Multidrug resistance in pathogenic Escherichia coli isolates from urinary tract infections in dogs, Spain.

Escherichia coli (E. coli) is a pathogen frequently isolated in cases of urinary tract infections (UTIs) in both humans and dogs and evidence exists that dogs are reservoirs for human infections. In addition, E. coli is associated to increasing antimicrobial resistance rates. This study focuses on the analysis of antimicrobial resistance and the presence of selected virulence genes in E. coli isolates from a Spanish dog population suffering from UTI. This collection of isolates showed an extremely high level of phenotypic resistance to 1st-3rd generation cephalosporins, followed by penicillins, fluoroquinolones and amphenicols. Apart from that, 13.46% of them were considered extended-spectrum beta-lactamase producers. An alarmingly high percentage (71.15%) of multidrug resistant isolates were also detected. There was a good correlation between the antimicrobial resistance genes found and the phenotypic resistance expressed. Most of the isolates were classified as extraintestinal pathogenic E. coli, and two others harbored virulence factors related to diarrheagenic pathotypes. A significant relationship between low antibiotic resistance and high virulence factor carriage was found, but the mechanisms behind it are still poorly understood. The detection of high antimicrobial resistance rates to first-choice treatments highlights the need of constant antimicrobial resistance surveillance, as well as continuous revision of therapeutic guidelines for canine UTI to adapt them to changes in antimicrobial resistance patterns.

Diagnosis in Scrapie: Conventional Methods and New Biomarkers.

Scrapie, a naturally occurring prion disease affecting goats and sheep, comprises classical and atypical forms, with classical scrapie being the archetype of transmissible spongiform encephalopathies. This review explores the challenges of scrapie diagnosis and the utility of various biomarkers and their potential implications for human prion diseases. Understanding these biomarkers in the context of scrapie may enable earlier prion disease diagnosis in humans, which is crucial for effective intervention. Research on scrapie biomarkers bridges the gap between veterinary and human medicine, offering hope for the early detection and improved management of prion diseases.

Detection of classical BSE prions in asymptomatic cows after inoculation with atypical/Nor98 scrapie.

The emergence of bovine spongiform encephalopathy (BSE) prions from atypical scrapie has been recently observed upon experimental transmission to rodent and swine models. This study aimed to assess whether the inoculation of atypical scrapie could induce BSE-like disease in cattle. Four calves were intracerebrally challenged with atypical scrapie. Animals were euthanized without clinical signs of prion disease and tested negative for PrPSc accumulation by immunohistochemistry and western blotting. However, an emergence of BSE-like prion seeding activity was detected during in vitro propagation of brain samples from the inoculated animals. These findings suggest that atypical scrapie may represent a potential source of BSE infection in cattle.

Update on Commonly Used Molecular Typing Methods for Clostridioides difficile.

This review aims to provide a comprehensive overview of the significant Clostridioides difficile molecular typing techniques currently employed in research and medical communities. The main objectives of this review are to describe the key molecular typing methods utilized in C. difficile studies and to highlight the epidemiological characteristics of the most prevalent strains on a global scale. Geographically distinct regions exhibit distinct strain types of C. difficile, with notable concordance observed among various typing methodologies. The advantages that next-generation sequencing (NGS) offers has changed epidemiology research, enabling high-resolution genomic analyses of this pathogen. NGS platforms offer an unprecedented opportunity to explore the genetic intricacies and evolutionary trajectories of C. difficile strains. It is relevant to acknowledge that novel routes of transmission are continually being unveiled and warrant further investigation, particularly in the context of zoonotic implications and environmental contamination.

Effect of Intramuscularly Administered Oxytetracycline or Enrofloxacin on Vancomycin-Resistant Enterococci, Extended Spectrum Beta-Lactamase- and Carbapenemase-Producing Enterobacteriaceae in Pigs.

Nowadays, there is a great concern about the prevalence of multidrug resistant Enterococcus spp. and Enterobacteriaceae in food-producing animals. The aim of this work was to evaluate the effect of oxytetracycline or enrofloxacin treatment on vancomycin-resistant enterococci (VRE), extended spectrum ß-lactamase (ESBL) and carbapenemase-producing Enterobacteriaceae in pigs. A total of 26 piglets were received and distributed in three groups. Group 1 was treated with enrofloxacin (N = 12), group 2 with oxytetracycline (N = 10) and group 3 did not receive any treatment (control group) (N = 4). A higher number of vancomycin-resistant E. faecium were recovered compared to E. faecalis. In the pigs treated with enrofloxacin, vancomycin resistant E. faecium was found in a higher percentage of animals than in the control group. ESBL-producing E. coli was not detected in rectal samples from control animals. However, it was detected in 17-20% of animals treated with oxytetracycline on days 6 to 17 and in 17-50% of the animals treated with enrofloxacin. Carbapenemase-producing E. coli was isolated in animals treated with oxytetracycline, but not in animals treated with enrofloxacin or in the control group. This study highlights that the use of oxytetracycline or enrofloxacin in food-producing animals could select ESBL and carbapenemase-producing E. coli. Further studies shall be needed to validate the results obtained, considering a more robust and extended experimental design.

SARS-CoV-2 Outbreak on a Spanish Mink Farm: Epidemiological, Molecular, and Pathological Studies.

Farmed minks have been reported to be highly susceptible to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and may represent a risk to humans. In this study, we describe the first outbreak of SARS-CoV-2 occurred on a mink farm in Spain, between June and July 2020, involving 92,700 animals. The outbreak started shortly after some farm workers became seropositive for SARS-CoV-2. Minks showed no clinical signs compatible with SARS-CoV-2 infection throughout the outbreak. Samples from 98 minks were collected for histopathological, serological, and molecular studies. Twenty out of 98 (20.4%) minks were positive by RT-qPCR and 82 out 92 (89%) seroconverted. This finding may reflect a rapid spread of the virus at the farm with most of the animals overcoming the infection. Additionally, SARS-CoV-2 was detected by RT-qPCR in 30% of brain samples from positive minks. Sequencing analysis showed that the mink sequences were not closely related with the other mink SARS-CoV-2 sequences available, and that this mink outbreak has its probable origin in one of the genetic variants that were prevalent in Spain during the first COVID-19 epidemic wave. Histological studies revealed bronchointerstitial pneumonia in some animals. Immunostaining of viral nucleocapsid was also observed in nasal turbinate tissue. Farmed minks could therefore constitute an important SARS-CoV-2 reservoir, contributing to virus spread among minks and humans. Consequently, continuous surveillance of mink farms is needed.

Resistance to colistin and production of extended-spectrum ß-lactamases and/or AmpC enzymes in Salmonella isolates collected from healthy pigs in Northwest Spain in two periods: 2008-2009 and 2018.

Salmonellosis is a common subclinical infection in pigs and therefore apparently healthy animals may represent a reservoir of antibiotic-resistant Salmonella for humans. This study estimates and characterizes resistance to two classes of antimicrobials considered of the highest priority within the critically important antimicrobials for humans, i.e. colistin (CR) and 3rd generation cephalosporins (3GC), on a collection of Salmonella isolates from pigs from two periods: between 2008 and 09, when colistin was massively used; and in 2018, after three years under a National Plan against Antibiotic Resistance. Prevalence of CR was low (6 out of 625; 0.96%; 95%CI: 0.44-2.1) in 2008-09 and associated mostly to the mcr-1 gene, which was detected in four S. 4,5,12:i:- isolates. Polymorphisms in the pmrAB genes were detected in a S. 9,12:-:- isolate. No CR was detected in 2018 out of 59 isolates tested. Among 270 Salmonella isolates considered for the assessment of resistance to 3GC in the 2008-2009 sampling, only one Salmonella Bredeney (0.37%; 95%CI: 0.07-2.1) showed resistance to 3GC, which was associated with the blaCMY-2 gene (AmpC producer). In 2018, six isolates out of 59 (10.2%; 95%CI: 4.7-20.5) showed resistance to 3GC, but only two different strains were identified (S. 4,12:i:- and S. Rissen), both confirmed as extended-spectrum ß-lactamases (ESBL) producers. The blaCTX-M-3 and blaTEM-1b genes in S. 4,12:i:- and the blaTEM-1b gene in S. Rissen seemed to be associated with this resistance. Overall, the prevalence of CR in Salmonella appeared to be very low in 2008-2009 despite the considerable use of colistin in pigs at that time, and seemed to remain so in 2018. Resistance to 3GC was even lower in 2008-2009 but somewhat higher in 2018. Resistance was mostly coded by genes associated with mobile genetic elements. Most serotypes involved in these antimicrobial resistances displayed a multidrug resistance pattern and were considered zoonotic.

Antimicrobial resistance among canine enteric Escherichia coli isolates and prevalence of attaching-effacing and extraintestinal pathogenic virulence factors in Spain.

The aim of this study was to estimate the prevalence of antimicrobial resistance (AMR) in Escherichia coli from a dog population in Spain and assess specific virulence factors. Susceptibility to 22 antimicrobials was tested along with the production of extended-spectrum ß-lactamases (ESBLs) and AmpC in faecal isolates from 100 dogs. Virulence-related genes associated with attaching and effacing E. coli (eae, Stx1, Stx2) and extraintestinal pathogenic E. coli - ExPEC - (papC, hlyA and cnf1) were detected by PCR. At least one kind of AMR was observed in 73% of the isolates. The highest prevalences corresponded to penicillin (45%), aminoglycoside (40%) and non-extended spectrum cephalosporin (39%) classes. Multidrug resistance (MDR) was observed in 53.4% of the resistant isolates. No resistance to colistin was found. Production of ESBL/AmpC enzymes was detected in 5% of E. coli. Shiga toxin-producing E. coli were not observed, enteropathogenic E. coli were identified in only 12% of them, and ExPEC were found in 25%. Dog faeces can be a source of E. coli strains potentially presenting a threat to humans through their virulence factors or AMR. The non-hygienic keeping of animals may increase the risk of colonisation of such pathogens in humans.

Plasmid-mediated metronidazole resistance in Clostridioides difficile.

Metronidazole was until recently used as a first-line treatment for potentially life-threatening Clostridioides difficile (CD) infection. Although cases of metronidazole resistance have been documented, no clear mechanism for metronidazole resistance or a role for plasmids in antimicrobial resistance has been described for CD. Here, we report genome sequences of seven susceptible and sixteen resistant CD isolates from human and animal sources, including isolates from a patient with recurrent CD infection by a PCR ribotype (RT) 020 strain, which developed resistance to metronidazole over the course of treatment (minimal inhibitory concentration [MIC] = 8 mg L-1). Metronidazole resistance correlates with the presence of a 7-kb plasmid, pCD-METRO. pCD-METRO is present in toxigenic and non-toxigenic resistant (n = 23), but not susceptible (n = 563), isolates from multiple countries. Introduction of a pCD-METRO-derived vector into a susceptible strain increases the MIC 25-fold. Our finding of plasmid-mediated resistance can impact diagnostics and treatment of CD infections.

Wild griffon vultures (Gyps fulvus) fed at supplementary feeding stations: Potential carriers of pig pathogens and pig-derived antimicrobial resistance?

The carriage of two important pathogens of pigs, that is enterotoxigenic Escherichia coli (ETEC) and Clostridioides difficile, was investigated in 104 cloacal samples from wild griffon vultures (Gyps fulvus) fed on pig carcasses at supplementary feeding stations (SFS), along with their level of antimicrobial resistance (AMR). E. coli was isolated from 90 (86.5%) samples, but no ETEC was detected, likely because ETEC fimbriae confer the species specificity of the pathogen. Resistance to at least one antimicrobial agent was detected in 89.9% of E. coli isolates, with AMR levels being extremely high (>70%) for tetracycline and streptomycin and very high (>50%) for ampicillin and sulfamethoxazole-trimethoprim. Resistance to other critically important antimicrobials such as colistin and extended-spectrum cephalosporins was 2.2% and 1.1%, respectively, and was encoded by the mcr-1 and blaSHV-12 genes. Multidrug resistance was displayed by 80% of the resistant E. coli, and blaSHV-12 gene shared plasmid with other AMR genes. In general, resistance patterns in E. coli from vultures mirrored those found in pigs. Clostridioides difficile was detected in three samples (2.9%); two of them belonged to PCR ribotype 078 and one to PCR ribotype 126, both commonly found in pigs. All C. difficile isolates were characterized by a moderate-to-high level of resistance to fluoroquinolones and macrolides but susceptible to metronidazole or vancomycin, similar to what is usually found in C. difficile isolates from pigs. Thus, vultures may contribute somewhat to the environmental dissemination of some pig pathogens through their acquisition from pig carcasses and, more importantly, of AMR for antibiotics of critical importance for humans. However, the role of vultures would likely be much lesser than that of disposing pig carcasses at the SFS. The monitoring of AMR, and particularly of colistin-resistant and ESBL-producing E. coli, should be considered in pig farms used as sources of carcasses for SFS.

Antimicrobial Susceptibilities and Phylogenetic Analyses of Enterococcus hirae Isolated from Broilers with Valvular Endocarditis.

Enterococcus hirae is a zoonotic Enterococcus species that causes opportunistic infections in both humans and animals and can be transmitted by contact with animals or through contaminated food. The aim of this study was to investigate the importance of E. hirae in broilers with endocarditis, as well as the antimicrobial resistance patterns and genetic relatedness of the isolates. A total of 477 three- to five-week-old broilers were studied during five fattening periods on a farm with mortality due to endocarditis. Endocarditis was observed in 27 chickens (5.66%), and samples were taken for pathological, microbiological, and molecular studies. Lesions were mainly found in the right atrioventricular valve and corresponded with a fibrinous endocarditis. Enterococcus hirae was identified in all cases. Pulsed-field gel electrophoresis results showed clonality among some isolates, with one pulsotype harboring 11 isolates that were found throughout the study. Most of the isolates showed multi-drug-resistant phenotypes. These results confirm that E. hirae is a significant cause of endocarditis in broilers, and suggest that broilers may be important carriers of antimicrobial-resistant E. hirae that might enter into the food chain.

Susceptibilidad antimicrobiana y análisis filogenético de Enterococcus hirae aislados de pollos de engorde con endocarditis valvular. Enterococcus hirae es una especie zoonótica de enterococo que provoca infecciones oportunistas en el hombre y en los animales y que puede transmitirse mediante el contacto con animales o a través de alimentos contaminados. El objetivo de este estudio fue la investigación de la importancia de E. hirae en pollos de engorde con endocarditis, así como el estudio de sus patrones de resistencia antimicrobiana y la relación genética entre los aislados. Se estudiaron 477 pollos de engorde de tres a cinco semanas de edad, durante cinco periodos de engorde, en una granja con historial de muertes por endocarditis. Se detectó endocarditis en 27 pollos (5.66%) y se recolectaron muestras para estudios histopatológicos, microbiológicos y moleculares. Las lesiones se observaron principalmente en la válvula atrioventricular derecha, correspondiendo con una endocarditis fibrinosa. En todos los casos se identificó E. hirae. Mediante electroforesis en gel de campo con pulsaciones se detectó clonalidad en algunos aislados, con once aislados agrupados en un pulsotipo, los cuales fueron detectados a lo largo de todo el estudio. La mayoría de los aislados presentaban fenotipos multirresistentes a varios antibióticos. Estos resultados confirman que E. hirae es una causa importante de endocarditis en pollos de engorde y que estos pueden ser portadores importantes de cepas multirresistentes de E. hirae, las cuales podrían entrar en la cadena alimentaria.

Incidence and characterization of Clostridium difficile in a secondary care hospital in Spain

Introduction: Clostridium difficile (C. difficile) is a major nosocomial infectious agent in hospitals. Previous studies have addressed the high proportion of infection episodes that are overlooked in health care facilities. Objective: the main aim of this study was to characterize C. difficile clinical cases that occurred in a secondary care hospital during a five-month period. Material and methods: for this purpose, a total of 137 stool samples from the same number of patients with diarrhea were analyzed for the presence of C. difficile by culture techniques. An enzyme immunoassay (EIA) test for the detection of C. difficile and its toxins was also used in 50 cases (36.5%) for diagnostic purposes. Results: a total of 14 (10.2%) C. difficile isolates were obtained, of which nine (64.3%) were toxigenic. A mean incidence of 3.2 episodes of C. difficile infections (CDI) per 10,000 patients-days was estimated for the study period. Around 56% of the CDI cases were determined as hospital-acquired, whereas 44% originated in the community. Among these, only two episodes (22.2%) were detected in the hospital by the EIA test, which indicated that the hospital CDI detection protocol needed to be revised. One unusual C. difficile isolate was negative for all toxin genes examined and also for the non-toxigenic strain assay, which highlights the need to perform genome sequencing to study its pathogenicity locus insertion site organization. A stable metronidazole-resistant C. difficile strain and three strains showing multidrug resistance were detected in this study, suggesting that C. difficile antimicrobial susceptibility surveillance programs should be established in this health-care facility

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Incidence and characterization of Clostridium difficile in a secondary care hospital in Spain.

INTRODUCTION: Clostridium difficile (C. difficile) is a major nosocomial infectious agent in hospitals. Previous studies have addressed the high proportion of infection episodes that are overlooked in health care facilities. OBJECTIVE: the main aim of this study was to characterize C. difficile clinical cases that occurred in a secondary care hospital during a five-month period. MATERIAL AND METHODS: for this purpose, a total of 137 stool samples from the same number of patients with diarrhea were analyzed for the presence of C. difficile by culture techniques. An enzyme immunoassay (EIA) test for the detection of C. difficile and its toxins was also used in 50 cases (36.5%) for diagnostic purposes. RESULTS: a total of 14 (10.2%) C. difficile isolates were obtained, of which nine (64.3%) were toxigenic. A mean incidence of 3.2 episodes of C. difficile infections (CDI) per 10,000 patients-days was estimated for the study period. Around 56% of the CDI cases were determined as hospital-acquired, whereas 44% originated in the community. Among these, only two episodes (22.2%) were detected in the hospital by the EIA test, which indicated that the hospital CDI detection protocol needed to be revised. One unusual C. difficile isolate was negative for all toxin genes examined and also for the non-toxigenic strain assay, which highlights the need to perform genome sequencing to study its pathogenicity locus insertion site organization. A stable metronidazole-resistant C. difficile strain and three strains showing multidrug resistance were detected in this study, suggesting that C. difficile antimicrobial susceptibility surveillance programs should be established in this health-care facility.