American crocodiles (Crocodylus acutus: Reptilia: Crocodilidae) visiting the facilities of a freshwater aquaculture of the Northern Pacific region, Costa Rica, carry tetracycline-resistant Escherichia coli.

Apex predators are exposed to antimicrobial compounds and resistant microbes, which accumulate at different trophic levels of the related ecosystems. The study aimed to characterize the presence and the antimicrobial resistance patterns of fecal Escherichia coli isolated from cloacal swab samples obtained from wild-living American crocodiles (Crocodylus acutus) (n = 53). Sampling was conducted within the distinctive context of a freshwater-intensive aquaculture farm in Costa Rica, where incoming crocodiles are temporarily held in captivity before release. Phenotypic antimicrobial susceptibility profiles were determined in all isolates, while resistant isolates were subjected to whole-genome sequencing and bioinformatics analyses. In total, 24 samples contained tetracycline-resistant E. coli (45.3%). Isolates carried either tet(A), tet(B), or tet(C) genes. Furthermore, genes conferring resistance to ß-lactams, aminoglycosides, fosfomycin, sulfonamides, phenicol, quinolones, trimethoprim, and colistin were detected in single isolates, with seven of them carrying these genes on plasmids. Genome sequencing further revealed that sequence types, prevalence of antibiotic resistance carriage, and antibiotic resistance profiles differed between the individuals liberated within the next 24 h after their capture in the ponds and those liberated from enclosures after longer abodes. The overall presence of tetracycline-resistant E. coli, coupled with potential interactions with various anthropogenic factors before arriving at the facilities, hinders clear conclusions on the sources of antimicrobial resistance for the studied individuals. These aspects hold significant implications for both the aquaculture farm's biosecurity and the planning of environmental monitoring programs using such specimens. Considering human-crocodile conflicts from the One Health perspective, the occurrence of antimicrobial resistance underscores the importance of systematical surveillance of antibiotic resistance development in American crocodiles.

Comamonas testosteroni as the cause of mortality in embryonated chicken eggs of breeding broiler hens in Costa Rica.

Mortality of chicken embryos and first-week chickens was reported in a commercial incubator company in Costa Rica. Six 1-day-old Cobb chickens and twenty-four embryonated chicken eggs were examined in the Laboratory of Avian Pathology and the Laboratory of Bacteriology of the National University of Costa Rica. Twelve dead-in-shell embryos showed maceration and were immersed in a putrid, turbid, slightly thick brown liquid. Additionally, the other 12 embryonated eggs had milky yellow-orange content. The livers of those embryos had congestion, haemorrhages and multifocal cream foci of necrosis. Granulocytic infiltration was observed in the bursa of Fabricius, myocardium, liver, lung and kidney. Livers and egg yolks from six embryonated chickens and all 1-day-old chickens were aseptically collected and cultured. In addition, tissues from six better conserved embryos and all 1-day-old chickens were fixed in buffered formalin and embedded in paraffin. Biochemical and molecular tests identified Comamonas testosteroni as the cause of the early, middle and late embryo mortality. As all the eggshells from the sampled embryonated eggs were dirty with soiled a fecal matter, contamination after manipulating the eggs was considered the source of infection. C. testosteroni is an environmental microorganism that has rarely been reported to cause human disease. To our knowledge, this is the first report of C. testosteroni causing mortality in a hatchery. Cleaning and disinfection using ozone were implemented in the hatchery to eliminate the embryo mortality associated with C. testosteroni.

A bvrR/bvrS Non-Polar Brucella abortus Mutant Confirms the Role of the Two-Component System BvrR/BvrS in Virulence and Membrane Integrity.

Brucella abortus is a bacterial pathogen causing bovine brucellosis worldwide. This facultative extracellular-intracellular pathogen can be transmitted to humans, leading to a zoonotic disease. The disease remains a public health concern, particularly in regions where livestock farming is present. The two-component regulatory system BvrR/BvrS was described by isolating the attenuated transposition mutants bvrR::Tn5 and bvrS::Tn5, whose characterization led to the understanding of the role of the system in bacterial survival. However, a phenotypic comparison with deletion mutants has not been performed because their construction has been unsuccessful in brucellae and difficult in phylogenetically related Rhizobiales with BvrR/BvrS orthologs. Here, we used an unmarked gene excision strategy to generate a B. abortus mutant strain lacking both genes, called B. abortus ∆bvrRS. The deletion was verified through PCR, Southern blot, Western blot, Sanger sequencing, and whole-genome sequencing, confirming a clean mutation without further alterations at the genome level. B. abortus ∆bvrRS shared attenuated phenotypic traits with both transposition mutants, confirming the role of BvrR/BvrS in pathogenesis and membrane integrity. This B. abortus ∆bvrRS with a non-antimicrobial marker is an excellent tool for continuing studies on the role of BvrR/BvrS in the B. abortus lifestyle.

If You're Not Confused, You're Not Paying Attention: Ochrobactrum Is Not Brucella.

Bacteria of the genus Brucella are facultative intracellular parasites that cause brucellosis, a severe animal and human disease. Recently, a group of taxonomists merged the brucellae with the primarily free-living, phylogenetically related Ochrobactrum spp. in the genus Brucella. This change, founded only on global genomic analysis and the fortuitous isolation of some opportunistic Ochrobactrum spp. from medically compromised patients, has been automatically included in culture collections and databases. We argue that clinical and environmental microbiologists should not accept this nomenclature, and we advise against its use because (i) it was presented without in-depth phylogenetic analyses and did not consider alternative taxonomic solutions; (ii) it was launched without the input of experts in brucellosis or Ochrobactrum; (iii) it applies a non-consensus genus concept that disregards taxonomically relevant differences in structure, physiology, population structure, core-pangenome assemblies, genome structure, genomic traits, clinical features, treatment, prevention, diagnosis, genus description rules, and, above all, pathogenicity; and (iv) placing these two bacterial groups in the same genus creates risks for veterinarians, medical doctors, clinical laboratories, health authorities, and legislators who deal with brucellosis, a disease that is particularly relevant in low- and middle-income countries. Based on all this information, we urge microbiologists, bacterial collections, genomic databases, journals, and public health boards to keep the Brucella and Ochrobactrum genera separate to avoid further bewilderment and harm.

Virulent Brucella nosferati infecting Desmodus rotundus has emerging potential due to the broad foraging range of its bat host for humans and wild and domestic animals.

Desmodus rotundus, vampire bats, transmit dangerous infections, and brucellosis is a hazardous zoonotic disease, two adversities that coexist in the subtropical and tropical areas of the American continent. Here, we report a 47.89% Brucella infection prevalence in a colony of vampire bats inhabiting the tropical rainforest of Costa Rica. The bacterium induced placentitis and fetal death in bats. Wide-range phenotypic and genotypic characterization placed the Brucella organisms as a new pathogenic species named Brucella nosferati sp. nov., isolated from bat tissues, including the salivary glands, suggesting feeding behavior might favor transmission to their prey. Overall analyses placed B. nosferati as the etiological agent of a reported canine brucellosis case, demonstrating its potential for infecting other hosts. To assess the putative prey hosts, we analyzed the intestinal contents of 14 infected and 23 non-infected bats by proteomics. A total of 54,508 peptides sorted into 7,203 unique peptides corresponding to 1,521 proteins were identified. Twenty-three wildlife and domestic taxa, including humans, were foraged by B. nosferati-infected D. rotundus, suggesting contact of this bacterium with a broad range of hosts. Our approach is appropriate for detecting, in a single study, the prey preferences of vampire bats in a diverse area, demonstrating its suitability for control strategies where vampire bats thrive. IMPORTANCE The discovery that a high proportion of vampire bats in a tropical area is infected with pathogenic Brucella nosferati and that bats forage on humans and many wild and domestic animals is relevant from the perspective of emerging disease prevention. Indeed, bats harboring B. nosferati in their salivary glands may transmit this pathogenic bacterium to other hosts. This potential is not trivial since, besides the demonstrated pathogenicity, this bacterium possesses all the required virulent arsenal of dangerous Brucella organisms, including those that are zoonotic for humans. Our work has settled the basis for future surveillance actions in brucellosis control programs where these infected bats thrive. Moreover, our strategy to identify the foraging range of bats may be adapted for exploring the feeding habits of diverse animals, including arthropod vectors of infectious diseases, and therefore of interest to a broader audience besides experts on Brucella and bats.

Prevalence Estimation, Antimicrobial Susceptibility, and Serotyping of Salmonella enterica Recovered from New World Non-Human Primates (Platyrrhini), Feed, and Environmental Surfaces from Wildlife Centers in Costa Rica.

Concern about zoonoses and wildlife has increased. Few studies described the role of wild mammals and environments in the epidemiology of Salmonella. Antimicrobial resistance is a growing problem associated with Salmonella that threatens global health, food security, the economy, and development in the 21st century. The aim of this study is to estimate the prevalence and identify antibiotic susceptibility profiles and serotypes of non-typhoidal Salmonella enterica recovered from non-human primate feces, feed offered, and surfaces in wildlife centers in Costa Rica. A total of 180 fecal samples, 133 environmental, and 43 feed samples from 10 wildlife centers were evaluated. We recovered Salmonella from 13.9% of feces samples, 11.3% of environmental, and 2.3% of feed samples. Non-susceptibility profiles included six isolates from feces (14.6%): four non-susceptible isolates (9.8%) to ciprofloxacin, one (2.4%) to nitrofurantoin, and one to both ciprofloxacin and nitrofurantoin (2.4%). Regarding the environmental samples, one profile was non-susceptible to ciprofloxacin (2.4%) and two to nitrofurantoin (4.8%). The serotypes identified included Typhimurium/I4,[5],12:i:-, S. Braenderup/Ohio, S. Newport, S. Anatum/Saintpaul, and S. Westhampton. The epidemiological surveillance of Salmonella and antimicrobial resistance can serve in the creation of strategies for the prevention of the disease and its dissemination throughout the One Health approach.

First Report of Campylobacter hepaticus Isolation in Laying Hens and Broiler Breeders with Spotty Liver Disease in Costa Rica.

Poultry producers in Costa Rica have informally reported a spotty liver disease-like syndrome for more than 20 yr. However, despite many attempts, the infectious agent responsible for this syndrome had not been identified. Therefore, following current knowledge of spotty liver disease diagnosis, we invited veterinarians and poultry producers to submit samples to the diagnostic laboratories of the Veterinary Medicine School, Universidad Nacional, to identify the infectious agent of this syndrome. Veterinarians and poultry producers were instructed to collect gallbladders and livers aseptically and send them for pathology examinations and bacterial cultures in less than 24 hr after collection. Samples were processed for standard histopathologic studies and cultured under aerophilic, anaerobic, and microaerophilic conditions. Campylobacter-like colonies were isolated and identified by biochemical and PCR tests. Here we report for the first time the isolation, biochemical characterization, and molecular confirmation of Campylobacter hepaticus in laying hens and broiler breeders with spotty liver disease in Costa Rica.

Nota de investigación- Primer reporte de aislamiento de Campylobacter hepaticus en gallinas de postura y reproductoras pesadas con necrosis hepática focal en Costa Rica. Los productores avícolas en Costa Rica han reportado extraoficialmente un síndrome similar a la necrosis hepática focal durante más de 20 años. Sin embargo, a pesar de muchos intentos, el agente infeccioso responsable de este síndrome no había sido identificado. Por ello, siguiendo los conocimientos actuales relacionados con la necrosis hepática focal, se invitó a los veterinarios y a los productores avícolas a enviar muestras a los laboratorios de diagnóstico de la Facultad de Medicina Veterinaria de la Universidad Nacional, para identificar el agente infeccioso de este síndrome. Se instruyó a los veterinarios y productores avícolas para recolectar vesículas biliares e hígados asépticamente y enviarlos para exámenes patológicos y para cultivos bacterianos en menos de 24 horas después de la recolección. Las muestras se procesaron para estudios histopatológicos estándar y se cultivaron en condiciones aerófilas, anaeróbicas y microaerófilas. Las colonias sugestivas de Campylobacter se aislaron e identificaron mediante pruebas bioquímicas y por PCR. Aquí se reporta por primera vez el aislamiento, caracterización bioquímica y confirmación molecular de Campylobacter hepaticus en gallinas de postura y reproductoras pesadas con la necrosis hepática focal en Costa Rica.

Passive epidemiological surveillance in wildlife in Costa Rica identifies pathogens of zoonotic and conservation importance.

Epidemiological surveillance systems for pathogens in wild species have been proposed as a preventive measure for epidemic events. These systems can minimize the detrimental effects of an outbreak, but most importantly, passive surveillance systems are the best adapted to countries with limited resources. Therefore, this research aimed to evaluate the technical and infrastructural feasibility of establishing this type of scheme in Costa Rica by implementing a pilot program targeting the detection of pathogens of zoonotic and conservation importance in wildlife. Between 2018 and 2020, 85 carcasses of free-ranging vertebrates were admitted for post-mortem and microbiology analysis. However, we encountered obstacles mainly related to the initial identification of cases and limited local logistics capacity. Nevertheless, this epidemiological surveillance scheme allowed us to estimate the general state of health of the country's wildlife by establishing the causes of death according to pathological findings. For instance, 60% (51/85) of the deaths were not directly associated with an infectious agent. Though in 37.6% (32/85) of these cases an infectious agent associated or not with disease was detected. In 27.1% (23/85) of the cases, death was directly related to infectious agents. Furthermore, 12.9% (11/85), the cause of death was not determined. Likewise, this wildlife health monitoring program allowed the detection of relevant pathogens such as Canine Distemper Virus, Klebsiella pneumoniae, Angiostrongylus spp., Baylisascaris spp., among others. Our research demonstrated that this passive surveillance scheme is cost-effective and feasible in countries with limited resources. This passive surveillance can be adapted to the infrastructure dedicated to monitoring diseases in productive animals according to the scope and objectives of monitoring wildlife specific to each region. The information generated from the experience of the initial establishment of a WHMP is critical to meeting the challenges involved in developing this type of scheme in regions with limited resources and established as hotspots for emerging infectious diseases.

Human neutrophils are resistant to Clostridioides difficile toxin B.

OBJECTIVE: The main objective of this study was to evaluate the glucosyltransferase activity of C. difficile TcdB on the activity of human PMNs. METHODS: To better understand the interaction between PMNs and TcdB, PMNs were treated with sub-lethal concentrations of TcdB. We evaluated: (i) the glucosylation of GTPases, (ii) the phagocytic and bactericidal activity, and (iii) PMNs activation (through quantification of TNF-α, IL-8, and expression of CD11b cell surface activation marker). RESULTS: We found that TcdB did not glucosylate RhoA and Rac1 GTPases and did not affect the phagocytic or bactericidal capacity of PMNs. Moreover, TcdB did not increase the production of TNF-α, IL-8, or the expression of activation marker CD11b. The only significant effect of TcdB on PMNs was the partial inhibition of TNF-α and IL-8 production and the diminished expression of CD11b induced by E. coli-LPS. CONCLUSION: Our results show that human PMNs are resistant to TcdB GTPase glucosyltransferase activity against RhoA and Rac1.

Platelet depletion does not alter the course of Brucella abortus infection in vivo.

Brucellosis is a bacterial disease of animals and a zoonotic infection. Thrombocytopenia is a common outcome in long-lasting brucellosis in humans. Likewise, ex vivo experiments have shown that platelets may play a role in Brucella abortus infections. Following these reports, we explored the course of brucellosis in thrombocytopenic mice, using the non-toxic low-molecular-weight aspercetin protein that depletes platelets in vivo. Aspercetin does not induce systemic hemorrhage or inflammation, and when injected into mice, it generates a rapid dose-dependent drop in platelet counts without affecting central organs, disrupting hematological parameters, or the proinflammatory cytokine profile. Compared to the B. abortus infected control group, the infected thrombocytopenic mice did not show significant differences in the hematological profiles, pathological score, spleen, liver histopathology, or bacterial loads. Except for IL-6, which was higher in the infected thrombocytopenic mice, the TNF-α, IFN-γ and IL-10 did not significantly differ with the PBS-infected group. The results indicate that platelets do not play a significant role in modulating Brucella infection in vivo at the early stages of infection, which is commensurate with the stealthy strategy followed by Brucella organisms at the onset of the disease.

Determination of streptomycin and doxycycline using LC/MS towards an effective treatment against an experimental Brucella abortus infection in mice.

Herein we described a versatile liquid chromatographic method for detection and quantification of the total levels of two antimicrobials [i.e., streptomycin (STM) and doxycycline (DOX)], in mice plasma and selected tissues, with the aid of a single quadrupole as a detection method. The method included a few sample preparation steps, including freeze-drying and in situ triphasic solvent-assisted defatting, precipitation, and extraction, allowing easy and fast tissue sample processing and avoiding analyte loss. Using a murine model, we demonstrated that mass spectrometry detects simultaneously and with high specificity two of the most widespread antimicrobials used against Brucellosis. An accurate [recoveries varied from 75.23 (bone marrow) to 101.33% (liver)] and sensitive (LoD in the ng g-1 range) method to assess STM and DOX in murine tissue, including subtherapeutic and therapeutic doses of the antimicrobials, was achieved. This validated method can be successfully used to monitor the depletion of STM and DOX in several mice tissues and plasma during metabolism after administration.

First Report of a Multidrug-Resistant ST58 Escherichia coli Harboring Extended-Spectrum Beta-Lactamase of the CTX-M-1 Class in a Fecal Sample of a Captive Baird's Tapir (Tapirus bairdii) in Costa Rica, Central America.

Objective: This study's main objective was to analyze the antibiotic susceptibility profile of Escherichia coli isolates obtained from a fecal sample of a captive Baird's tapir (Tapirus bairdii) in Costa Rica. Materials and Methods: The fecal sample was collected inside the enclosure on March 3, 2017, right after the animal defecated. Samples were cultured on MacConkey agar plates nonsupplemented and supplemented with 2 µg/mL of cefotaxime. Bacterial identification and antibiotic susceptibility were performed with the Vitek 2 Compact System and the Kirby Bauer disk diffusion method, respectively. Polymerase chain reaction amplification was performed to detect blaCTX-M beta-lactamase genes. Resistant isolates were subjected to whole-genome sequencing (WGS). Results: After evaluating several antibiotic classes, a multidrug-resistant E. coli strain with extended-spectrum beta-lactamase phenotype was isolated. Resistance to cefotaxime, cefepime, ampicillin, ampicillin/sulbactam, and tetracycline was detected. WGS analysis showed the presence of blaCTX-M-1, blaTEM-1B, and tet(B) genes. The presence of IncN plasmids and Col156 was also detected. Conclusion: Our findings are according with the notion that animals' high density enhances the spread of resistant determinants in a captive environment in a limited space, where the likelihood of direct or indirect contact with other animals and humans is more frequent.

Brucella abortus S19 GFP-tagged vaccine allows the serological identification of vaccinated cattle.

Bovine brucellosis induces abortion in cows, produces important economic losses, and causes a widely distributed zoonosis. Its eradication was achieved in several countries after sustained vaccination with the live attenuated Brucella abortus S19 vaccine, in combination with the slaughtering of serologically positive animals. S19 induces antibodies against the smooth lipopolysaccharide (S-LPS), making difficult the differentiation of infected from vaccinated bovines. We developed an S19 strain constitutively expressing the green fluorescent protein (S19-GFP) coded in chromosome II. The S19-GFP displays similar biological characteristics and immunogenic and protective efficacies in mice to the parental S19 strain. S19-GFP can be distinguished from S19 and B. abortus field strains by fluorescence and multiplex PCR. Twenty-five heifers were vaccinated withS19-GFP (5×109 CFU) by the subcutaneous or conjunctival routes and some boosted with GFP seven weeks thereafter. Immunized animals were followed up for over three years and tested for anti-S-LPS antibodies by both the Rose Bengal test and a competitive ELISA. Anti-GFP antibodies were detected by an indirect ELISA and Western blotting. In most cases, anti-S-LPS antibodies preceded for several weeks those against GFP. The anti-GFP antibody response was higher in the GFP boosted than in the non-boosted animals. In all cases, the anti-GFP antibodies persisted longer, or at least as long, as those against S-LPS. The drawbacks and potential advantages of using the S19-GFP vaccine for identifying vaccinated animals in infected environments are discussed.

Effect of the use of probiotic Bacillus subtilis (QST 713) as a growth promoter in broilers: an alternative to bacitracin methylene disalicylate.

The global poultry trend toward the more responsible use of antibiotics is becoming recurrent and has demanded the need to generate new natural alternatives. Probiotics have gained importance as an option to use as growth promoters. This study aimed to evaluate Bacillus subtillis QST713 as a substitute for an antibiotic growth promoter (BMD). A total of 150 male broilers were assigned to three dietary treatments: 1) control diet (CO), 2) control diet + 500 g/t of BMD (AGP), and 3) control diet + 100 g/t of B. subtilis QST713 (PB), respectively. Each treatment was monitored for 5 wk for the productive variables: body weight, accumulated feed consumption, food conversion, and European efficiency factor. At the end of each week, fresh fecal samples were cultured and quantified for E. coli, Enterococcus spp., and Lactobacillus spp. At the end of the trial, blood samples were analyzed for hemogram and intestinal samples (anterior portion) for histomorphometry. The data were statistically analyzed with an analysis of variance and subjected to a least significant difference test (Tukey). The zootechnical yields were similar in the AGP and PB groups (P ˃ 0.05); both superior to the control group. In the hematological profiles, no difference was observed between the experimental groups. E. coli and Enterococcus counts were significantly lower (P ˂ 0.05), and Lactobacillus counts were significantly (P ˂ 0.05) higher in the PB group, relative to CO and AGP groups. No differences (P ˃ 0.05) were found in bacterial counts between the CO and AGP groups. The intestinal mucosa and villi in the PB group were significantly (P ˂ 0.05) longer and with less deeper crypts than CO and AGP groups. We conclude that B. subtillis QST713, used at the suggested commercial dose (100 g/ton), is an effective growth-promoting alternative to BMD that modulates the microbiota and intestinal architecture, thus producing zootechnical yields consistent with BMD.

A Sinorhizobium meliloti and Agrobacterium tumefaciens ExoR ortholog is not crucial for Brucella abortus virulence.

Brucella is a facultative extracellular-intracellular pathogen that belongs to the Alphaproteobacteria class. Precise sensing of environmental changes and a proper response mediated by a gene expression regulatory network are essential for this pathogen to survive. The plant-related Alphaproteobacteria Sinorhizobium meliloti and Agrobacterium tumefaciens also alternate from a free to a host-associated life, where a regulatory invasion switch is needed for this transition. This switch is composed of a two-component regulatory system (TCS) and a global inhibitor, ExoR. In B. abortus, the BvrR/BvrS TCS is essential for intracellular survival. However, the presence of a TCS inhibitor, such as ExoR, in Brucella is still unknown. In this work, we identified a genomic sequence similar to S. meliloti exoR in the B. abortus 2308W genome, constructed an exoR mutant strain, and performed its characterization through ex vivo and in vivo assays. Our findings indicate that ExoR is related to the BvrR phosphorylation state, and is related to the expression of known BvrR/BrvS gene targets, such as virB8, vjbR, and omp25 when grown in rich medium or starving conditions. Despite this, the exoR mutant strain showed no significant differences as compared to the wild-type strain, related to resistance to polymyxin B or human non-immune serum, intracellular replication, or infectivity in a mice model. ExoR in B. abortus is related to BvrR/BvrS as observed in other Rhizobiales; however, its function seems different from that observed for its orthologs described in A. tumefaciens and S. meliloti.

Canine brucellosis in Costa Rica reveals widespread Brucella canis infection and the recent introduction of foreign strains.

Brucellosis is a prevalent disease in Costa Rica (CR), with an increasing number of human infections. Close to half of homes in CR have one or more dogs, corresponding to ∼1.4 million canines, most of them in the Central Valley within or near the cities of San José, Heredia, and Alajuela. From 302 dog sera collected from this region, 19 were positive for Brucella canis antigens, and five had antibodies against smooth lipopolysaccharide, suggesting infections by both B. canis and other Brucella species. B. canis strains were isolated in the Central Valley from 26 kennel dogs and three pet dogs, all displaying clinical signs of canine brucellosis. We detected three recent introductions of different B. canis strains in kennels: two traced from Mexico and one from Panama. Multiple locus-variable number tandem repeats (MLVA-16) and whole-genome sequencing (WGSA) analyses showed that B. canis CR strains comprise three main lineages. The tree topologies obtained by WGSA and MLVA-16 just partially agreed, indicating that the latter analysis is not suitable for phylogenetic studies. The fatty acid methyl ester analysis resolved five different B. canis groups, showing less resolution power than the MLVA-16 and WGSA. Lactobacillic acid was absent in linages I and II but present in linage III, supporting the recent introductions of B. canis strains from Mexico. B. canis displaying putative functional cyclopropane synthase for the synthesis of lactobacillic acid are phylogenetically intertwined with B. canis with non-functional protein, indicating that mutations have occurred independently in the various lineages.

Multidrug-Resistant CTX-M and CMY-2 Producing Escherichia coli Isolated from Healthy Household Dogs from the Great Metropolitan Area, Costa Rica.

Objective: This study aimed to determine the prevalence of fecal carriage of antibiotic-resistant Escherichia coli of healthy household dogs with an emphasis on extended-spectrum ß-lactamases (ESBL), AmpC-type ß-lactamases and resistance to quinolones. Materials and Methods: Rectal swabs were collected from 74 dogs without any clinical evidence of gastrointestinal disease. Samples were cultured on MacConkey agar plates and MacConkey supplemented with 2 µg/mL cefotaxime or 5 µg/mL ciprofloxacin. Isolates were identified with Vitek 2 Compact and susceptibility testing performed by Kirby Bauer disk diffusion method. Minimal inhibitory concentration (MIC) was done on isolates resistant to cefotaxime, ciprofloxacin, and nalidixic acid. PCR amplification was performed to detect CTX-M and CMY-2. Isolates positive for CTX-M and/or CMY-2 were selected for whole-genome sequencing. Results: Multiresistance was detected in 56% of the isolates. A high percentage of resistance was detected for cefazolin (63%), ampicillin (54%), streptomycin (49%), nalidixic acid (42%) and tetracycline (38%). The MIC50 and MIC90 for isolates resistant to cefotaxime (24%) was determined as 16 and >250 µg/mL, respectively; for ciprofloxacin (18%), 125 and 250 µg/mL, respectively. ESBL (CTX-M type) and AmpC (CMY-2 type) were detected in 6 (7.1%) and 14 (19%) of the isolates, respectively. Whole-genome sequence analysis showed high genetic diversity in most of the isolates and a large variety of resistance mechanisms, including mobile genetic elements. Conclusion: The frequency of multidrug-resistant E. coli is worrying, mainly because of the presence of many isolates producing ESBL and AmpC ß-lactamases. Based on the "One Health" concept, considering the relationships between animals, humans, and the environment, these data support the notion that companion animals are important reservoirs of multidrug-resistant bacteria.

The Role of Neutrophils in Brucellosis.

Brucellosis is a bacterial disease of domestic animals and humans. The pathogenic ability of Brucella organisms relies on their stealthy strategy and their capacity to replicate within host cells and to induce long-lasting infections. Brucella organisms barely induce neutrophil activation and survive within these leukocytes by resisting microbicidal mechanisms. Very few Brucella-infected neutrophils are found in the target organs, except for the bone marrow, early in infection. Still, Brucella induces a mild reactive oxygen species formation and, through its lipopolysaccharide, promotes the premature death of neutrophils, which release chemokines and express "eat me" signals. This effect drives the phagocytosis of infected neutrophils by mononuclear cells that become thoroughly susceptible to Brucella replication and vehicles for bacterial dispersion. The premature death of the infected neutrophils proceeds without NETosis, necrosis/oncosis, or classical apoptosis morphology. In the absence of neutrophils, the Th1 response exacerbates and promotes bacterial removal, indicating that Brucella-infected neutrophils dampen adaptive immunity. This modulatory effect opens a window for bacterial dispersion in host tissues before adaptive immunity becomes fully activated. However, the hyperactivation of immunity is not without a price, since neutropenic Brucella-infected animals develop cachexia in the early phases of the disease. The delay in the immunological response seems a sine qua non requirement for the development of long-lasting brucellosis. This property may be shared with other pathogenic alphaproteobacteria closely related to Brucella We propose a model in which Brucella-infected polymorphonuclear neutrophils (PMNs) function as "Trojan horse" vehicles for bacterial dispersal and as modulators of the Th1 adaptive immunity in infection.

Persistence of Brucella abortus lineages revealed by genomic characterization and phylodynamic analysis.

Brucellosis, caused by Brucella abortus, is a major disease of cattle and humans worldwide distributed. Eradication and control of the disease has been difficult in Central and South America, Central Asia, the Mediterranean and the Middle East. Epidemiological strategies combined with phylogenetic methods provide the high-resolution power needed to study relationships between surveillance data and pathogen population dynamics, using genetic diversity and spatiotemporal distributions. This information is crucial for prevention and control of disease spreading at a local and worldwide level. In Costa Rica (CR), the disease was first reported at the beginning of the 20th century and has not been controlled despite many efforts. We characterized 188 B. abortus isolates from CR recovered from cattle, humans and water buffalo, from 2003 to 2018, and whole genome sequencing (WGS) was performed in 95 of them. They were also assessed based on geographic origin, date of introduction, and phylogenetic associations in a worldwide and national context. Our results show circulation of five B. abortus lineages (I to V) in CR, phylogenetically related to isolates from the United States, United Kingdom, and South America. Lineage I was dominant and probably introduced at the end of the 19th century. Lineage II, represented by a single isolate from a water buffalo, clustered with a Colombian sample, and was likely introduced after 1845. Lineages III and IV were likely introduced during the early 2000s. Fourteen isolates from humans were found within the same lineage (lineage I) regardless of their geographic origin within the country. The main CR lineages, introduced more than 100 years ago, are widely spread throughout the country, in contrast to new introductions that seemed to be more geographically restricted. Following the brucellosis prevalence and the farming practices of several middle- and low-income countries, similar scenarios could be found in other regions worldwide.

Salmonellosis detection and evidence of antibiotic resistance in an urban raccoon population in a highly populated area, Costa Rica.

Wild animals are involved in zoonotic disease transmission cycles. These are generally complex and poorly understood, especially among animals adapted to life in human ecosystems. Raccoons are reservoirs and effective carriers for infectious agents such as Salmonella throughout different environments and contribute to the transference of resistance genes. This study examined the presence of circulating Salmonella sp. in a population of raccoons in a tropical urban environment and evaluated resistance to antibiotics commonly used to treat salmonellosis. A total of 97 raccoons of different ages and sex were included in this study. 49% (38-60 CI) of the faecal samples were positive for Salmonella spp. The study identified 15 circulating serovars with the most prevalent being S. Hartford (7/15), S. Typhimurium (4/15) and S. Bovismorbificans (4/15). These serovars correspond to the serovars detected in humans with clinical symptoms in Costa Rica. 9.5% of the Salmonella strains recovered demonstrated ciprofloxacin resistance, and 7.1% showed resistance to nalidixic acid. This study provides evidence of multiple Salmonella serovars circulating in a population of urban raccoons in Costa Rica. Furthermore, the study confirms the existence of antimicrobial resistance to two antibiotics used to treat human salmonellosis. The findings emphasize the role of the raccoon as a reservoir of Salmonella in the Greater Metropolitan Area of Costa Rica (GAM) and stress the need for active monitoring of the presence and possible spread in antibiotic resistance due to this peri-domestic carnivore.