M. kansasii genotypes (I-VII), genotypes I and II are most prevalent and have been associated with human disease, whereas the other five (III-VII) genotypes are predominantly of environmental origin and are believed to be non-pathogenic. Subtyping of M. kansasii serves as a valuable tool to guide clinicians in pursuing diagnosis and to initiate the proper timely treatment. Most of the previous rapid diagnostic tests for mycobacteria employing the matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) technology focused on species-level identification. The purpose of this study was to establish MALDI-TOF MS reference spectra database for discrimination of M. kansasii at the genotype level. A panel of 32 strains, representatives of M. kansasii genotypes I-VI were selected, whole cell proteins extracted and measured with MALDI-TOF MS. A unique main spectra (MSP) library was created using MALDI Biotyper Compass Explorer software. The spectra reproducibility was assessed by computing composite correlation index and MSPs cross-matching. One hundred clinical M. kansasii isolates used for testing of the database resulted in 90% identification at genus-level, 7% identification at species-level and 2% identification was below the threshold of log score value 1.7, of which all were correct at genotype level. One strain could not be identified. On the other hand, 37% of strains were identified at species level, 40% at genus level and 23% was not identified with the manufacturer's database. The MALDI-TOF MS was proven a rapid and robust tool to detect and differentiate between M. kansasii genotypes. It is concluded that MALDI-TOF MS has a potential to be incorporated into the routine diagnostic workflow of M. kansasii and possibly other NTM species.