Rhodococcus Equi: Challenges to Treat Infections and to Mitigate Antimicrobial Resistance.

Rhodococcus equi, a gram-positive facultative intracellular pathogen and a soil saprophyte, is one of the most common causes of pneumonia in young foals. It poses a threat to the economy in endemic horse-breeding farms and to animal welfare annually. Many farms use thoracic ultrasonographic screening and antimicrobial treatment of subclinically affected foals as a preventive measure against severe R. equi infections. The wide use antimicrobials to treat subclinically affected foals has contributed to the emergence of multidrug resistant (MDR)-R. equi in both clinical isolates from sick foals and in the environment of horse-breeding farms. Alternatives to treat foals infected with MDR-R. equi are scarce and the impact of the emergence of MDR-R. equi in the environment of farms is still unknown. The aim of this review is to discuss the emergence of MDR-R. equi in the United States and the challenges faced to guide antimicrobial use practices. Reduction of antimicrobial use at horse-breeding farms is essential for the preservation of antimicrobial efficacy and, ultimately, human, animal, and environmental health.

Rhodococcus equi-What is New This Decade?

Foals become infected shortly after birth; most develop subclinical pneumonia and 20% to 30% develop clinical pneumonia that requires treatment. It is now well established that the combination of screening programs based on thoracic ultrasonography and treatment of subclinical foals with antimicrobials has led to the development of resistant Rhodococcus equi strains. Thus, targeted treatment programs are needed. Administration of R equi-specific hyperimmune plasma shortly after birth is beneficial as foals develop less severe pneumonia but does not seem to prevent infection. This article provides a summary of clinically relevant research published during this past decade.

Construction of live-attenuated Trueperella pyogenes by antibiotic treatment and sequential passage: methods for vaccine development.

Trueperella pyogenes (T. pyogenes) is a zoonotic pathogen that is cause a variety of pyogenic diseases in animals. The complex pathogenicity and various virulence factors are important challenges to produce an effective vaccine. According to previous trials, inactivated whole-cell bacteria or recombinant vaccines were unsuccessful in preventing disease. Thus, this study aims to introduce a new vaccine candidate based on a live-attenuated platform. For this purpose, first T. pyogenes was subjected to sequential passage (SP) and antibiotic treatment (AT) to lose their pathogenicity. Second, Plo and fimA expressions as virulence genes were evaluated by qPCR and then mice were challenged with bacteria from SP and AT culture by intraperitoneal route. Compared to the control group (T. pyogenes-wild type), plo and fimA gene expressions were downregulated and vaccinated mice have a normal spleen appearance in contrast to the control group. In addition, there was no significant difference between bacterial count from spleen, liver, heart and peritoneal fluid in vaccinated mice and the control group. In conclusion, this study introduces a new T. pyogenes vaccine candidate based on a live-attenuated strategy that mimics natural infection without pathogenicity for further investigation on vaccines against T. pyogenes infections.

Involvement of the E3 ubiquitin ligase Cblb in host defense and evaluation of transcriptome during Trueperella pyogenes infection.

Trueperella pyogenes (T. pyogenes) is a versatile and ingenious bacterium that causes severe suppurative injuries in lots of economically important ruminants. The underlying pathogenesis of T. pyogenes infection remains poorly understood. In the current study, we performed transcriptome sequencing of mouse blood tissue infected with T. pyogenes. A total of 36.73 G clean data were collected, and 136 differentially expressed genes were obtained in the infection group compared to the control group. In addition, we found that the E3 ubiquitin ligase Cblb exhibited significant upregulation in the infection groups compared to the control group. Mechanistically, T. pyogenes infection markedly enhanced the expression of Cblb and regulated the host defense response. Inhibiting Cblb expression with Cblb siRNA impaired the inflammatory response and reduced the effect of phagocytosis in RAW264.7 murine macrophages. Intriguingly, overexpression of Cblb induced a strong inflammatory response and enhanced phagocytosis against T. pyogenes infection in macrophages. More importantly, the overexpression of Cblb significantly reduced the bacterial load and protected mice from the T. pyogenes infections. Therefore, our findings reveal that Cblb is a novel and potential regulator in response to T. pyogenes infection and shed new light on the development of promising treatments against T. pyogenes-related diseases.

Epidemiology and in vitro antimicrobial susceptibility of aerobic Actinomycetales in a clinical setting / Epidemiología y sensibilidad in vitro de especies de Actinomycetales aerobios en la rutina asistencial

Introduction: The incidence of infections caused by aerobic actinomycetes is increasing. Recent changes in taxonomy and the variability in susceptibility patterns among species make necessary a proper identification and antibiotic susceptibility testing. Material and methods: Fifty-three strains of aerobic actinomycetes were identified by MALDI-TOF MS using the VITEK MS Mycobacterium/Nocardia kit (bioMérieux, France) in a tertiary hospital in Spain during a six-year period. Antimicrobial susceptibility testing of the isolates was performed using the Sensititre Rapmycoi microdilution panel (Thermo Fisher Scientific, Massachusetts, USA). Results: Forty strains of Nocardia spp. were identified in the study, being N. farcinica and N. cyriacigeorgica the most prevalent ones. All isolates were susceptible to linezolid and the resistance to amikacin was only observed in one isolate of Gordonia sputi. Resistance to cotrimoxazole was only found in five isolates. Conclusions: Routine identification and antimicrobial susceptibility testing of aerobic actinomycetes is advisable for an efficient identification of species and effective treatment.(AU)

Introducción: La incidencia de infecciones por actinomicetos aerobios está aumentando. Los recientes cambios en la taxonomía y la variabilidad en la sensibilidad entre especies hacen necesaria una identificación y estudio de sensibilidad adecuados. Material y métodos: Se identificaron 53 cepas de actinomicetos aerobios mediante MALDI-TOF utilizando el kit VITEK-MS Mycobacterium/Nocardia (bioMérieux, Francia) en un hospital terciario español durante seis años. Los estudios de sensibilidad de los aislados se realizaron utilizando el panel de microdilución Sensititre Rapmycoi (Thermo Fisher Scientific, Massachusetts, EE. UU.). Resultados: Se identificaron 40 cepas de Nocardia spp., siendo Nocardia farcinica y Nocardia cyriacigeorgica las más prevalentes. Todos los aislados fueron sensibles a linezolid, y solo se detectó resistencia a amikacina en un aislado de Gordonia sputi. Solo se encontró resistencia al cotrimoxazol en cinco aislados. Conclusiones: Es aconsejable realizar la identificación de rutina y las pruebas de sensibilidad antimicrobiana de los actinomicetos aerobios para conseguir una identificación eficiente de las especies y un tratamiento eficaz.(AU)

Tuberculosis with cavities? Rapid diagnosis of Rhodococcus equi pulmonary infection with cavities by acid-fast staining: A case report.

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.

Rhodococcus equi-Derived Extracellular Vesicles Promoting Inflammatory Response in Macrophage through TLR2-NF-κB/MAPK Pathways.

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-1ß, 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.

International Spread of Multidrug-Resistant Rhodococcus equi.

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.

Fitness cost conferred by the novel erm(51) and rpoB mutation on environmental multidrug resistant-Rhodococcus equi.

Rhodococcus equi is a common cause of severe pneumonia in foals. Emergence of macrolide-resistant R. equi isolated from foals and their environment has been reported in the United States. A novel erm(51) gene was recently identified in R. equi in soil from horse farms in Kentucky. Our objective was to determine the effect of the erm(51) gene and associated rpoB mutation on the fitness of multidrug resistant-R. equi (MDR-R. equierm(51)+, rpoB+) under different nutrient conditions. Bacterial growth curves were generated for 3 MDR-R. equierm(51)+, rpoB+ isolates and 3 wild-type (WTN) R. equi isolates recovered from environmental samples of farms in central Kentucky. Growth was measured over 30.5 h in brain-heart infusion broth (BHI), minimal medium (MM), and minimal medium without iron (MM-I). All isolates had significantly (P < 0.05) higher growth in BHI compared to either MM or MM-I. MDR-R. equierm(51)+, rpoB+ exhibited significantly lower growth compared to WTN isolates in BHI (nutrient-rich condition), but not in either MM or MM-I (nutrient-restricted conditions). This study indicates that under nutrient-rich conditions fitness of MDR-R. equierm(51)+, rpoB+ is reduced relative to susceptible isolates; however, under nutrient-restricted conditions MDR-R. equierm(51)+, rpoB+ isolates grow similarly to susceptible isolates. These findings indicate that MDR-R. equierm(51)+, rpoB+ might be outcompeted by susceptible isolates in nature when practices to reduce antimicrobial pressure, such as reducing antimicrobial use in foals, are implemented. But it also raises the concern that these resistant genotypes might persist in the environment of horse-breeding farms in the face of selective pressures such as antimicrobials or nutrient restriction.

Arcanobacterium haemolyticum: A case series.

Arcanobacterium haemolyticum formerly known as Corynebacteria haemolyticum is a Gram positive bacilli. It is a fastidious, facultative anerobic, catalase negative, beta haemolytic and non motile bacterium. Gram positive bacilli are usually considered to be non-pathogenic as the majority are part of normal flora of human skin and mucous membranes. Hence, diagnosis of such infection and its treatment may be delayed by a failure of recognition. However, this bacterium has been implicated in wound, superficial and deep-seated soft tissue infections, endocarditis, osteomyelitis, meningitis, pneumonia, and also septicemia. The early diagnostic evaluation of this organism is emphasized. We report a case series which illustrates the significance of Arcanobacterium haemolyticum in skin and soft tissue infections.

Virulence plasmids in clinical isolates of Rhodococcus equi from sick foals in the Netherlands.

Clinical samples from 123 foals with suspected rhodococcosis submitted to the Veterinary Microbiological Diagnostic Centre of the Faculty of Veterinary Medicine between 1993 and 2006 were tested for the presence of the virulence gene vapA. Of the 123 samples, 120 were vapA-positive and 3 vapA-negative Rhodococcus equi were isolated. The 120 vapA-positive R. equi were isolated from 70 tracheal wash, 19 lung tissues, 7 lymph nodes, 6 synovial fluids, 13 abscesses or pus and single isolates from the uterus, gut, cerebrospinal fluid, abdomen fluid and faeces. Of the 120 isolates, 46 were from Dutch warmblood horses, 23 from Friesian horses, 14 from Trotters, 4 from Holsteiners, 3 from Arab breed, 2 from ponies, 1 from a Welsh pony and 27 from undefined breed horses. Using plasmid profile analysis of the 120 isolates, 117 isolates contained the 85-kb type I plasmid, 2 contained the 87-kb type I plasmid and 1 contained the novel 52-kb non-mobilizable virulence plasmid reported recently. These results showed that the virulent R. equi strains harbouring a virulence plasmid of 85-kb type I or 87-kb type I, which have been detected in clinical isolates from five European countries, are widespread in the Netherlands. This is the first report of plasmid types of clinical R. equi isolates in the Netherlands.

An Autobioluminescent Method for Evaluating In Vitro and In Vivo Growth of Rhodococcus equi.

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.

Arsenicicoccus cauae sp. nov., isolated from the blood of a pediatric gastroenteritis patient.

A Gram-stain-positive coccus was isolated from the blood of a paediatric patient suffering from gastroenteritis. The taxonomic position of this catalase-positive, non-motile, non-spore-forming facultative anaerobe designated as strain MKL-02T was investigated using a polyphasic approach. Colonies grown on tryptic soy agar with 10 % sheep blood were circular, creamy yellow, and convex. Phylogenetic analysis based on 16S rRNA gene and whole-genome sequences revealed that this strain was most closely related to Arsenicicoccus bolidensis CCUG 47306T within the cluster of the genus Arsenicicoccus. Average nucleotide identity and digital DNA-DNA hybridization values between strain MKL-02T and A. bolidensis DSM 15745T, A. dermatophillus DSM 25571T and A. piscis DSM 22760T were 89.5 and 37.0 %, 79.6 and 22.4 %, and 75.9 and 21.0 %, respectively. The genomic size of strain MKL-02T was 3 423 857 bp with a 72.7 mol% G+C content. Growth was observed at 10-45 °C (optimum, 37-40 °C) and pH 6.0-10.0 (optimum, pH 7.0), in the presence of 0-10 % (w/v) NaCl (optimum, 0.5 %). Cells of strain MKL-02T were non-motile cocci and 0.50-0.60 µm long, as determined by transmission electron microscopy. The strain was catalase-positive and oxidase-negative. The major fatty acid type (>10 % of total) was C15 : 0. The polar lipid profile consisted of two unidentified phospholipids, three unidentified lipids and an unidentified aminophospholipid. The strain contained MK-8 (H4) as the predominant menaquinone. Based on phylogenetic and phenotypic considerations, it is proposed that strain MKL-02T be classified as a new species, named Arsenicicoccus cauae sp. nov. The type strain is MKL-02T (=NCCP 16967T=JCM 34624T).

Fecal concentration of Rhodococcus equi determined by quantitative polymerase chain reaction of rectal swab samples to differentiate foals with pneumonia from healthy foals.

BACKGROUND: Diagnostic accuracy of real-time, quantitative PCR (qPCR) assays to quantify virulent Rhodococcus equi using rectal swab samples has not been systematically evaluated. OBJECTIVE: To evaluate the accuracy of qPCR of rectal swab samples to differentiate foals with pneumonia from healthy foals of similar age from the same environment. ANIMALS: One hundred privately owned foals born in 2021 from 2 farms in New York. METHODS: An incident case-control study design was used. Rectal swabs were collected from all foals diagnosed with R. equi pneumonia at 2 horse-breeding farms (n = 47). Eligible pneumonia cases (n = 39) were matched by age to up to 2 healthy (n = 53) control foals; rectal swabs were collected from control foals on the day of diagnosis of the index case. DNA was extracted from fecal swabs and the concentration of virulent R. equi (ie, copy numbers of the virulence-associated protein A gene [vapA] per 100 ng fecal DNA) was estimated by qPCR. RESULTS: The area under the ROC curve for qPCR of fecal swabs was 83.7% (95% CI, 74.9-92.6). At a threshold of 14 883 copies of vapA per 100 ng fecal DNA, specificity of the assay was 83.0% (95% CI, 71.7-92.4) and sensitivity was 79.5% (95% CI, 66.7-92.3). CONCLUSIONS AND CLINICAL IMPORTANCE: Although fecal concentrations of virulent R. equi are significantly higher in pneumonic foals than healthy foals of similar age in the same environment, qPCR of rectal swabs as reported here lacks adequate diagnostic accuracy for clinical use.

Association of pneumonia with concentrations of virulent Rhodococcus equi in fecal swabs of foals before and after intrabronchial infection with virulent R. equi.

BACKGROUND: Intragastric administration of virulent Rhodococcus equi protects foals against subsequent experimental intrabronchial (IB) infection, but it is unknown whether R. equi naturally ingested by foals contributes to their susceptibility to pneumonia. HYPOTHESIS: Fecal concentration of virulent R. equi before IB infection with R. equi is positively associated with protection from pneumonia in foals. ANIMALS: Twenty-one university-owned foals. METHODS: Samples were collected from experimental studies. Five foals were gavaged with live, virulent R. equi (LVRE) at age 2 and 4 days; the remaining 16 foals were not gavaged with LVRE (controls). Fecal swabs were collected from foals at ages 28 days, immediately before IB infection. Foals were monitored for clinical signs of pneumonia, and fecal swabs were collected approximately 2 weeks after IB infection. Swabs were tested by quantitative PCR for concentration of virulent R. equi (ie, copy numbers of the virulence-associated protein A gene [vapA] per 100 ng fecal DNA). RESULTS: Fecal concentrations of virulent R. equi (vapA) before IB infection were significantly (P < .05) lower in control foals (25 copies/100 ng DNA [95% CI, 5 to 118 copies/100 ng DNA) that developed pneumonia (n = 8) than in healthy control foals (n = 8; 280 copies/100 ng DNA; 95% CI, 30 to 2552 copies/100 ng DNA) or those gavaged with LVRE (707 copies/100 ng DNA, 95% CI, 54 to 9207 copies/100 ng DNA). CONCLUSIONS AND CLINICAL IMPORTANCE: Greater natural ingestion of LVRE might contribute to protection against pneumonia among foals.

The type of anticoagulant used for plasma collection affects in vitro Rhodococcus equi assays.

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.

A rare case of male Fournier's gangrene with mixed Actinomyces turicensis infection.

BACKGROUND: Fournier's gangrene (FG), a urological emergency with high mortality, is an infectious necrotizing fasciitis of the perineal and genital regions. The majority of FG is caused by polymicrobial organisms involving mixed aerobes and anaerobes but rarely reveals Actinomyces species. CASE PRESENTATION: We report a healthy 67-year-old Asian male who presented with rapidly progressive painful swelling of the scrotum. Clinically diagnosed with FG, the patient underwent an emergency radical debridement, followed by broad-spectrum antibiotics and negative pressure wound therapy. The identification of the causative microorganisms showed Actinomyces turicensis and the antibiotic treatment was adjusted accordingly. After wound bed preparation, we took split-thickness skin grafts to cover the scrotal wound. Active management to minimize faecal contamination was applied throughout the whole course of treatment and repair. The patient was satisfied with the outcome. This was an extremely rare case of A. turicensis as the main causative pathogen of FG. CONCLUSIONS: FG due to Actinomyces species is rarely reported, but we should still consider this pathogenic microorganism that has long been neglected.