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Simple Summary: Rhodococcus equi infection causes life-threatening bacterial pneumonia in foals, resulting in significant economic losses to equine farms. Due to the lack of an effective vaccine, infected foals receive intensive antibiotic treatment. However, the emergence of antibiotic-resistant bacteria in recent years has become problematic. Thus, appropriate use of antibiotics is required. Horses that die from R. equi infection tend to show specific patterns of IgG subtype responses. Therefore, this study focused on qualitative differences in antibody responses to R. equi between resistant and susceptible foals as a means of predicting R. equi susceptibility. These findings may be useful in predicting which foals require treatment. Rhodococcus equi is an intracellular bacterium that causes suppurative pneumonia in foals. T-helper (Th) 1 cells play an important role in the protective response against R. equi. In mice and humans, the directionality of IgG switching reflects the polarization of Th-cell responses, but this has not been fully elucidated in horses. In this 4-year study, we classified R. equi-infected foals into surviving and non-surviving group and investigated differences in IgG subclass response to virulence-associated protein A, the main virulence factor of R. equi, between the groups. IgGa, IgGb, and IgG(T) titers were significantly higher in the non-surviving group compared with the surviving group. The titers of IgGa and IgG(T), IgGb and IgG(T), and IgGa and IgGb, respectively, were positively correlated, and the IgG(T)/IgGb ratio in the non-surviving group was significantly higher than that in the surviving group. The IgG(T) titer tended to increase more than the IgGa and IgGb titers in the non-surviving group compared with the surviving group. Our findings suggest that the IgG(T) bias in IgG subclass responses reflects the immune status, which exacerbates R. equi infection. [ABSTRACT FROM AUTHOR]

Background: Rhodococcus equi (R. equi) is a Gram-positive zoonotic pathogen that frequently leads to illness and death in young horses (foals). This study presents the complete genome sequence of R. equi strain BJ13, which was isolated from a thoroughbred racehorse breeding farm in Beijing, China. Results: The BJ13 genome has a length of 5.30 Mb and consists of a complete chromosome and a plasmid measuring 5.22 Mb and 0.08 Mb, respectively. We predicted 4,929 coding gene open reading frames, along with 52 tRNAs and 12 rRNAs. Through analysis of mobile genetic elements, we identified 6 gene islands and 1 prophage gene. Pathogenic system analysis predicted the presence of 418 virulence factors and 225 drug resistance genes. Secretion system analysis revealed the prediction of 297 secreted proteins and 1,106 transmembrane proteins. BJ13 exhibits genomic features, virulence-associated genes, potential drug resistance, and a virulence plasmid structure that may contribute to the evolution of its pathogenicity. Lastly, the pathogenicity of the isolated strain was assessed through animal experiments, which resulted in inflammatory reactions or damage in the lungs, liver, and spleen of mice. Moreover, by the 7th day post-infection, the mortality rate of the mice reached 50.0%, indicating complex immune regulatory mechanisms, including overexpression of IL-10 and increased production of pro-inflammatory cytokines like TNF-α. These findings validate the strong pathogenicity of the isolated strain and provide insights for studying the pathogenic mechanisms of Rhodococcus equi infection. Conclusions: The complete genome sequence of R. equi strain BJ13 provides valuable insights into its genomic characteristics, virulence potential, drug resistance, and secretion systems. The strong pathogenicity observed in animal experiments underscores the need for further investigation into the pathogenic mechanisms of R. equi infection. [ABSTRACT FROM AUTHOR]

Abstract Background Rhodococcus equi (R. equi) is a Gram-positive zoonotic pathogen that frequently leads to illness and death in young horses (foals). This study presents the complete genome sequence of R. equi strain BJ13, which was isolated from a thoroughbred racehorse breeding farm in Beijing, China. Results The BJ13 genome has a length of 5.30 Mb and consists of a complete chromosome and a plasmid measuring 5.22 Mb and 0.08 Mb, respectively. We predicted 4,929 coding gene open reading frames, along with 52 tRNAs and 12 rRNAs. Through analysis of mobile genetic elements, we identified 6 gene islands and 1 prophage gene. Pathogenic system analysis predicted the presence of 418 virulence factors and 225 drug resistance genes. Secretion system analysis revealed the prediction of 297 secreted proteins and 1,106 transmembrane proteins. BJ13 exhibits genomic features, virulence-associated genes, potential drug resistance, and a virulence plasmid structure that may contribute to the evolution of its pathogenicity. Lastly, the pathogenicity of the isolated strain was assessed through animal experiments, which resulted in inflammatory reactions or damage in the lungs, liver, and spleen of mice. Moreover, by the 7th day post-infection, the mortality rate of the mice reached 50.0%, indicating complex immune regulatory mechanisms, including overexpression of IL-10 and increased production of pro-inflammatory cytokines like TNF-α. These findings validate the strong pathogenicity of the isolated strain and provide insights for studying the pathogenic mechanisms of Rhodococcus equi infection. Conclusions The complete genome sequence of R. equi strain BJ13 provides valuable insights into its genomic characteristics, virulence potential, drug resistance, and secretion systems. The strong pathogenicity observed in animal experiments underscores the need for further investigation into the pathogenic mechanisms of R. equi infection.

Abstract Rhodococcus equi (R. equi) is a zoonotic opportunistic pathogen that mainly causes fatal lung and extrapulmonary abscesses in foals and immunocompromised individuals. To date, no commercial vaccine against R. equi exists. We previously screened all potential vaccine candidates from the complete genome of R. equi using a reverse vaccinology approach. Five of these candidates, namely ABC transporter substrate-binding protein (ABC transporter), penicillin-binding protein 2 (PBD2), NlpC/P60 family protein (NlpC/P60), esterase family protein (Esterase), and M23 family metallopeptidase (M23) were selected for the evaluation of immunogenicity and immunoprotective effects in BALB/c mice model challenged with R. equi. The results showed that all five vaccine candidate-immunized mice experienced a significant increase in spleen antigen-specific IFN-γ- and TNF-α-positive CD4 + and CD8 + T lymphocytes and generated robust Th1- and Th2-type immune responses and antibody responses. Two weeks after the R. equi challenge, immunization with the five vaccine candidates reduced the bacterial load in the lungs and improved the pathological damage to the lungs and livers compared with those in the control group. NlpC/P60, Esterase, and M23 were more effective than the ABC transporter and PBD2 in inducing protective immunity against R. equi challenge in mice. In addition, these vaccine candidates have the potential to induce T lymphocyte memory immune responses in mice. In summary, these antigens are effective candidates for the development of protective vaccines against R. equi. The R. equi antigen library has been expanded and provides new ideas for the development of multivalent vaccines.

Rhodococcus equi, predominantly recognized as an opportunistic pathogen affecting immunocompromised hosts, and Brucella, a widespread zoonotic bacterium, infrequently co-infect immunocompetent adults, thereby posing a distinctive diagnostic challenge. Here, we describe a case involving a 53-year-old male with a history of goat farming, who presented with persistent chest tightness, cough, and notable weight loss, absent fever. Radiological and bronchoscopic assessments showed a right hilar mass, extensive vertebral destruction, and bronchial lesions, deviating from the typical symptoms associated with either pathogen. Laboratory analyses confirmed a co-infection involving R. equi and Brucella. Initial therapy with levofloxacin and vancomycin proved ineffective; however, a subsequent treatment regimen comprising azithromycin, etimicin, minocycline, and moxifloxacin resulted in substantial clinical improvement. This case accentuates the intricacies involved in diagnosing and managing atypical co-infections in immunocompetent individuals and underscores the importance of careful microbiological testing to inform effective therapeutic strategies.

Rhodococcus equi (R. equi) is a zoonotic opportunistic pathogen that mainly causes fatal lung and extrapulmonary abscesses in foals and immunocompromised individuals. To date, no commercial vaccine against R. equi exists. We previously screened all potential vaccine candidates from the complete genome of R. equi using a reverse vaccinology approach. Five of these candidates, namely ABC transporter substrate-binding protein (ABC transporter), penicillin-binding protein 2 (PBD2), NlpC/P60 family protein (NlpC/P60), esterase family protein (Esterase), and M23 family metallopeptidase (M23) were selected for the evaluation of immunogenicity and immunoprotective effects in BALB/c mice model challenged with R. equi. The results showed that all five vaccine candidate-immunized mice experienced a significant increase in spleen antigen-specific IFN-γ- and TNF-α-positive CD4 + and CD8 + T lymphocytes and generated robust Th1- and Th2-type immune responses and antibody responses. Two weeks after the R. equi challenge, immunization with the five vaccine candidates reduced the bacterial load in the lungs and improved the pathological damage to the lungs and livers compared with those in the control group. NlpC/P60, Esterase, and M23 were more effective than the ABC transporter and PBD2 in inducing protective immunity against R. equi challenge in mice. In addition, these vaccine candidates have the potential to induce T lymphocyte memory immune responses in mice. In summary, these antigens are effective candidates for the development of protective vaccines against R. equi. The R. equi antigen library has been expanded and provides new ideas for the development of multivalent vaccines. [ABSTRACT FROM AUTHOR]

To investigate the etiological role of vapB-positive Rhodococcus equi in pigs, R. equi was isolated from the submaxillary lymph nodes with or without macroscopically detectable lesions of apparently healthy growing-finishing pigs at a slaughterhouse in Toyama Prefecture, Japan. R. equi was isolated from 57 (24.6%) of 232 pigs with macroscopically detectable lymph node lesions, and 56 (98.2%) of the 57 isolates were vapB-positive. R. equi was isolated from 10 (2.4%) of 420 pigs without lymph node lesions, and six (60%) of the 10 isolates were vapB-positive. Plasmid DNA was isolated from the 62 vapB-positive isolates and digested with EcoRI and NsiI to obtain the plasmid profile. Fifty-two (83.9%), three (4.8%), and four (6.5%) isolates contained pVAPB subtypes 1, 2, and 3, respectively, while the remaining three isolates were of pVAPB subtypes 9, 13, and 14, respectively. Twelve specimens from lymph nodes with macroscopically detectable lesions were randomly selected for histopathological staining. Granulomatous lesions resembling tuberculosis were found in 11 of the 12 specimens, and the remaining specimen showed typical foci of malakoplakia in the lymph node. The isolation rates of R. equi and vapB-positive R. equi from lymph nodes with macroscopically detectable lesions were significantly higher (P<0.05) than those of lymph nodes without lesions, suggesting an etiologic association between vapB-positive R. equi and macroscopically detectable granulomatous lesions in porcine submaxillary lymph nodes. Previous reports on the prevalence of vapB-positive R. equi in pigs are reviewed and discussed. [ABSTRACT FROM AUTHOR]

OBJECTIVE Design and evaluate immune responses of neonatal foals to a mRNA vaccine expressing the virulence-associated protein A (VapA) of Rhodococcus equi. ANIMALS Cultured primary equine respiratory tract cells; Serum, bronchoalveolar lavage fluid (BALF), and peripheral blood mononuclear cells (PBMCs) from 30 healthy Quarter Horse foals. METHODS VapA expression was evaluated by western immunoblot in cultured equine bronchial cells transfected with 4 mRNA constructs encoding VapA. The mRNA construct with greatest expression was used to immunize foals at ages 2 and 21 days in 5 groups: (1) 300 μg nebulized mRNA (n = 6); (2) 600 μg nebulized mRNA (n = 4); (3) 300 μg mRNA administered intramuscularly (IM) (n = 5); (4) 300 μg VapA IM (positive controls; n = 6); or (5) nebulized water (negative controls; n = 6). Serum, BALF, and PBMCs were collected at ages 3, 22, and 35 days and tested for relative anti-VapA IgG1, IgG4/7, and IgA activities using ELISA and cell-mediated immunity by ELISpot. RESULTS As formulated, nebulized mRNA was not immunogenic. However, a significant increase in anti-VapA IgG4/7 activity (P < .05) was noted exclusively in foals immunized IM with VapA mRNA by age 35 days. The proportion of foals with anti-VapA IgG1 activity > 30% of positive control differed significantly (P = .0441) between negative controls (50%; 3/6), IM mRNA foals (100%; 5/5), and IM VapA (100%; 6/6) groups. Natural exposure to virulent R equi was immunogenic in some negative control foals. CLINICAL RELEVANCE Further evaluation of the immunogenicity and efficacy of IM mRNA encoding VapA in foals is warranted. [ABSTRACT FROM AUTHOR]

Simple Summary: The role of the environment as a reservoir and source of antimicrobial resistance (AMR) is not fully understood. In this report, we explore the consequences of historical antimicrobial use in horse-breeding farms on the accumulation of antimicrobial residues in the environment and its impact on Rhodococcus equi populations carrying antimicrobial resistance genes (AMRGs). Understanding these relationships is important because the environment is the main source of R. equi infections of young horses. Moreover, the higher concentration of transferable AMRGs in the soil contributes to the emergence of pathogenic organisms carrying multidrug resistance that can affect humans and animals. This report reinforces how farm practices can have One Health impacts and suggests future research to investigate ways to mitigate the spread of AMR. Antimicrobial residues excreted in the environment following antimicrobial treatment enhance resistant microbial communities in the environment and have long-term effects on the selection and maintenance of antimicrobial resistance genes (AMRGs). In this study, we focused on understanding the impact of antimicrobial use on antimicrobial residue pollution and antimicrobial resistance (AMR) in the environment of horse-breeding farms. Rhodococcus equi is an ideal microbe to study these associations because it lives naturally in the soil, exchanges AMRGs with other bacteria in the environment, and can cause disease in animals and humans. The environment is the main source of R. equi infections in foals; therefore, higher levels of multidrug-resistant (MDR) R. equi in the environment contribute to clinical infections with MDR R. equi. We found that macrolide residues in the environment of horse-breeding farms and the use of thoracic ultrasonographic screening (TUS) for early detection of subclinically affected foals with R. equi infections were strongly associated with the presence of R. equi carrying AMRGs in the soil. Our findings indicate that the use of TUS contributed to historically higher antimicrobial use in foals, leading to the accumulation of antimicrobial residues in the environment and enhancing MDR R. equi. [ABSTRACT FROM AUTHOR]

Abstract Background Malakoplakia is a rare chronic granulomatous disease characterized by the presence of Michaelis-Gutmann bodies (MGBs) within histiocytic aggregates. It predominantly affects immunocompromised individuals, including those living with Human Immunodeficiency Virus (HIV). Case Presentation We present a unique case of bifocal malakoplakia in a 49-year-old man, previously with Coronavirus disease 2019 (COVID-19) and HIV positive, presented with respiratory symptoms, weight loss, and lymphadenopathy. He had various infections including Non-Tuberculous Mycobacteria (NTM), Cytomegalovirus (CMV), and Candida, with evolving lung and gastrointestinal issues. Despite treatment attempts, he deteriorated due to respiratory distress, multi-organ failure, and coagulopathy, leading to his unfortunate demise. Conclusion This report presents a distinctive and complex case of malakoplakia in an HIV-positive patient, a rare inflammatory disorder originally described by Michaelis and Gutmann in 1902. The hallmark Michaelis-Gutmann organisms were observed, confirming the diagnosis. While typically affecting the urinary tract, this case demonstrates the exceptional ability of malakoplakia to manifest in various organ systems, including pulmonary, gastrointestinal, and more. Although Escherichia coli is a prevalent associated pathogen, the exact cause remains elusive. Treatment, often involving surgical excision and antibiotic therapy, underscores the challenging nature of managing this condition in immunocompromised individuals.

Rhodococcus equi is an intracellular bacterium that causes suppurative pneumonia in foals. T-helper (Th) 1 cells play an important role in the protective response against R. equi. In mice and humans, the directionality of IgG switching reflects the polarization of Th-cell responses, but this has not been fully elucidated in horses. In this 4-year study, we classified R. equi-infected foals into surviving and non-surviving group and investigated differences in IgG subclass response to virulence-associated protein A, the main virulence factor of R. equi, between the groups. IgGa, IgGb, and IgG(T) titers were significantly higher in the non-surviving group compared with the surviving group. The titers of IgGa and IgG(T), IgGb and IgG(T), and IgGa and IgGb, respectively, were positively correlated, and the IgG(T)/IgGb ratio in the non-surviving group was significantly higher than that in the surviving group. The IgG(T) titer tended to increase more than the IgGa and IgGb titers in the non-surviving group compared with the surviving group. Our findings suggest that the IgG(T) bias in IgG subclass responses reflects the immune status, which exacerbates R. equi infection.

Background: Malakoplakia is a rare chronic granulomatous disease characterized by the presence of Michaelis-Gutmann bodies (MGBs) within histiocytic aggregates. It predominantly affects immunocompromised individuals, including those living with Human Immunodeficiency Virus (HIV). Case Presentation: We present a unique case of bifocal malakoplakia in a 49-year-old man, previously with Coronavirus disease 2019 (COVID-19) and HIV positive, presented with respiratory symptoms, weight loss, and lymphadenopathy. He had various infections including Non-Tuberculous Mycobacteria (NTM), Cytomegalovirus (CMV), and Candida, with evolving lung and gastrointestinal issues. Despite treatment attempts, he deteriorated due to respiratory distress, multi-organ failure, and coagulopathy, leading to his unfortunate demise. Conclusion: This report presents a distinctive and complex case of malakoplakia in an HIV-positive patient, a rare inflammatory disorder originally described by Michaelis and Gutmann in 1902. The hallmark Michaelis-Gutmann organisms were observed, confirming the diagnosis. While typically affecting the urinary tract, this case demonstrates the exceptional ability of malakoplakia to manifest in various organ systems, including pulmonary, gastrointestinal, and more. Although Escherichia coli is a prevalent associated pathogen, the exact cause remains elusive. Treatment, often involving surgical excision and antibiotic therapy, underscores the challenging nature of managing this condition in immunocompromised individuals. [ABSTRACT FROM AUTHOR]

Forty-five foals with Rhodococcus equi infection and pneumonia symptoms were classified into a surviving group and a dead group. Using serum samples, the oxidative stress index (OSI) was determined at the first visit and the follow-up visit. The OSI of the surviving group was significantly lower at the follow-up than that at the first visit. No significant difference was observed between the OSI of the dead group at the first and follow-up visits. In the surviving group, treatment at the first visit mitigated inflammation and reduced OSI. However, in the dead group, poor response to the treatment provided at the first visit led to continued inflammation, and no change was observed the OSI. [ABSTRACT FROM AUTHOR]

Decades of antimicrobial overuse to treat respiratory disease in foals have promoted the emergence and spread of zoonotic multidrug-resistant (MDR) Rhodococcus equi worldwide. Three main R. equi MDR clonal populations—2287, G2106, and G2017—have been identified so far. However, only clones 2287 and G2016 have been isolated from sick animals, with clone 2287 being the main MDR R. equi recovered. The genetic mechanisms that make this MDR clone superior to the others at infecting foals are still unknown. Here, we performed a deep genetic characterization of the accessory genomes of 207 R. equi isolates, and we describe IME2287, a novel genetic element in the accessory genome of clone 2287, potentially involved in the maintenance and spread of this MDR population over time. IME2287 is a putative self-replicative integrative mobilizable element (IME) carrying a DNA replication and partitioning operon and genes encoding its excision and integration from the R. equi genome via a serine recombinase. Additionally, IME2287 encodes a protein containing a Toll/interleukin-1 receptor (TIR) domain that may inhibit TLR-mediated NF-kB signaling in the host and a toxin–antitoxin (TA) system, whose orthologs have been associated with antibiotic resistance/tolerance, virulence, pathogenicity islands, bacterial persistence, and pathogen trafficking. This new set of genes may explain the success of clone 2287 over the other MDR R. equi clones. [ABSTRACT FROM AUTHOR]

Background: Rhodococcus equi is a Gram-positive microorganism that causes infections, particularly in immunocompromised patients. Treatment duration can be prolonged. While vancomycin is an effective drug in this scenario, its use may lead to renal damage. Studies have shown that continuous vancomycin infusion appears to be a safe strategy in terms of adverse effects compared to bolus administration. Case description: We present the case of a 71-year-old female liver transplant recipient. After being diagnosed with a mediastinal infection caused by Rhodococcus equi with poor response to initial therapy, she required 12 months of continuous intravenous domiciliary infusion of vancomycin combined with oral levofloxacin and rifampicin. There was no drug-related complication throughout the follow-up. Conclusions: The use of continuous vancomycin infusion has emerged as a safer, more efficient, and cost-effective alternative to intermittent administration. We want to emphasise the uniqueness of this case, where despite the unprecedented treatment duration, no adverse effects occurred.

ABSTRACT Since 2015, virulence-associated protein N (vapN)-positive Rhodococcus equi has been isolated from pyogenic lesions in ruminants in multiple countries, including Japan, supporting the widespread distribution of this pathogen in ruminants, and suggesting that VapN is responsible for the pathogenicity of this infection. However, no immunological diagnostic method has been established for this infection. In this study, we attempted to produce anti-VapN monoclonal antibodies and apply them to immunostaining methods. Mice were immunized intraperitoneally or subcutaneously with 5 µg of recombinant VapN (rVapN). After cell fusion and cloning by limiting dilution, we generated three anti-VapN antibody-producing hybridomas (4H4, 5C3, and 5G10). Next, VapN-based immunostaining was performed using a mouse model inoculated with rVapN, the JCM94-3 (VapN-producing) strain, or the JID03-27 (low-producing) strain. Positive reactions were observed in necrotic lesions in the liver and spleen, especially in the rVapN-treated and JCM94-3-treated groups. Immunostaining of goats (n = 2) and cattle (n = 3) presumed to be infected with vapN-positive R. equi was also positive in organs carrying necrotic lesions including the lungs, lymph nodes, abomasum, and ribs. The reactivity of immunostaining differed among the antibodies, with the 4H4 antibody exhibiting the best reactivity and allowing detection at low concentrations. However, the reactivity of the other two antibodies was improved by antigen retrieval treatment. Several known diseases cause necrotizing granuloma formation, and the immunological diagnostic method established in this study can be used to distinguish R. equi infection from similar diseases or identify infection in previously missed cases. IMPORTANCE Rhodococcus equi can cause infection in ruminants, and its pathogenicity is suggested to be associated with VapN. Despite its wide distribution, no immunological diagnostic method has been developed for VapN-producing R. equi. Against this background, we attempted to develop monoclonal antibodies targeting VapN and assess their application in immunostaining. In the study, mice were immunized with recombinant VapN, and cell fusion and cloning by limiting dilution permitted the generation of three antibody-producing hybridomas. The utility of the antibodies produced from the hybridomas in immunostaining was demonstrated using an infected mouse model, and the antibodies were further applied to previously reported cases of R. equi infection in goats and cattle. Although the 4H4 antibody induced the strongest reactions, the reactivity of two other antibodies was improved by antigen retrieval. Our monoclonal antibodies will be utilized to support the definitive diagnosis of suspected R. equi infection, including cases that were previously missed.

Rhodococcus equi is a Gram-positive coccobacillus that falls within the category of aerobic actinomycetes. The Rhodococcus genus belongs to the nocardioform bacteria group. This microorganism has been found in various settings, including natural environments, animals, and particularly in individuals with compromised immune systems, such as those living with HIV. Notably, there is an increasing number of reports concerning R. equi infections in transplant recipients and even individuals with a normally functioning immune system.Traditionally, R. equi has been primarily associated with pulmonary infections, but there is a growing body of evidence documenting its involvement in extrapulmonary infections. In this report, we present a case involving a newly diagnosed HIV patient who experienced R. equi -induced necrotizing pneumonia, bacteremia, and a brain abscess in newly diagnosed HIV patient. It is important to note that a direct Gram stain may potentially lead to misclassification of such microorganisms as contaminants. Microbiologists should therefore prioritize the careful examination of colony morphology, biochemical reactions, and consider the limitations of automated machine databases. Furthermore, they should correlate their identification findings with clinical data to ensure optimal patient care and management, especially in the context of an immunocompromised state.

Antimicrobial residues excreted in the environment following antimicrobial treatment enhance resistant microbial communities in the environment and have long-term effects on the selection and maintenance of antimicrobial resistance genes (AMRGs). In this study, we focused on understanding the impact of antimicrobial use on antimicrobial residue pollution and antimicrobial resistance (AMR) in the environment of horse-breeding farms. Rhodococcus equi is an ideal microbe to study these associations because it lives naturally in the soil, exchanges AMRGs with other bacteria in the environment, and can cause disease in animals and humans. The environment is the main source of R. equi infections in foals; therefore, higher levels of multidrug-resistant (MDR) R. equi in the environment contribute to clinical infections with MDR R. equi. We found that macrolide residues in the environment of horse-breeding farms and the use of thoracic ultrasonographic screening (TUS) for early detection of subclinically affected foals with R. equi infections were strongly associated with the presence of R. equi carrying AMRGs in the soil. Our findings indicate that the use of TUS contributed to historically higher antimicrobial use in foals, leading to the accumulation of antimicrobial residues in the environment and enhancing MDR R. equi.

ABSTRACT Genomics allows accurately pinpointing microbial ancestry for taxonomic purposes, yet there is currently no consensus to genus definition based on genome data. Different metrics and criteria are used, with an increasing trend toward genera over-splitting. Here, we report a method for prokaryotic genus assignment that combines normalized tree clustering and network analysis of several genomic relatedness indices. Objectivity is maximized by linear application of the same partitioning thresholds across an entire middle/high taxonomic rank context (order level), with the classical (“pre-genomic”) genera as demarcation reference, ensuring continuity in taxonomy and nomenclature. We tested the method with the Mycobacteriales, where recent examples of genus fragmentation divided Mycobacterium into five genera, or made Rhodococcus paraphyletic by creating the genus Prescottella for the sublineage containing the animal and human pathogen, Rhodococcus equi. Our approach did not support the mycobacterial five-genus split or the Prescottella nested genus, but identified a basal branch in each of the mycobacterial and rhodococcal radiations warranting genus status (Mycobacteroides, and novel genus Rhodococcoides for the Rhodococcus fascians clade, respectively). The median average amino acid identity (AAI) between the demarcated genera was 56% to 59%, consistent with the

A Rodococose é uma enfermidade de caráter zoonótico causada pelo microrganismo Rhodococcus equi, sendo a principal causa de broncopneumonia em potros com menos de seis meses de idade. Essa afecção compreende altas taxas de morbidade e de mortalidade nos equinos, principalmente quando afeta potros com idade inferior a um mês de vida. Em humanos, esta doença ocorre apenas àqueles indivíduos imunossuprimidos. O Rhodococcus equi é uma bactéria gram-positiva oportunista, intracelular facultativa que se multiplica no interior dos macrófagos e tem como caraterística ser endêmica em propriedades que alojam equinos pela facilidade em se multiplicar no solo, um dos fatores responsáveis pela alta distribuição da doença. O diagnóstico precoce por meio de exames hematológicos, PCR e exames de imagem como ultrassom e raio-x de tórax são imprescindíveis para o prognóstico da doença. O tratamento é baseado no uso de antimicrobianos, entretanto, a bactéria é altamente resistente, o que dificulta a ação dos medicamentos instituídos. O objetivo deste trabalho foi discorrer sobre a alta incidência da infecção por Rhodococcus equi em potros e apresentar um caso de Rodococose crônica em uma potra de dois meses de idade, descrevendo principalmente o histórico clínico e os medicamentos utilizados durante o tratamento até a resolução do quadro clínico e alta do animal.

Rhodococcus equi (R. equi) is a zoonotic opportunistic pathogen that can cause life-threatening infections. The rapid evolution of multidrug-resistant R. equi and the fact that there is no currently licensed effective vaccine against R. equi warrant the need for vaccine development. Reverse vaccinology (RV), which involves screening a pathogen’s entire genome and proteome using various web-based prediction tools, is considered one of the most effective approaches for identifying vaccine candidates. Here, we performed a pangenome analysis to determine the core proteins of R. equi. We then used the RV approach to examine the subcellular localization, host and gut flora homology, antigenicity, transmembrane helices, physicochemical properties, and immunogenicity of the core proteins to select potential vaccine candidates. The vaccine candidates were then subjected to epitope mapping to predict the exposed antigenic epitopes that possess the ability to bind with major histocompatibility complex I/II (MHC I/II) molecules. These vaccine candidates and epitopes will form a library of elements for the development of a polyvalent or universal vaccine against R. equi. Sixteen R. equi complete proteomes were found to contain 6,238 protein families, and the core proteins consisted of 3,969 protein families (∼63.63% of the pangenome), reflecting a low degree of intraspecies genomic variability. From the pool of core proteins, 483 nonhost homologous membrane and extracellular proteins were screened, and 12 vaccine candidates were finally identified according to their antigenicity, physicochemical properties and other factors. These included four cell wall/membrane/envelope biogenesis proteins; four amino acid transport and metabolism proteins; one cell cycle control, cell division and chromosome partitioning protein; one carbohydrate transport and metabolism protein; one secondary metabolite biosynthesis, transport and catabolism protein; and one defense mechanism protein. All 12 vaccine candidates have an experimentally validated 3D structure available in the protein data bank (PDB). Epitope mapping of the candidates showed that 16 MHC I epitopes and 13 MHC II epitopes with the strongest immunogenicity were exposed on the protein surface, indicating that they could be used to develop a polypeptide vaccine. Thus, we utilized an analytical strategy that combines pangenome analysis and RV to generate a peptide antigen library that simplifies the development of multivalent or universal vaccines against R. equi and can be applied to the development of other vaccines.

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Abstract Background Early detection of Rhodococcus equi pneumonia in foals is essential for horse health and for veterinarians. Objectives This study aimed to demonstrate the usefulness of assessing the serum concentration of acute‐phase proteins (APPs) in the early diagnosis of pneumonia. Methods The study evaluated APPs in 19 Arabian foals with R. equi pneumonia and compared them with 18 normal Arabian foals in equestrian clubs in Tabriz, Iran. Affected foals were identified through history, clinical findings and bacterial culture of tracheal washing. Biochemical methods and polymerase chain reaction tests were performed by examining the 16S rRNA and vapA genes to confirm the diagnosis of bacterial isolates. Blood samples were taken from all sick and healthy horses, and their serum was isolated. APPs in the serum were measured in all the samples. Results Rhodococcosis increased the serum concentration of haptoglobin (Hp) and serum amyloid A (SAA) (p < 0.001). The relationship between SAA and Hp was meaningful in the infected group (r = 0.933) but not in the healthy group. In cases where there are clinical findings of R. equi pneumonia, the concentration of SAA and Hp can help the effectiveness of treatment.‐ Conclusions Serum concentration analysis of APPs can be helpful in early diagnosis and successfully treating foals with R. equi pneumonia.

Rhodococcus equi is a zoonotic opportunistic pathogen that mainly infects immunodeficient individuals, such as those with HIV infection. In R. equi-infected individuals, serious lung lesions can develop and death may result without appropriate antiviral treatment. This bacterium is rare in clinic and there is little information regarding its diagnosis and treatment. To improve our understanding, this case report describes the diagnosis and treatment of a patient with HIV complicated with R. equi infection from Ditan Hospital, Beijing, China.

A multidrug-resistant clone of the animal and human pathogen Rhodococcus equi, MDR-RE 2287, has been circulating among equine farms in the United States since the 2000s. We report the detection of MDR-RE 2287 outside the United States. Our finding highlights the risk for MDR-RE spreading internationally with horse movements.

The objective of this study was to evaluate the prevalence of involvement of common viral organisms R. equi and K. pneumoniae and their clinicopathological and radiological features in respiratory disease of Malaysian domestic cats. A total of 34 feline cases with acute/chronic infectious respiratory disease signs were followed prospectively to investigate respiratory disease due to R. equi and K. pneumoniae and their relationship with concurrent viral infections in disease manifestation. All sampled cats (n = 27) were positive for FCoV antibodies and negative for FeLV. A significantly high antibody titer for FCV in n = 26 cases was also noticed. A single sample of pyothorax from a 3-months-old, non-vaccinated kitten was positive for R. equi. Bronchopneumonia with severe infiltration of the polymorphs and mononuclear inflammatory cells were prominent features of lungs histopathology from the kitten positive for R. equi. K. pneumoniae subsp. pneumoniae was confirmed from tracheal swabs of two cats. Histologically, the tracheal tissues of the two cats positive for K. pneumoniae were normal. In diagnostic imaging, epicenter of the infectious URT disease was nasal conchae rostrally and nasal turbinates caudally, however for infectious LRT disease was bronchial tree. Conclusively, infectious respiratory disease is a complex illness in cats, predominantly for unvaccinated kittens and young adult cats, especially those kept in multi-cat household or shelter environments because of the involvement of multiple bacterial and viral organisms as primary or secondary invaders. Clinicians should not preclude feline rhodococcosis from differentials, especially in kittens with pyothorax and less than one year of age. Unlike R. equi, K. pneumoniae has the potential to colonize URT of cats which might be disseminating further to cause LRT disease. [ABSTRACT FROM AUTHOR]

Decades of antimicrobial overuse to treat respiratory disease in foals have promoted the emergence and spread of zoonotic multidrug-resistant (MDR) Rhodococcus equi worldwide. Three main R. equi MDR clonal populations—2287, G2106, and G2017—have been identified so far. However, only clones 2287 and G2016 have been isolated from sick animals, with clone 2287 being the main MDR R. equi recovered. The genetic mechanisms that make this MDR clone superior to the others at infecting foals are still unknown. Here, we performed a deep genetic characterization of the accessory genomes of 207 R. equi isolates, and we describe IME2287, a novel genetic element in the accessory genome of clone 2287, potentially involved in the maintenance and spread of this MDR population over time. IME2287 is a putative self-replicative integrative mobilizable element (IME) carrying a DNA replication and partitioning operon and genes encoding its excision and integration from the R. equi genome via a serine recombinase. Additionally, IME2287 encodes a protein containing a Toll/interleukin-1 receptor (TIR) domain that may inhibit TLR-mediated NF-kB signaling in the host and a toxin–antitoxin (TA) system, whose orthologs have been associated with antibiotic resistance/tolerance, virulence, pathogenicity islands, bacterial persistence, and pathogen trafficking. This new set of genes may explain the success of clone 2287 over the other MDR R. equi clones.

Pneumonia is one of the most frequent diseases in foals between one and six months of age. Thereby Rhodococcus equi (R. equi) and Streptococcus equi subsp. zooepidemicus (Strep. zoo.) are regarded as the most common pathogens causing bacterial respiratory infections. The aim of the current study was to determine in which treatment group a faster reduction of fecal excretion of R. equi can be achieved during therapy. The study was designed as a prospective, randomized and blind study. 160 foals older than four weeks of age with moderate (abscess score 10–15cm) to severe (abscess score >15cm) pneumonia from a Warmblood stud were examined, randomly assigned to two treatment groups and initially treated for two weeks. Foals of group one (R/T), including 87 foals, were treated with rifampin, in a 10mg/ kg dose orally once daily, in combination with tulathromycin, in a 2.5mg/kg dose intramuscularly or intravenously once a week. According to the routine treatment of the foals on the study farm, there was a weekly rotation of IM and IV administration during the treatment. In the second group (R/A), 73 foals were treated with rifampin and azithromycin, both in a 10mg/kg dose orally once daily. All foals were subjected to a weekly weight control using a measuring tape from the start of therapy in order to ensure exact dosages. The success of the treatment was monitored once weekly based on the findings of the clinical examination and the sonographically determined pulmonary abscess score. Twenty-nine randomly chosen foals (R/T n=14; R/A n=15) of the two study groups were examined endoscopically on the day of diagnosis and tracheobronchial secretion (TBS) was sampled to identify the bacteria involved by culture. In addition, a native sample of the feces was obtained from each foal and a quantitative, culturally specific isolation and a polymerase chain reaction (PCR) for R. equi were performed. Samples from 29 foals were taken from July to September 2019. If R. equi was found in the feces, the foals were sampled once weekly with a rectal swab until the expected end of treatment (two weeks after the start of treatment) for R. equi PCR. In eight of the 14 foals (57,1%) from the R/T group and four of the 15 foals (26.6%) from the R/A group R. equi was identified in the tracheobronchial secretions and in the feces at the day of diagnosis. Other pathogens as Streptococcus equi subsp. equi, Bordetella bronchiseptica, Escherichia coli and Staphylococcus aureus were identified in the airway sample of very few foals of both treatment groups. Season seemed to influence the incidence of pathogens in sick foals. The exact test by Fisher showed that R. equi was detected significantly more frequently (P=0.0025) in the very warm and dry early summer months, while Strep. zoo. was isolated more frequently in late summer (P=0.0007). In the tracheobronchial secretions of foals with moderate to severe pneumonia, R. equi was detected significantly more frequently in high bacterial counts than in the feces (P<0.001). On the other hand, when a high number of R. equi was found in the feces, the number of that pathogen in the tracheobronchial secretions was also high. Our hypothesis, that R. equi is often isolated concurrently to Strep. zoo. detection was rejected with the two-sided test by Fisher (P=0,05). Two weeks after starting antimicrobial treatment, no foal showed positive pathogen detection in the feces anymore. Based on the results of the current study it can be assumed, that both active antimicrobial combinations are equally effective and that R. equi was eliminated in the gastrointestinal tract 14 days after starting treatment. [ABSTRACT FROM AUTHOR]

Background: Early detection of Rhodococcus equi pneumonia in foals is essential for horse health and for veterinarians. Objectives: This study aimed to demonstrate the usefulness of assessing the serum concentration of acute‐phase proteins (APPs) in the early diagnosis of pneumonia. Methods: The study evaluated APPs in 19 Arabian foals with R. equi pneumonia and compared them with 18 normal Arabian foals in equestrian clubs in Tabriz, Iran. Affected foals were identified through history, clinical findings and bacterial culture of tracheal washing. Biochemical methods and polymerase chain reaction tests were performed by examining the 16S rRNA and vapA genes to confirm the diagnosis of bacterial isolates. Blood samples were taken from all sick and healthy horses, and their serum was isolated. APPs in the serum were measured in all the samples. Results: Rhodococcosis increased the serum concentration of haptoglobin (Hp) and serum amyloid A (SAA) (p < 0.001). The relationship between SAA and Hp was meaningful in the infected group (r = 0.933) but not in the healthy group. In cases where there are clinical findings of R. equi pneumonia, the concentration of SAA and Hp can help the effectiveness of treatment.‐ Conclusions: Serum concentration analysis of APPs can be helpful in early diagnosis and successfully treating foals with R. equi pneumonia. [ABSTRACT FROM AUTHOR]

Simple Summary: For decades, Rhodococcus equi infections remain one of the most common causes of death in foals before weaning, coming from endemic studs. The pathogen can create many problems other than its classical form—pneumonia. Such issues are described in the clinical cases presented in this paper. The first case concerns the potential contribution of rhodococcal infection to a grave outcome in a prematurely born foal lost as a yearling. Another presents so-called "extrapulmonary disorders" (EPD) and a theory of inherited immunodeficiency in a breeding dam line from one stud. The third case is connected with a suspected atypical location of the pulmonary abscess. The last example is associated with drug-induced anhidrosis. These clinical pictures may be considered rare or interesting and worth presenting in a scientific report. This article aims to present several interesting and less typical courses of Rhodococcus equi infections in foals, collected during the 2019–2021 foaling seasons in some Polish studs. The study was conducted by the Division of Veterinary Epidemiology and Economics, Warsaw University of Life Sciences—SGGW, and concentrated on ultrasonographic contribution to diagnostics and treatment of the disease. Among many standard cases of rhodococcal pneumonia, some rare ones occurred. The aforementioned issues include the potential contribution of rhodococcal infection to a grave outcome in a prematurely born filly, lost as a yearling, so-called "extrapulmonary disorders" (EPD), a hypothesis of inherited immunodeficiency with grave outcome in a breeding dam line from one stud, and macrolide-induced anhidrosis. The main benefit of this report would be to supplement the general picture of clinical rhodococcosis. [ABSTRACT FROM AUTHOR]

Abstract Objective The efficacy of Rhodococcus equi-specific hyperimmune plasma (HIP) is usually evaluated in vitro. Anticoagulants (AC) used for plasma collection can negatively impact bacterial replication but their effect on R. equi growth has not been evaluated. The aim of this study was to establish the effect that AC routinely used in veterinary medicine (ACD, K2EDTA, Li Heparin, and Na Citrate) have on in vitro R. equi growth. To assess this, in vitro assays commonly used to test HIP efficacy (direct effect on microorganism and macrophage infection), were performed using each AC and non-treated bacteria. Results There was no direct effect of ACD, Li Heparin or Na Citrate on R. equi growth. These AC significantly (p

Rhodococcus equi (R. equi) is a Gram-positive coccobacillus that causes pneumonia in foals of less than 3 months, which have the ability of replication in macrophages. The ability of R. equi persist in macrophages is dependent on the virulence plasmid pVAPA. Gram-positive extracellular vesicles (EVs) carry a variety of virulence factors and play an important role in pathogenic infection. There are few studies on R. equi-derived EVs (R. equi-EVs), and little knowledge regarding the mechanisms of how R. equi-EVs communicate with the host cell. In this study, we examine the properties of EVs produced by the virulence strain R. equi 103+ (103+-EVs) and avirulenct strain R. equi 103− (103−-EVs). We observed that 103+-EVs and 103−-EVs are similar to other Gram-positive extracellular vesicles, which range from 40 to 260 nm in diameter. The 103+-EVs or 103−-EVs could be taken up by mouse macrophage J774A.1 and cause macrophage cytotoxicity. Incubation of 103+-EVs or 103−-EVs with J774A.1 cells would result in increased expression levels of IL-1β, IL-6, and TNF-α. Moreover, the expression of TLR2, p-NF-κB, p-p38, and p-ERK were significantly increased in J774A.1 cells stimulated with R. equi-EVs. In addition, we presented that the level of inflammatory factors and expression of TLR2, p-NF-κB, p-p38, and p-ERK in J774A.1 cells showed a significant decreased when incubation with proteinase K pretreated-R. equi-EVs. Overall, our data indicate that R. equi-derived EVs are capable of mediating inflammatory responses in macrophages via TLR2-NF-κB/MAPK pathways, and R. equi-EVs proteins were responsible for TLR2-NF-κB/MAPK mediated inflammatory responses in macrophage. Our study is the first to reveal potential roles for R. equi-EVs in immune response in R. equi-host interactions and to compare the differences in macrophage inflammatory responses mediated by EVs derived from virulent strain R. equi and avirulent strain R. equi. The results of this study have improved our knowledge of the pathogenicity of R. equi. [ABSTRACT FROM AUTHOR]

A multidrug-resistant clone of the animal and human pathogen Rhodococcus equi, MDR-RE 2287, has been circulating among equine farms in the United States since the 2000s. We report the detection of MDR-RE 2287 outside the United States. Our finding highlights the risk for MDR-RE spreading internationally with horse movements. [ABSTRACT FROM AUTHOR]

Background. Surveillance of antimicrobial resistance (AMR) among veterinary pathogens is necessary to identify clinically relevant patterns of AMR and to inform antimicrobial use practices. Streptococcus equi subsp. zooepidemicus and Rhodococcus equi are bacterial pathogens of major clinical importance in horses and are frequently implicated in respiratory tract infections. The objectives of this study were to describe antimicrobial resistance patterns and identify predictors of AMR and multidrug resistance (MDR) (resistance to three or more antimicrobial classes) among equine S. zooepidemicus and R. equi isolates. Methods. Antimicrobial susceptibility data from equine specimens submitted to the University of Kentucky Veterinary Diagnostic Laboratory between 2012 and 2017 were used in the study. Temporal trends in AMR andMDRwere assessed using the Cochran-Armitage test. Logistic regression was used to identify associations between patient characteristics and the following outcomes: (a) MDR among S. zooepidemicus isolates, and (b) resistance to macrolides and ansamycins (rifampin) among R. equi isolates. Logistic regression was also used to investigate whether resistance of S. zooepidemicus and R. equi isolates to an antimicrobial class could be predicted by resistance to other drug classes. Results. The vast majority of S. zooepidemicus (99.6%) and R. equi isolates (83%) were resistant to at least one antimicrobial agent, but no significant temporal trends in AMR were observed. Approximately half (53.3%) of the S. zooepidemicus isolates were multidrug-resistant, and there was a significant (p<0:001) increasing temporal trend of MDR among S. zooepidemicus isolates. Resistance to penicillin, which is typically recommended for treatment of suspected S. zooepidemicus infections, also increased during the study period, from 3.3% to 9.5%. Among R. equi isolates, 19.2% were resistant to one or more macrolide antibiotics, 24% were resistant to rifampin, and 15.6% were resistant to both macrolide(s) and rifampin. For both organisms, resistance to an antimicrobial class could be predicted based on resistance profiles to other drug classes. For instance, significant (p<0:01) predictors of β-lactam resistance among S. zooepidemicus isolates included resistance to macrolides (Odds Ratio (OR) D 14.7) and ansamycins (OR D 9.3). Resistance to phenicols (OR D 3.7) and ansamycins (OR D 19.9) were associated with higher odds of macrolide resistance among R. equi isolates. Conclusions. The increase in MDR among S. zooepidemicus isolates is concerning. The observed levels of resistance to macrolides and rifampin among R. equi are also worrisome given the limited number of antimicrobials available for treatment of this organism. The findings of this study highlight the importance of ongoing surveillance of AMR to guide treatment decisions and directions for future research. [ABSTRACT FROM AUTHOR]

Background Surveillance of antimicrobial resistance (AMR) among veterinary pathogens is necessary to identify clinically relevant patterns of AMR and to inform antimicrobial use practices. Streptococcus equi subsp. zooepidemicus and Rhodococcus equi are bacterial pathogens of major clinical importance in horses and are frequently implicated in respiratory tract infections. The objectives of this study were to describe antimicrobial resistance patterns and identify predictors of AMR and multidrug resistance (MDR) (resistance to three or more antimicrobial classes) among equine S. zooepidemicus and R. equi isolates. Methods Antimicrobial susceptibility data from equine specimens submitted to the University of Kentucky Veterinary Diagnostic Laboratory between 2012 and 2017 were used in the study. Temporal trends in AMR and MDR were assessed using the Cochran-Armitage test. Logistic regression was used to identify associations between patient characteristics and the following outcomes: (a) MDR among S. zooepidemicus isolates, and (b) resistance to macrolides and ansamycins (rifampin) among R. equi isolates. Logistic regression was also used to investigate whether resistance of S. zooepidemicus and R. equi isolates to an antimicrobial class could be predicted by resistance to other drug classes. Results The vast majority of S. zooepidemicus (99.6%) and R. equi isolates (83%) were resistant to at least one antimicrobial agent, but no significant temporal trends in AMR were observed. Approximately half (53.3%) of the S. zooepidemicus isolates were multidrug-resistant, and there was a significant (p

Rhodococcus equi is a conditionally pathogenic bacterium widely distributed in soil, water, and marine environments, which can cause respiratory infections, pleurisy, blood and even bone marrow infections in immunocompromised people, and particularly in patients with acquired immunodeficiency syndrome (AIDS). This case report describes a patient with initially suspicion of tuberculosis (TB) as an outpatient in a TB clinic. However, laboratory findings identified R. equi in his sputum sample based on a positive acid-fast stain, which was highly suggestive of a pulmonary infection caused by R. equi. The patient was subsequently admitted to the respiratory unit for treatment. Once the source of infection was identified, the patient was treated with a combination of antibiotics for 2 weeks and was discharged with a significant improvement in symptoms.

Simple Summary: Serious consequences of septicemic bacterial infections include the formation of purulent and pyogranulomatous inflammation resulting in abscesses in inner organs. Different bacteria are known to cause these infections in livestock. In this study, we report in detail on a case of a fatal Rhodococcus (R.) equi infection in an alpaca (Vicugna pacos), to our knowledge, for the first time. R. equi is a member of the actinomycetes, a bacterial group known to contain several pathogenic bacteria. R. equi primarily affects equine foals and other domestic animals, but also humans, which renders this bacterium a zoonotic agent. The rhodococcal infection of the alpaca reported herein caused septicemia, resulting in emaciation and severe lesions in the lungs and heart. The onset of infection was presumably caused by aspiration pneumonia, resulting in abscesses exclusively in the lungs. The R. equi isolate proved to be pathogenic, based on the virulence gene vapA encoding the virulence-associated protein A. Antibiotic susceptibility testing revealed a susceptibility to doxycycline, erythromycin, gentamycin, neomycin, rifampicin, trimethoprim/sulfamethoxazole, tetracycline and vancomycin. This report of an R. equi infection in an alpaca makes clear that we still have knowledge gaps about bacterial infectious diseases in alpacas and potential zoonotic impacts. Therefore, the determination of pathogenic, zoonotic bacteria in alpacas is essential for treatment and preventive measures with respect to sustaining the health, welfare and productivity of this camelid species. Rhodococcus (R.) equi is a pathogen primarily known for infections in equine foals, but is also present in numerous livestock species including New World camelids. Moreover, R. equi is considered an emerging zoonotic pathogen. In this report, we describe in detail a fatal rhodococcal infection in an alpaca (Vicugna pacos), to our best knowledge, for the first time. The alpaca died due to a septicemic course of an R. equi infection resulting in emaciation and severe lesions including pyogranulomas in the lungs and pericardial effusion. The onset of the infection was presumably caused by aspiration pneumonia. R. equi could be isolated from the pyogranulomas in the lung and unequivocally identified by MALDI-TOF MS analysis and partial sequencing of the 16S rRNA gene, the 16S-23S internal transcribed spacer (ITS) region and the rpoB gene. The isolate proved to possess the vapA gene in accordance with tested isolates originating from the lungs of infected horses. The R. equi isolates revealed low minimal inhibitory concentrations (MIC values) for doxycycline, erythromycin, gentamycin, neomycin, rifampicin, trimethoprim/sulfamethoxazole, tetracycline and vancomycin in antibiotic susceptibility testing. Investigations on the cause of bacterial, especially fatal, septicemic infections in alpacas are essential for adequately addressing the requirements for health and welfare issues of this New World camelid species. Furthermore, the zoonotic potential of R. equi has to be considered with regard to the One Health approach. [ABSTRACT FROM AUTHOR]

Simple Summary: Multidrug-resistant (MDR) Rhodococcus equi (R. equi) is rapidly spreading across the US in equine breeding farms, causing devastating untreatable disease in foals. There is a critical need for new diagnostic tools that can identify R. equi and its antibiotic resistance profile accurately and fast. This project aimed to develop and standardize a novel multiplex quantitative real-time PCR (qPCR) to detect R. equi and its most clinically relevant antimicrobial resistance genes directly from equine respiratory samples. We have designed three pairs of oligos (a.k.a primers or oligonucleotides) to identify R. equi and MLSB (macrolide, lincosamide, streptogramin B) resistance genes erm(46) and erm(51) that can be used in single-plex and multiplex qPCR assays. Furthermore, our new qPCR shows high sensitivity and specificity in in-silico analysis and when tested in mock equine respiratory samples. Therefore, we believe it can be used for a fast-preliminary diagnosis of R. equi and the simultaneous prediction of its most critical resistant profile. The new molecular diagnostic tool presented here will shorten the waiting time from the moment the practitioner sees the equid patient until it is diagnosed and appropriately treated. R. equi is an important veterinary pathogen that takes the lives of many foals every year. With the emergence and spread of MDR R. equi to current antimicrobial treatment, new tools that can provide a fast and accurate diagnosis of the disease and antimicrobial resistance profile are needed. Here, we have developed and analytically validated a multiplex qPCR for the simultaneous detection of R. equi and related macrolide resistance genes in equine respiratory samples. The three sets of oligos designed in this study to identify R. equi housekeeping gene choE and macrolide resistance genes erm(46) and erm(51) showed high analytic sensitivity with a limit of detection (LOD) individually and in combination below 12 complete genome copies per PCR reaction, and an amplification efficiency between 90% and 147%. Additionally, our multiplex qPCR shows high specificity in in-silico analysis. Furthermore, it did not present any cross-reaction with normal flora from the equine respiratory tract, nor commonly encountered respiratory pathogens in horses or other genetically close organisms. Our new quantitative PCR is a trustable tool that will improve the speed of R. equi infection diagnosis, as well as helping in treatment selection. [ABSTRACT FROM AUTHOR]

Seven genomes of Corynebacterium pseudotuberculosis biovar equi were sequenced on the Ion Torrent PGM platform, generating high-quality scaffolds over 2.35 Mbp. This bacterium is the causative agent of disease known as "pigeon fever" which commonly affects horses worldwide. The pangenome of biovar equi was calculated and two phylogenomic approaches were used to identify clustering patterns within Corynebacterium genus. Furthermore, other comparative analyses were performed including the prediction of genomic islands and prophages, and SNP-based phylogeny. In the phylogenomic tree, C. pseudotuberculosis was divided into two distinct clades, one formed by nitrate non-reducing species (biovar ovis) and another formed by nitrate-reducing species (biovar equi). In the latter group, the strains isolated from California were more related to each other, while the strains CIP 52.97 and 1/06-A formed the outermost clade of the biovar equi. A total of 1,355 core genes were identified, corresponding to 42.5% of the pangenome. This pangenome has one of the smallest core genomes described in the literature, suggesting a high genetic variability of biovar equi of C. pseudotuberculosis. The analysis of the similarity between the resistance islands identified a higher proximity between the strains that caused more severe infectious conditions (infection in the internal organs). Pathogenicity islands were largely conserved between strains. Several genes that modulate the pathogenicity of C. pseudotuberculosis were described including peptidases, recombination enzymes, micoside synthesis enzymes, bacteriocins with antimicrobial activity and several others. Finally, no genotypic differences were observed between the strains that caused the three different types of infection (external abscess formation, infection with abscess formation in the internal organs, and ulcerative lymphangitis). Instead, it was noted that there is a higher phenetic correlation between strains isolated at California compared to the other strains. Additionally, high variability of resistance islands suggests gene acquisition through several events of horizontal gene transfer.

Objective To isolate and identify Rhodococcus equi(R.equi) from clinical specimens for potential relationship between antibiotic sensitivity and efficacy of antibiotic chemotherapy. Methods From April 2019 to April 2020, 17 cases patients with positive isolation of R. equi were grouped as test one and and 17 cases of patients underwent empirical treatment only were collected as the control group. Results The efficacy of antibiotic chemotherapy with sensitivity test (test group) showed significantly better than that of the control group. Conclusions Rational use of antibiotics chemotherapy supported by sensitivity test can significantly improve the effect of chemotherapy.

Abstract Rhodococcus equi is a Gram‐positive coccobacillus bacterium commonly found on dry surface soil of farming and livestock properties. This organism is a rare cause of subacute, necrotizing infection, and mostly causes cavitary pneumonia and lung abscess, especially in immunocompromised hosts with advanced human immunodeficiency virus (HIV) infection.

ABSTRACT A previously reported method for evaluating the intracellular growth of Rhodococcus equi using enhanced green fluorescent protein is unsuitable for the quantitative evaluation of the entire sample because the signal can be detected only in the excitation region. Therefore, we created an autobioluminescent R. equi using luciferase (luxABCDE). First, we connected luxABCDE to the functional promoter PaphII and introduced it into the chromosomes of ATCC33701 and ATCC33701_P-. Luminescence was detected in both transformants, and a correlation between the bacterial number and luminescence intensity in the logarithmic phase was observed, indicating that luxABCDE is functionally and quantitatively expressed in R. equi. The luminescence of ATCC33701 was significantly higher than that of ATCC33701_P- at 24 h after infection with J774A.1. Next, RNA-Seq analysis of ATCC33701 to search for endogenous high-expression promoters resulted in the upstream sequences of RS29370, RS41760, and vapA being selected as candidates. Luminescence was detected in each transformant expressing the luxABCDE using these upstream sequences. We examined the luminescence intensity by coexpressing the frp gene, an enhancer of the luciferase reaction, with luxABCDE. The luminescence intensity of the coexpressing transformant was significantly enhanced in J774A.1 compared with the non-coexpressing transformant. Finally, we examined the luminescence in vivo. The luminescence signals in the organs peaked on the third day following the administration of ATCC33701 derivatives in mice, but no luminescence signal was detected when the ATCC33701_P- derivative was administered. The autologous bioluminescent method described herein will enhance the in vitro and in vivo quantitative analysis of R. equi proliferation. IMPORTANCE We established an autologous bioluminescent strain of R. equi and a method to evaluate its proliferation in vitro and in vivo quantitatively. This method overcomes the weakness of the fluorescence detection system that only measures the site of excitation light irradiation. It is expected to be used as an in vitro and in vivo growth evaluation method with excellent quantitative properties. In addition, it was suggested that the selection of a promoter that expresses luxABCDE could produce a luminescence with high intensity. Although this method needs further improvement, such as creating transformants that can maintain high luminescence intensity regardless of environmental changes such as temperature fluctuations, it is possible to observe bacterial growth over time in mice without killing them. Therefore, this method can be used to not only evaluate the pathogenicity of various wild and gene-deficient strains but also to screen preventive and therapeutic methods such as vaccines.

The objective of this study was to evaluate the prevalence of involvement of common viral organisms R. equi and K. pneumoniae and their clinicopathological and radiological features in respiratory disease of Malaysian domestic cats. A total of 34 feline cases with acute/chronic infectious respiratory disease signs were followed prospectively to investigate respiratory disease due to R. equi and K. pneumoniae and their relationship with concurrent viral infections in disease manifestation. All sampled cats (n = 27) were positive for FCoV antibodies and negative for FeLV. A significantly high antibody titer for FCV in n = 26 cases was also noticed. A single sample of pyothorax from a 3-months-old, non-vaccinated kitten was positive for R. equi. Bronchopneumonia with severe infiltration of the polymorphs and mononuclear inflammatory cells were prominent features of lungs histopathology from the kitten positive for R. equi. K. pneumoniae subsp. pneumoniae was confirmed from tracheal swabs of two cats. Histologically, the tracheal tissues of the two cats positive for K. pneumoniae were normal. In diagnostic imaging, epicenter of the infectious URT disease was nasal conchae rostrally and nasal turbinates caudally, however for infectious LRT disease was bronchial tree. Conclusively, infectious respiratory disease is a complex illness in cats, predominantly for unvaccinated kittens and young adult cats, especially those kept in multi-cat household or shelter environments because of the involvement of multiple bacterial and viral organisms as primary or secondary invaders. Clinicians should not preclude feline rhodococcosis from differentials, especially in kittens with pyothorax and less than one year of age. Unlike R. equi, K. pneumoniae has the potential to colonize URT of cats which might be disseminating further to cause LRT disease.

Objective: The efficacy of Rhodococcus equi-specific hyperimmune plasma (HIP) is usually evaluated in vitro. Anticoagulants (AC) used for plasma collection can negatively impact bacterial replication but their effect on R. equi growth has not been evaluated. The aim of this study was to establish the effect that AC routinely used in veterinary medicine (ACD, K2EDTA, Li Heparin, and Na Citrate) have on in vitro R. equi growth. To assess this, in vitro assays commonly used to test HIP efficacy (direct effect on microorganism and macrophage infection), were performed using each AC and non-treated bacteria. Results: There was no direct effect of ACD, Li Heparin or Na Citrate on R. equi growth. These AC significantly (p < 0.05) delayed growth for 12 h following opsonization. The number of R. equi colonies after macrophage infection was significantly (p < 0.05) lower 72 h post-opsonization with Na Citrate. K2EDTA inhibited the formation of R. equi colonies by 12 h in all the assays. In conclusion, AC should be taken into consideration when interpreting in vitro results as their negative effect on bacterial growth may be mistakenly interpreted as HIP efficacy. ACD and Li Heparin appear more appropriate for the selected assays. [ABSTRACT FROM AUTHOR]

Rhodococcus equi is a zoonotic pathogen that can cause fatal disease in patients who are immunocompromised. At present, the epidemiology and pathogenic mechanisms of R. equi infection are not clear. This study characterized the genomes of 53 R. equi strains from different sources. Pan-genome analysis showed that all R. equi strains contained 11481 pan genes, including 3690 core genes and 602 ~ 1079 accessory genes. Functional annotation of pan genome focused on the genes related to basic lifestyle, such as the storage and expression of metabolic and genetic information. Phylogenetic analysis based on pan-genome showed that the R. equi strains were clustered into six clades, which was not directly related to the isolation location and host source. Also, a total of 84 virulence genes were predicted in 53 R. equi strains. These virulence factors can be divided into 20 categories related to substance metabolism, secreted protein and immune escape. Meanwhile, six antibiotic resistance genes (RbpA, tetA (33), erm (46), sul1, qacEdelta 1 and aadA9) were detected, and all strains carried RbpA related to rifamycin resistance. In addition, 28 plasmids were found in the 53 R. equi strains, belonging to Type-A (n = 14), Type-B (n = 8) and Type-N (n = 6), respectively. The genetic structures of the same type of plasmid were highly similar. In conclusion, R. equi strains show different genomic characteristics, virulence-related genes, potential drug resistance and virulence plasmid structures, which may be conducive to the evolution of its pathogenesis. [ABSTRACT FROM AUTHOR]

Rhodococcus equi (R. equi) was identified among the most relevant antimicrobial‐resistant (AMR) bacteria in the EU for horses in a previous scientific opinion. Thus, it has been assessed according to the criteria of the Animal Health Law (AHL), in particular criteria of Article 7 on disease profile and impacts, Article 5 on its eligibility to be listed, Annex IV for its categorisation according to disease prevention and control rules as in Article 9 and Article 8 for listing animal species related to the bacterium. The assessment has been performed following a methodology previously published. The outcome is the median of the probability ranges provided by the experts, which indicates whether each criterion is fulfilled (lower bound ≥ 66%) or not (upper bound ≤ 33%), or whether there is uncertainty about fulfilment. Reasoning points are reported for criteria with uncertain outcome. According to the assessment here performed, it is uncertain whether AMR R. equi can be considered eligible to be listed for Union intervention according to Article 5 of the AHL (10–66% probability). According to the criteria in Annex IV, for the purpose of categorisation related to the level of prevention and control as in Article 9 of the AHL, the AHAW Panel concluded that the bacterium does not meet the criteria in Sections 1 and 2 (Categories A and B; 5–10% and 10–33% probability of meeting the criteria, respectively), and the AHAW Panel is uncertain whether it meets the criteria in Sections 3, 4 and 5 (Categories C, D and E; 10–66% probability of meeting the criteria in all three categories). The animal species to be listed for AMR R. equi according to Article 8 criteria are mainly horses and other species belonging to the Perissodactyla and Artiodactyla orders. [ABSTRACT FROM AUTHOR]