Comparison of two selective media for the recovery, isolation, enumeration and differentiation of Rhodococcus equi

The use of selective media to facilitate the isolation of Rhodococcus equi from environmental and clinical samples has aided studies of the ecology of R. equi and the epidemiology of disease caused by R. equi. Here, we compared the efficacy of two selective media (NANAT and modified CAZ-NB) for the recovery of six defined strains of R. equi and for the isolation and enumeration of both avirulent and virulent R. equi from 60 paired soil samples from horse farms using colony blotting and DNA hybridisation. No difference was found between the two media in the recoverability of defined strains of R. equi or the proportion of soil cultures positive for R. equi or virulent R. equi. NANAT medium was significantly less inhibitory of bacterial growth from soil culture compared to mCAZ-NB (P = 0.001), but there was no difference between the media in the number of R. equi colonies recovered. Soil cultured on mCAZ-NB medium yielded a significantly greater number of virulent R. equi colonies than NANAT (P = 0.03). The proportion of R. equi that were virulent in soil cultures on mCAZ-NB (32%) was more than three times that seen in cultures on NANAT (9%). Thus modified CAZ-NB appeared to be a better selective media for studies where the optimal recovery of virulent R. equi is required, such as in studies of the gastrointestinal carriage of virulent R. equi and of subclinically infected foals.     

Development of a new selective medium for isolation of methicillin-resistant Staphylococcus aureus

A new selective medium containing cephem antibiotics was developed for isolation of methicillin-resistant Staphylococcus aureus (MRSA). MRSA colonies on a medium containing ceftazidime (CAZ) were most easily identifiable and a medium containing cefoperazone (CPZ) was superior in suppressing the growth of other bacteria. With the medium containing a couple of CAZ and CPZ, MRSA and methicillin-resistant coagulase-negative staphylococci (MRCNS) were detected from 2 and 1 of 15 chicken meat samples respectively. The MRSA and MRCNS recovery test showed that the medium was effective for MRSA isolation, suppressing the growth of other bacteria efficiently. These results suggested that the medium containing a couple of CAZ and CPZ was useful for MRSA detection from foods and animals.     

A new selective medium for the isolation of Listeria monocytogenes.

A new selective medium for the isolation of Listeria monocytogenes (Lm), is described. The medium contained propolis, nalidixic acid, polymyxin B and rivanol as selective substances. The new medium (propolis-agar) was compared with two other selective media and one nonselective medium. No inhibitory effect was found on the 6 strains of Lm tested, and Lm was easily isolated from a mixture of Lm and contaminating bacteria. The selective effect was better than for the two other selective media tested.     

Selective medium containing fosfomycin, nalidixic acid, and culture supernatant of Rhodococcus equi for isolation of Corynebacterium pseudotuberculosis.

FNR medium containing fosfomycin, nalidixic acid, bovine blood and culture supernatant of Rhodococcus equi was prepared by the present authors, and the medium did not inhibit growth of Corynebacterium pseudotuberculosis but completely hampered the growth of Bacillus subtilis, Staphylococcus aureus, and Escherichia coli. The culture supernatant of R. equi facilitated detection of suspected colonies of C. pseudotuberculosis due to synergistic hemolysis. Rate of isolation of the organisms (from the trachea, larynx and nasal cavity of 16 slaughtered sheep with caseous abscess in the lung) was higher with FNR, the selective medium, than with nonselective medium. The selective medium was thus found to be useful for isolation of C. pseudotuberculosis from sheep.     

Evaluation of a selective medium for isolation of Haemophilus pleuropneumoniae.

Crystal violet, lincomycin, spectinomycin and bacitracin were evaluated as selective agents in media for isolation of Haemophilus pleuropneumoniae. No single antimicrobial agent or combination of two or more inhibited all non-Haemophilus strains (Escherichia coli, Pasteurella haemolytica, Pasteurella multocida, Streptococcus faecalis, Streptococcus equisimilis and Staphylococcus aureus) without marked suppression of 16 H. pleuropneumoniae strains. A medium containing 1 micrograms/mL of crystal violet, 1 microgram/mL of lincomycin, 8 micrograms/mL of spectinomycin and 128 micrograms/mL of bacitracin inhibited one E. coli strain and the Gram-positive strains while H. pleuropneumoniae strains were suppressed to a minor degree only. Haemophilus pleuropneumoniae was isolated on the selective medium on three occasions from the nose or pharynx of two out of eight experimentally inoculated pigs. Haemophilus pleuropneumoniae was recovered from the nose of only two pigs at necropsy and from tonsil of one, whereas the lower airways in most pigs and the lung lesions in all pigs were positive. There was no advantage to using the selective medium for the recovery of H. pleuropneumoniae at necropsy from these eight experimentally infected pigs, probably because other bacteria were absent or present in very low numbers in the tissues with H. pleuropneumoniae. The isolation rate on selective medium was higher than the rate on non-selective medium (p less than or equal to 0.1; chi 2 test) when the airways of slaughtered pigs were cultured. This was likely due to a high degree of contamination. Dry swabs placed in tryptone yeast extract with nicotinamide-adenine-dinucleotide gave a significantly higher recovery rate than commercial Culturette swabs in modified Stuart's transport medium.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification and differentiation of avirulent and virulent Rhodococcus equi using selective media and colony blotting DNA hybridization to determine their concentrations in the environment

Selective agar media have been used for many years to facilitate the isolation of Rhodococcus equi from environmental and clinical samples. However, characterisation of R. equi still requires the use of immunochemical or polymerase chain reaction (PCR) analysis to differentiate between virulent and avirulent isolates. Here, we describe a novel method to detect and differentiate between R. equi isolates using colony blotting and DNA hybridization. Radiolabelled PCR product derived from the R. equi rrnA gene and specific hybridization conditions enabled differentiation of colonies of R. equi from environmental species, whilst radiolabelled PCR product derived from the R. equi vapA gene, under specific hybridization conditions, allowed differentiation between avirulent and virulent R. equi. This technique has the potential to be used for quantitative screening of large environmental and clinical samples for both avirulent and virulent R. equi. Its use in ecological and epidemiological studies of R. equi has the potential to improve understanding of the relationship between the environment, the foal and the disease.     

Use of a selective sulfite-reducing medium for the isolation of Clostridium perfringens]

The effect of sulphite-reduction in 33 sample bacterial strains was tested. With regard to the capacity of reducing sulphite in modified sulphite-reduction media in a wide scale of bacterial strains the possibility of an application of selective media with an addition of various concentrations of antibiotic solutions was checked. A concentration of 750 microgram of D-cycloserine per 1 ml of the sulphite-reduction medium appeared to be the most advantageous for the isolation and detection of sulphite-reductive clostridia, above all of Clostridium perfringens. This concentration ensured also a sufficient inhibition of undesirable bacteria without any affection of the growth and capacity of Clostridium perfringens to reduce sulphite in the applied medium.     

Surgical management of Rhodococcus equi metaphysitis in a foal

A chronic Rhodococcus equi metaphysitis involving the distal growth plate of the left third metatarsal bone had induced a longstanding lameness in a young foal. Abnormal hematologic values included mild anemia, hyperfibrinogemia, mild leukocytosis, and neutrophilia. Radiography of the distal portion of MT3 revealed a radiolucent zone on the medial aspect of the growth plate, and small pieces of bone suggestive of sequestra. Treatment with erythromycin estolate and rifampin, aggressive surgical debridement, and cancellous bone grafting helped resolve the bone infection.     

Association of disease with isolation and virulence of Rhodococcus equi from farm soil and foals with pneumonia

OBJECTIVE: To determine whether isolation and virulence of Rhodococcus equi from soil and infected foals are associated with clinical disease. DESIGN: Cross-sectional and case-control study. SAMPLE POPULATION: R equi isolates from 50 foals with pneumonia and soil samples from 33 farms with and 33 farms without a history of R equi infection (affected and control, respectively). PROCEDURE: R equi was selectively isolated from soil samples. Soil and clinical isolates were evaluated for virulence-associated protein antigen plasmids (VapA-P) and resistance to the beta-lactam antibiotics penicillin G and cephalothin. Microbiologic cultures and VapA-P assays were performed at 2 independent laboratories. RESULTS: VapA-P was detected in 49 of 50 (98%) clinical isolates; there was complete agreement between laboratories. Rhodococcus equi was isolated from soil on 28 of 33 (84.8%) affected farms and 24 of 33 (72.7%) control farms, but there was poor agreement between laboratories. Virulence-associated protein antigen plasmids were detected on 14 of 66 (21.2%) farms by either laboratory, but results agreed for only 1 of the 14 VapA-P-positive farms. We did not detect significant associations between disease status and isolation of R equi from soil, detection of VapA-P in soil isolates, or resistance of soil isolates to beta-lactam antibiotics. No association between beta-lactam antibiotic resistance and presence of VapA-P was detected. CONCLUSIONS AND CLINICAL RELEVANCE: On the basis of soil microbiologic culture and VapA-P assay results, it is not possible to determine whether foals on a given farm are at increased risk of developing disease caused by R equi.     

Rhodococcus equi (Prescottella equi) vaccines; the future of vaccine development

For decades researchers have been targeting prevention of Rhodococcus equi (Rhodococcus hoagui/Prescottella equi) by vaccination and the horse breeding industry has supported the ongoing efforts by researchers to develop a safe and cost effective vaccine to prevent disease in foals. Traditional vaccines including live, killed and attenuated (physical and chemical) vaccines have proved to be ineffective and more modern molecular‐based vaccines including the DNA plasmid, genetically attenuated and subunit vaccines have provided inadequate protection of foals. Newer, bacterial vector vaccines have recently shown promise for R. equi in the mouse model. This article describes the findings of key research in R. equi vaccine development and looks at alternative methods that may potentially be utilised.     

Prevalence,antimicrobial resistance profile and comparison of selective plating media for the isolation of Salmonella in backyard chickens from Entre Rios,Argentina

This study was conducted to estimate the apparent prevalence of Salmonella spp. in birds kept under backyard system in Entre Ríos, Argentina, and determine the performance of two selective plating media used for Salmonella isolation, and the antimicrobial resistance of the isolated. Also, the association of farms characteristics with Salmonella presence was evaluated. A total of 657 backyard chickens and 15 gooses were sampled one time by cloacal swab, belonging to 51 and one family farms, respectively, and four counties in Entre Rios state from April 2014 to May 2015. Only four samples from backyard chickens belonged to three family farms from Uruguay County were positive to Salmonella spp., so the apparent prevalence was 0.6% for this kind of chicken. Four serovars were isolated (Salmonella ser. Lille, S. ser. Newport, S. ser. Enteritidis and S. ser. Rissen), which were susceptible to all antibiotics tested with the exception of erythromycin. For Hektoen enteric agar and brilliant green agar, relative specificity and positive predictive value were 1, and the relative sensitivity and negative predictive value did not show any difference between them. The agreement was very good between these two plating media. None of the variables studied could be selected to calculate the risk factors associated with Salmonella isolation because p > .15. Although the prevalence of Salmonella spp. is low in backyard birds in Entre Rios, the presence of S. ser. Enteritidis should not be discounted, because it is found in the county that concentrates a large population of intensive poultry production in the state.     

Comparison of concentrations of Rhodococcus equi and virulent R. equi in air of stables and paddocks on horse breeding farms in a temperate climate

REASONS FOR PERFORMING STUDY: Rhodococcoccus equi is a significant cause of bronchopneumonia in foals worldwide. Infection of the lungs is believed to result from inhalation of virulent R. equi in dust from contaminated environments. A measure of infectious risk in an environment is the level of airborne contamination. OBJECTIVES: To assess and compare the level of airborne virulent R. equi in paddocks and stables. METHODS: Air samples were collected sequentially over the 2003 foaling season from the paddocks and stables on 3 Irish horse breeding farms affected by R. equi pneumonia. Colony blotting and DNA hybridisation techniques allowed quantitation of virulent R. equi. RESULTS: The odds of detecting airborne virulent R. equi in stables were 173 times greater than in paddocks. The median airborne concentration of virulent R. equi was significantly higher (P < 0.001) in stables than in paddocks on all farms. These observations suggested that stables were high-risk areas for infection. CONCLUSIONS AND POTENTIAL RELEVANCE: Our results indicate that contaminated stables are a significant risk factor in the epidemiology of R. equi pneumonia on horse-breeding farms in a temperate climate, such as in Ireland. Management strategies that improve the air hygiene of stables, through better ventilation, use of less fragile bedding material and the use of fogging agents to reduce the airborne concentration of virulent R. equi, may reduce the incidence and severity of R. equi pneumonia on farms.     

Diagnosis and therapy of Rhodococcus equi infection in the horse

Infection with Rhodococcus equi is an important cause of pneumonia in foals, but other organ systems may also be affected. The intracellular presence of R. equi and the formation of granulomatous and suppurative inflammatory tissue mean that prolonged treatment is needed. The pharmacological properties of the combination of erythromycin and rifampicin have improved the survival of foals infected with R. equi; however, erythromycin can cause adverse reactions in foals and mares, which has prompted the search for alternative therapies. The combination of azithromycin or clarithromycin with rifampicin seems to be a promising alternative. However these combinations are expensive and adverse effects remain to be determined, especially in the dams of treated foals. Thus correct diagnosis and appropriate use of drugs are essential for the treatment of R. equi infection in foals.     

Resistance studies of erythromycin and rifampin for Rhodococcus equi over a 10-year period

This study sought to determine whether an increase in resistance of Rhodococcus equi to the antibiotics rifampin and erythromycin occurred over a 10-year period. This was carried out by the use of E test strips for rifampin and erythromycin to determine the MIC (minimum inhibitory concentration) values of Rhodococcus equi to this combination of antibiotics.The findings of this study indicated that there was an increase in resistance of Rhodococcus equi to rifampin and erythromycin over the 10-year period. The MIC for rifampin increased from 0.081 μg/ml in 1996 to 0.187 μg/ml in 2006 and from 0.258 μg/ml for erythromycin during the years prior to 2000 to 0.583 μg/ml in 2006.This finding suggests that there may be a problem in the treatment of Rhodococcus equi infections in foals in the future, particularly as the number of drugs available for treatment of Rhodococcus equi infection is limited because of the intracellular capabilities of this bacterium. Antibiotics used in its treatment have to be able to penetrate the polysaccharide cell wall of Rhodococcus equi as well as the alveolar macrophages in which the bacterium is capable of surviving.