Epidemiology and Molecular Basis of Multidrug Resistance in Rhodococcus equi.

The development and spread of antimicrobial resistance are major concerns for human and animal health. The effects of the overuse of antimicrobials in domestic animals on the dissemination of resistant microbes to humans and the environment are of concern worldwide. Rhodococcus equi is an ideal model to illustrate the spread of antimicrobial resistance at the animal-human-environment interface because it is a natural soil saprophyte that is an intracellular zoonotic pathogen that produces severe bronchopneumonia in many animal species and humans. Globally, R. equi is most often recognized as causing severe pneumonia in foals that results in animal suffering and increased production costs for the many horse-breeding farms where the disease occurs. Because highly effective preventive measures for R. equi are lacking, thoracic ultrasonographic screening and antimicrobial chemotherapy of subclinically affected foals have been used for controlling this disease during the last 20 years. The resultant increase in antimicrobial use attributable to this "screen-and-treat" approach at farms where the disease is endemic has likely driven the emergence of multidrug-resistant (MDR) R. equi in foals and their environment. This review summarizes the factors that contributed to the development and spread of MDR R. equi, the molecular epidemiology of the emergence of MDR R. equi, the repercussions of MDR R. equi for veterinary and human medicine, and measures that might mitigate antimicrobial resistance at horse-breeding farms, such as alternative treatments to traditional antibiotics. Knowledge of the emergence and spread of MDR R. equi is of broad importance for understanding how antimicrobial use in domestic animals can impact the health of animals, their environment, and human beings.

The novel and transferable erm(51) gene confers macrolides, lincosamides and streptogramins B (MLSB ) resistance to clonal Rhodococcus equi in the environment.

The use of mass antimicrobial treatment has been linked to the emergence of antimicrobial resistance in human and animal pathogens. Using whole-genome single-molecule real-time (SMRT) sequencing, we characterized genomic variability of multidrug-resistant Rhodococcus equi isolated from soil samples from 100 farms endemic for R. equi infections in Kentucky. We discovered the novel erm(51)-encoding resistance to MLSB in R. equi isolates from soil of horse-breeding farms. Erm(51) is inserted in a transposon (TnErm51) that is associated with a putative conjugative plasmid (pRErm51), a mobilizable plasmid (pMobErm51), or both enabling horizontal gene transfer to susceptible organisms and conferring high levels of resistance against MLSB in vitro. This new resistant genotype also carries a previously unidentified rpoB mutation conferring resistance to rifampicin. Isolates carrying both vapA and erm(51) were rarely found, indicating either a recent acquisition of erm(51) and/or impaired survival when isolates carry both genes. Isolates carrying erm(51) are closely related genetically and were likely selected by antimicrobial exposure in the environment.

Antimicrobial susceptibility patterns of Rhodococcus equi from necropsied foals with rhodococcosis.

Mainstay therapy for rhodococcosis in foals is the combination of rifampicin and a macrolide. While emergence of resistance to rifampicin and macrolides has been reported, studies demonstrating the development of resistance to such drugs is limited in necropsied foals with rhodococcosis. In this study, the foal necropsy records between 01/01/2011 and 08/30/2019 were reviewed for culture-positive R. equi with MICs and, whether or not the affected foals received any mainstay dual therapy before their deaths. Resistance to antimicrobials in the R. equi isolates from necropsied foals were then compared between treated foals with dual therapy and untreated foals to determine the association between the administration of antimicrobials and development of the drug resistance. In a total of 256 R. equi isolates from each of the 256 necropsied foals with rhodococcosis, rifampicin, azithromycin, clarithromycin and erythromycin showed high rates of resistance, 22.65 %, 16.01 %, 14.84 % and 15.23 %, respectively. The most active antimicrobials exhibiting MIC50/90 values were imipenem, doxycycline, amikacin and gentamicin including in the rifampicin- and macrolides-resistant R. equi isolates. Based on the treatment histories available for the 114 necropsied foals with rhodococcosis, R. equi isolates resistant to rifampicin, and macrolides were significantly more isolated from treated foals with mainstay dual therapy compared to untreated foals. Despite dual therapy, development of resistance against rifampicin and macrolides warrants evaluation of new treatment protocols in foals.

Comparing PFGE, MLST, and WGS in monitoring the spread of macrolide and rifampin resistant Rhodococcus equi in horse production.

BACKGROUND: Rhodococcus equi (R. equi) infections are endemic in many horse facilities in the United States resulting significant economic loses annually. Currently, there is no commercial vaccine available and the emergence of isolates that are resistant to the current treatment and prophylaxis using antibiotics prompts closer surveillance of this pathogen. OBJECTIVE: This study compares three different genotyping techniques, Pulsed Field Gel Electrophoresis (PFGE), Multilocus Sequence Typing (MLST) and whole genome SNP-based phylogeny to determine the most accurate method to monitor the spread of macrolide-and-rifampin-resistant R. equi. METHODS: 16 macrolide and rifampin-resistant and 6 susceptible R. equi and their Illumina Miseq whole genome sequences were used in this study. The isolates were sub-typed by PFGE with VspI and a dendrogram based on their similarities generated. Additionally, three phylogenetic trees were constructed using CSI phylogeny on (i) whole genome sequences (WGS), (ii) in silico MLST sequences and (iii) MLST sequences obtained after PCR-amplification and Sanger sequencing. RESULTS: PFGE identified 18 different genetic profiles and grouped the 22 isolates into 3 clusters independently of their susceptibilities. The phylogenetic trees built from WGS and MLST data showed similar topology, separating the isolates into 2 major clades in accordance with their susceptibility profiles (susceptible and resistant). However, only the trees generated with next generation sequencing data could detect the clonality of the resistant isolates.

A Common Practice of Widespread Antimicrobial Use in Horse Production Promotes Multi-Drug Resistance.

The practice of prophylactic administration of a macrolide antimicrobial with rifampin (MaR) to apparently healthy foals with pulmonary lesions identified by thoracic ultrasonography (i.e., subclinically pneumonic foals) is common in the United States. The practice has been associated epidemiologically with emergence of R. equi resistant to MaR. Here, we report direct evidence of multi-drug resistance among foals treated with MaR. In silico and in vitro analysis of the fecal microbiome and resistome of 38 subclinically pneumonic foals treated with either MaR (n = 19) or gallium maltolate (GaM; n = 19) and 19 untreated controls was performed. Treatment with MaR, but not GaM, significantly decreased fecal microbiota abundance and diversity, and expanded the abundance and diversity of antimicrobial resistance genes in feces. Soil plots experimentally infected with Rhodococcus equi (R. equi) and treated with MaR selected for MaR-resistant R. equi, whereas MaR-susceptible R. equi out-competed resistant isolates in GaM-treated or untreated plots. Our results indicate that MaR use promotes multi-drug resistance in R. equi and commensals that are shed into their environment where they can persist and potentially infect or colonize horses and other animals.

Clonal Confinement of a Highly Mobile Resistance Element Driven by Combination Therapy in Rhodococcus equi.

Antibiotic use has been linked to changes in the population structure of human pathogens and the clonal expansion of multidrug-resistant (MDR) strains among healthcare- and community-acquired infections. Here we present a compelling example in a veterinary pathogen, Rhodococcus equi, the causative agent of a severe pulmonary infection affecting foals worldwide. We show that the erm(46) gene responsible for emerging macrolide resistance among equine R. equi isolates in the United States is part of a 6.9-kb transposable element, TnRErm46, actively mobilized by an IS481 family transposase. TnRErm46 is carried on an 87-kb conjugative plasmid, pRErm46, transferable between R. equi strains at frequencies up to 10-3 The erm(46) gene becomes stabilized in R. equi by pRErm46's apparent fitness neutrality and wholesale TnRErm46 transposition onto the host genome. This includes the conjugally exchangeable pVAPA virulence plasmid, enabling the possibility of cotransfer of two essential traits for survival in macrolide-treated foals in a single mating event. Despite its high horizontal transfer potential, phylogenomic analyses show that erm(46) is paradoxically confined to a specific R. equi clone, 2287. R. equi 2287 also carries a unique rpoBS531F mutation conferring high-level resistance to rifampin, systematically administered together with macrolides against rhodococcal pneumonia on equine farms. Our data illustrate that under sustained combination therapy, several independent "founder" genetic events are concurrently required for resistance, limiting not only its emergence but also, crucially, horizontal spread, ultimately determining multiresistance clonality.IMPORTANCE MDR clades arise upon acquisition of resistance traits, but the determinants of their clonal expansion remain largely undefined. Taking advantage of the unique features of Rhodococcus equi infection control in equine farms, involving the same dual antibiotic treatment since the 1980s (a macrolide and rifampin), this study sheds light into the determinants of multiresistance clonality and the importance of combination therapy in limiting the dissemination of mobile resistance elements. Clinically effective therapeutic alternatives against R. equi foal pneumonia are currently lacking, and the identified macrolide-rifampin MDR clone 2287 has serious implications. Still at early stages of evolution and local spread, R. equi 2287 may disseminate globally, posing a significant threat to the equine industry and, also, public health due to the risk of zoonotic transmission. The characterization of the 2287 clone and its resistance determinants will enable targeted surveillance and control interventions to tackle the emergence of MDR R. equi.

Prevalence and risk factors associated with emergence of Rhodococcus equi resistance to macrolides and rifampicin in horse-breeding farms in Kentucky, USA.

The combination of a macrolide and rifampicin has been the mainstay of therapy in foals with Rhodococcus equi pneumonia for decades. Recent studies suggest that mass antimicrobial treatment of subclinically affected foals over time has selected for antimicrobial resistance. Our objective was to estimate the prevalence of R. equi strains resistant to macrolides and rifampicin at horse breeding farms in Kentucky. A hundred breeding farms in Kentucky were surveyed and R. equi were cultured from soil samples. Data were analyzed with logistic regression and generalized linear modeling (P < 0.05). Seventy-six percent (76%) of farms yielded resistant R. equi, and resistance to macrolides and rifampicin was associated with their use at farms. The present study is the first to report the prevalence and distribution of resistant isolates in the environment of farms in Kentucky, USA. Collectively, previous reports and the data presented herein provide irrefutable evidence of emerging antimicrobial resistance in R. equi with alarming prevalence. Widespread dissemination and maintenance of resistance genes in the environment where many other pathogenic bacteria exist is a concern for both animal and human health.

Effect of Macrolide and Rifampin Resistance on Fitness of Rhodococcus equi during Intramacrophage Replication and In Vivo.

The soil-dwelling, saprophytic actinomycete Rhodococcus equi is a facultative intracellular pathogen of macrophages and causes severe bronchopneumonia when inhaled by susceptible foals. Standard treatment for R. equi disease is dual-antimicrobial therapy with a macrolide and rifampin. Thoracic ultrasonography and early treatment with antimicrobials prior to the development of clinical signs are used as means of controlling endemic R. equi infection on many farms. Concurrently with the increased use of macrolides and rifampin for chemoprophylaxis and the treatment of subclinically affected foals, a significant increase in the incidence of macrolide- and rifampin-resistant R. equi isolates has been documented. Previously, our laboratory demonstrated decreased fitness of R. equi strains that were resistant to macrolides, rifampin, or both, resulting in impaired in vitro growth in iron-restricted media and in soil. The objective of this study was to examine the effect of macrolide and/or rifampin resistance on intracellular replication of R. equi in equine pulmonary macrophages and in an in vivo mouse infection model in the presence and absence of antibiotics. In equine macrophages, the macrolide-resistant strain did not increase in bacterial numbers over time and the dual macrolide- and rifampin-resistant strain exhibited decreased proliferation compared to the susceptible isolate. In the mouse model, in the absence of antibiotics, the susceptible R. equi isolate outcompeted the macrolide- or rifampin-resistant strains.

Identification of macrolide- and rifampicin-resistant Rhodococcus equi in environmental samples from equine breeding farms in central Kentucky during 2018.

Rhodococcus equi causes severe pneumonia in foals and is most often recognized in people as an opportunistic pathogen. Longitudinal studies examining antimicrobial-resistant R. equi from environmental samples are lacking. We hypothesized that antimicrobial-resistant R. equi would be detectable in the ground (pasture soil or stall bedding) and air at breeding farms with previous documentation of foals infected with resistant isolates, and that concentrations of resistant isolates would increase over time during the foaling season. In this prospective cohort study, ground and air samples were collected from stalls and paddocks in January, March, May and July of 2018 at 10 horse-breeding farms with history of foal pneumonia attributed to macrolide- or Rifampicin-resistant R. equi. Environmental samples were cultured in the presence and absence of macrolides and Rifampicin to select for resistant organisms. Data were analyzed with linear mixed-effects and Hurdle models. Concentrations of total R. equi in bedding or air of stalls were significantly (P < 0.05) higher in January than other months. The proportion of resistant R. equi in soil samples from paddocks was significantly (P < 0.05) higher than stall bedding during all months. For each month, air samples from paddocks had a significantly (P < 0.05) higher proportion of resistant isolates than those from stalls. Fifty-five percent of resistant soil isolates and 34% of resistant air isolates were considered virulent by identification of the vapA gene. Concentrations of resistant R. equi isolates did not increase over time during the foaling season. Antimicrobial-resistant R. equi can persist in the environment at farms with a history of pneumonia caused by resistant R. equi infections, and exposure to resistant isolates in paddocks and stalls appears stable during the foaling season. Resistant isolates in the environment not only pose a risk for disease but also can serve as a repository for dissemination of resistance genes.

In vitro antimicrobial susceptibility of equine clinical isolates from France, 2006-2016.

OBJECTIVES: This study aimed to analyse antimicrobial susceptibility evolution of equine pathogens isolated from clinical samples from 2006-2016. METHODS: A collection of 25 813 bacterial isolates was studied, clustered according to their origins (respiratory tract, cutaneous, genital and other), and analysed for their antimicrobial susceptibility using the disk diffusion method. RESULTS: The most frequently isolated pathogens were group C Streptococci (27.6%), Escherichia coli (20.0%), Staphylococcus aureus (7.8%), Pseudomonas aeruginosa (4.0%), Enterobacter spp. (3.4%), Klebsiella pneumoniae (2.4%), and Rhodococcus equi (1.8%). Of the isolates, 9512 were from respiratory samples (36.8%), 7689 from genital origin (29.8%), and 4083 from cutaneous samples (15.8%). Over the 11-year period, the frequency of multidrug-resistant (MDR) strains fluctuated between 6.4-20.4% for group C Streptococci and 17-37.7% for Klebsiella pneumoniae. From 2006-2009, 24.5-43.0% of Staphylococcus aureus isolates were MDR; after 2009 the level did not exceeded 27.6%. For Escherichia coli and Enterobacter spp., these levels were mostly >30.0% until 2012, but significantly decreased thereafter (22.5-26.3%). CONCLUSIONS: This study is the first large-scale analysis of equine pathogens, by the number of samples and duration of study. The results showed high levels of MDR strains and the need to support veterinary antimicrobial stewardship to encourage proper use of antibiotics.

Effect of Macrolide and Rifampin Resistance on the Fitness of Rhodococcus equi.

Rhodococcus equi is a leading cause of severe pneumonia in foals. Standard treatment is dual antimicrobial therapy with a macrolide and rifampin, but the emergence of macrolide- and rifampin-resistant R. equi isolates is an increasing problem. The objective of this study was to determine the effect of macrolide and/or rifampin resistance on fitness of R. equi Three unique isogenic sets were created, each consisting of four R. equi strains, as follows: a susceptible parent isolate, strains resistant to macrolides or rifampin, and a dual macrolide- and rifampin-resistant strain. Each isogenic set's bacterial growth curve was generated in enriched medium, minimal medium (MM), and minimal medium without iron (MM-I). Bacterial survival in soil was analyzed over 12 months at -20°C, 4°C, 25°C, and 37°C, and the ability of these strains to retain antimicrobial resistance during sequential subculturing was determined. Insertion of the mobile element conferring macrolide resistance had minimal effect on in vitro growth. However, two of three rpoB mutations conferring rifampin resistance resulted in a decreased growth rate in MM. In soil, macrolide- or rifampin-resistant R. equi strains exhibited limited growth compared to that of the susceptible R. equi isolate at all temperatures except -20°C. During subculturing, macrolide resistance was lost over time, and two of three rpoB mutations reverted to the wild-type form. The growth of rifampin-resistant R. equi colonies is delayed under nutrient restriction. In soil, possession of rifampin or macrolide resistance results in decreased fitness. Both macrolide and rifampin resistance can be lost after repeated subculturing.IMPORTANCE This work advances our understanding of the opportunistic environmental pathogen Rhodococcus equi, a disease agent affecting horses and immunocompromised people. R. equi is one of the most common causes of severe pneumonia in young horses. For decades, the standard treatment for R. equi pneumonia in horses has been dual antimicrobial therapy with a macrolide and rifampin; effective alternatives to this combination are lacking. The World Health Organization classifies these antimicrobial agents as critically important for human medicine. Widespread macrolide and rifampin resistance in R. equi isolates is a major emerging problem for the horse-breeding industry and might also adversely impact human health if resistant strains infect people or transfer resistance mechanisms to other pathogens. This study details the impact of antimicrobial resistance on R. equi fitness, a vital step for understanding the ecology and epidemiology of resistant R. equi isolates, and will support development of novel strategies to combat antimicrobial resistance.

Emergence of Resistance to Macrolides and Rifampin in Clinical Isolates of Rhodococcus equi from Foals in Central Kentucky, 1995 to 2017.

The objective of this study was to determine the prevalence of Rhodococcus equi strains resistant to macrolides and rifampin over time in clinical samples from foals submitted to diagnostic laboratories in central Kentucky. We performed a retrospective observational study of all clinical samples from foals that were submitted to veterinary diagnostic laboratories in Kentucky between January 1995 and December 2017. Samples were included if the R. equi bacterium was cultured and tested for in vitro susceptibility to erythromycin or rifampin. In vitro susceptibility testing to erythromycin was available for 2,169 isolates of R. equi, while susceptibility testing to both erythromycin and rifampin was available for 1,681 isolates. Rifampin resistance was first detected in 2000, and erythromycin resistance was first detected in 2004. Between 1995 and 2006, the proportion of resistant isolates of R. equi was 0.7% for erythromycin and 2.3% for rifampin. There was a significant (P < 0.001) increase in the proportion of resistant R. equi between 2007 and 2017, with 13.6% of isolates being resistant to erythromycin and 16.1% being resistant to rifampin. Between 2007 and 2017, isolates of R. equi resistant to erythromycin or rifampin were significantly less likely to be isolated from feces than from the respiratory tract, other soft tissues, or musculoskeletal infections. The considerable increase in the prevalence of isolates of R. equi resistant to macrolides and rifampin since 2007 is of concern for both human and animal health.

Absceso glúteo por Rhodococcus Equi: presentación atípica de enfermedad emergente / Gluteus abscess due to RhodococcusEqui: atypical presentation of emerging disease

Resumen Rhodococcusequi, bacteria aislada principalmente en potros, se considera en la actualidad un patógeno zoonótico emergente en seres humanos, la mayoría inmunocomprometidos, coincidiendo con la epidemia de VIH y los avances en la medicina de trasplantes y la quimioterapia del cáncer, lo que justifica la revisión sobre su patogenia, características clínicas, diagnóstico y tratamiento. Más frecuente en hombres que en mujeres, el inicio de las infecciones es generalmente insidioso y los síntomas que presentan varían según el sitio de la infección, la enfermedad respiratoria, caracterizada por neumonía necrosante es la manifestación más común, y tiende a seguir un curso subagudo, mientras las personas inmunocompetentes son afectadas con baja frecuencia y suelen presentarse con enfermedad localizada. Las infecciones de la piel o partes blandas, incluyendo abscesos, son poco frecuentes incluso en huéspedes inmunocomprometidos y están usualmente relacionadas con contaminación ambiental. El laboratorio de microbiología juega un papel clave en la identificación del organismo, ya que este puede confundirse con flora normal de la piel. El caso que se presenta corresponde a una paciente adulta inmunocompetente, con infección de herida quirúrgica por R equiluego de una cirugía estética, requirió tratamiento antibiótico, múltiples lavados quirúrgicos y debridamientos, más drenaje de absceso, con mejoría del proceso infeccioso. Este caso sugiere la importancia clínica de la identificación precisa de patógenos inusuales, capaces de entorpecer la evolución normal de un proceso quirúrgico y de producir un alto costo de atención de salud y el alcance medidas preventivas relacionadas con la vigilancia de la salud.

Abstract Rhodococcusequi, a bacterium isolated mainly in foals, is currently considered an emerging zoonotic pathogen in humans, most of them immunocompromised, coinciding with the HIV epidemic and advances in transplant medicine and cancer chemotherapy, which justifies the review on its pathogenesis, clinical characteristics, diagnosis and treatment. More frequent in men than in women, the onset of infections is usually insidious and the symptoms they present vary according to the site of infection, the respiratory disease, characterized by necrotizing pneumonia is the most common manifestation, and tends to follow a subacute course , while immunocompetent people are affected with low frequency and usually have localized disease. Infections of the skin or soft tissues, including abscesses, are rare even in immunocompromised hosts and are usually related to environmental contamination. The microbiology laboratory plays a key role in the identification of the organism, since it can be confused with normal flora of the skin. The case presented corresponds to an adult immunocompetent patient, with surgical wound infection by R equiafter cosmetic surgery, required antibiotic treatment, multiple surgical washes and debridements, and abscess drainage, with improvement of the infectious process. This case suggests the clinical importance of the precise identification of unusual pathogens, capable of obstructing the normal evolution of a surgical process and of producing a high cost of health care and the scope of preventive measures related to health surveillance.

In vitro performances of novel co-spray-dried azithromycin/rifampicin microparticles for Rhodococcus equi disease treatment.

This work was aimed at providing clues on the in vitro performances of novel azithromycin/rifampicin combinations, in the form of co-spray-dried microparticles (AZM/RIF MP), against Rhodococcus equi, an animal and emerging human pathogen found responsible for worrying zoonosis. Various AZM/RIF combinations were spray-dried and characterized for their morphology and size. Susceptibility studies included determination of MIC, MBC, Fractional Inhibitory/Bactericidal Concentration Indexes and intracellular activity in R. equi-infected THP-1 cells. Cytotoxicity was tested on BEAS-2B cells through MTT assay and combination index assessment for drug interaction. Spray-dried MP were collapsed and 3-10 times smaller than commercial powders. Drug combinations showed an enhancement of in vitro antibacterial activity with a remarkable synergistic bactericidal effect. Azithromycin MP and AZM/RIF MP 2:1 led to a CFU reduction of >90% up to 4 days after treatment at all tested concentrations (p = 0.001) but AZM/RIF MP 2:1 were at least four-fold more potent than AZM MP alone. IC50 values of >100 mg/L supported low cytotoxicity of drug combinations and the combination index suggested an antagonistic toxic effect. Co-spray-drying enhanced powder dispersibility and solubility, which may improve bioavailability as well as provide administration alternatives. The novel AZM/RIF MP combinations could result a valid platform to develop new treatment strategies against R. equi infections in animals and humans.

Isolated Mediastinal Lymphadenopathy Caused by Rhodococcus equi Infection.

Mediastinal lymphadenopathy is common finding in thoracic surgery, and it often requires morphologic confirmation to establish the definitive diagnosis. The most frequent diagnoses are metastatic lung cancer, sarcoidosis, lymphoma, tuberculosis, and other causes of granulomatous infections. Rhodococcus equi is a rare pathogen in humans that mostly affects immunocompromised patients. This report presents a case with isolated mediastinal lymphadenopathy caused by Rhodococcus equi infection in a 71-year-old immunocompetent patient.

Effects of priming with cytokines on intracellular survival and replication of Rhodococcus equi in equine macrophages.

Rhodococcus equi is a common cause of pneumonia in foals and an opportunistic pathogen in immunosuppressed people. The ability of R. equi to survive and replicate in macrophages is the basis of its pathogenicity. Limited knowledge about the role of cytokines in host defense against R. equi comes from studies in mice and the role of cytokines in intracellular survival of R. equi in equine macrophages is unknown. The objectives of this study were to determine the effect of priming with interferon (IFN)-γ, interleukin (IL)-1ß, IL-4, IL-6, IL-10, or tumor necrosis factor (TNF)-α at various concentrations on intracellular survival of virulent R. equi in equine monocyte-derived macrophages (MDM), and to determine the effects of various combinations of the same cytokines on intracellular survival of R. equi. MDM from 10 adult horses were primed with recombinant equine cytokines at doubling concentrations ranging from 25 to 200 ng/mL prior to infection with virulent R. equi. Priming with IFN-γ, TNF-α, or IL-6 significantly decreased intracellular replication of R. equi compared to unprimed monolayers. In contrast, priming with IL-10 or IL-1ß significantly increased intracellular replication of R. equi. Pairwise combinations of the cytokines listed above did not results in synergism or antagonism. This study demonstrated that IFN-γ, TNF-α, or IL-6 improved equine MDM function against R. equi whereas IL-1ß or IL-10 were detrimental.

Antimicrobial Resistance in Rhodococcus equi.

Pneumonia caused by Rhodococcus equi remains an important cause of disease and death in foals. The combination of a macrolide (erythromycin, azithromycin, or clarithromycin) with rifampin has been the recommended treatment for foals with clinical signs of infection caused by R. equi since the early 1980s with, until recently, only rare reports of resistance. Resistance to macrolides and rifampin in isolates of R. equi cultured from horses is increasing, with isolates resistant to all macrolides and rifampin now being cultured from up to 40% of infected foals at some farms. This text reviews the available data regarding antimicrobial resistance in R. equi, with emphasis on the molecular mechanisms of the recent emergence of resistance to macrolides and rifampin in equine isolates of R. equi.

Efficacy of Tulathromycin for the Treatment of Foals with Mild to Moderate Bronchopneumonia.

BACKGROUND: There is conflicting data regarding the efficacy of tulathromycin for the treatment of foals with bronchopneumonia. HYPOTHESES: Tulathromycin is effective for the treatment of bronchopneumonia in foals and noninferior to the combination of azithromycin and rifampin. ANIMALS: A total of 240 foals on a farm endemic for infections caused by Rhodococcus equi. METHODS: In a controlled, randomized, and double-blinded clinical trial, foals with ultrasonographic pulmonary lesions (abscess score 10-15 cm) were allocated to 3 groups: 1-tulathromycin IM q 7 days (n = 80); 2-azithromycin-rifampin, orally q24h (n = 80); or 3-untreated controls (n = 80). Physical examination and thoracic ultrasonography were performed by individuals unaware of treatment group assignment. Foals that worsened were considered treatment failures and removed from the study. RESULTS: The proportion of foals that recovered was significantly higher for foals treated with tulathromycin (70 of 79) or azithromycin-rifampin (76 of 80) compared to that of control foals (22 of 80). The difference in the percentage of efficacy of azithromycin-rifampin versus tulathromycin was 6.4% (90% CI = -0.72-13.5%). Given that the confidence interval crossed the predetermined noninferiority limit of 10%, the null hypothesis that the response rate in the azithromycin-rifampin group is superior to that of the tulathromycin group could not be rejected. Resolution of ultrasonographic lesions occurred faster in foals treated with azithromycin-rifampin than in foals treated with tulathromycin. CONCLUSION AND CLINICAL IMPORTANCE: Tulathromycin was effective for the treatment of bronchopneumonia in foals at this farm but not as effective as the combination of azithromycin-rifampin.

Differences in Rhodococcus equi Infections Based on Immune Status and Antibiotic Susceptibility of Clinical Isolates in a Case Series of 12 Patients and Cases in the Literature.

Rhodococcus equi is an unusual zoonotic pathogen that can cause life-threatening diseases in susceptible hosts. Twelve patients with R. equi infection in Kentucky were compared to 137 cases reported in the literature. Although lungs were the primary sites of infection in immunocompromised patients, extrapulmonary involvement only was more common in immunocompetent patients (P < 0.0001). Mortality in R. equi-infected HIV patients was lower in the HAART era (8%) than in pre-HAART era (56%) (P < 0.0001), suggesting that HAART improves prognosis in these patients. Most (85-100%) of clinical isolates were susceptible to vancomycin, clarithromycin, rifampin, aminoglycosides, ciprofloxacin, and imipenem. Interestingly, there was a marked difference in susceptibility of the isolates to cotrimoxazole between Europe (35/76) and the US (15/15) (P < 0.0001). Empiric treatment of R. equi infection should include a combination of two antibiotics, preferably selected from vancomycin, imipenem, clarithromycin/azithromycin, ciprofloxacin, rifampin, or cotrimoxazole. Local antibiograms should be checked prior to using cotrimoxazole due to developing resistance.

Chloroquine inhibits Rhodococcus equi replication in murine and foal alveolar macrophages by iron-starvation.

Rhodococcus equi preferentially infects macrophages causing pyogranulomatous pneumonia in young foals. Both the vapA and rhbC genes are up-regulated in an iron (Fe)-deprived environment, such as that found within macrophages. Chloroquine (CQ) is a drug widely used against malaria that suppresses the intracellular availability of Fe in eukaryotic cells. The main objective of this study was to evaluate the ability of CQ to inhibit replication of virulent R. equi within murine (J774A.1) and foal alveolar macrophages (AMs) and to verify whether the mechanism of inhibition could be Fe-deprivation-dependent. CQ effect on R. equi extracellular survival and toxicity to J774A.1 were evaluated. R. equi survival within J774A.1 and foal AMs was evaluated under CQ (10 and 20µM), bovine saturated transferrin (bHTF), and bovine unsaturated transferrin (bATF) exposure. To explore the action mechanism of CQ, the superoxide anion production, the lysozyme activity, as well as the relative mRNA expression of vapA and rhbC were examined. CQ at≤20µM had no effect on R. equi extracellular multiplication and J774A.1 viability. Exposure to CQ significantly and markedly reduced survival of R. equi within J774A.1 and foal AMs. Treatment with bHTF did not reverse CQ effect on R. equi. Exposure to CQ did not affected superoxide anion production or lysozyme activity, however vapA and rhbC expression was significantly increased. Our results reinforce the hypothesis that intracellular availability of Fe is required for R. equi survival, and our initial hypothesis that CQ can limit replication of R. equi in J774A.1 and foal AMs, most likely by Fe starvation.