The Formation, Persistence, and Resistance to Disinfectant of the Erysipelothrix piscisicarius Biofilm.

Erysipelothrix piscisicarius is an emergent pathogen in fish aquaculture, particularly in the ornamental fish trade. Very little is known on the biology of this pathogen; however, the recurrence of infection and disease outbreaks after removing the fish from a system and disinfecting the tank suggest its environmental persistence. Moreover, biofilm lifestyle in E. piscisicarius has been suspected but not previously shown. The purpose of this study was to investigate the formation of biofilms on an abiotic surface in Erysipelothrix spp. We used hydroxyapatite-coated plastic pegs to demonstrate the attachment, growth, and persistence of E. piscisicarius on abiotic surfaces in both fresh and marine environments and to investigate the susceptibility of this pathogen to different disinfectants that are used in the aquaculture industry. E. piscisicarius formed biofilms that persisted significantly longer than planktonic cells did in both freshwater and saltwater over a period of 120 h (P = 0.004). The biofilms were also more resistant to disinfectants than the planktonic cells were. Hydrogen peroxide was the most effective disinfectant against E. piscisicarius, and it eradicated the biofilms and planktonic cells at the recommended concentrations. In contrast, Virkon and bleach were able to eradicate only the planktonic cells. This information should be taken into consideration when developing biosecurity protocols in aquaculture systems, aquariums, and private collections.

Production of bacteriophage-encoded endolysin, LysP11, in Nicotiana benthamiana and its activity as a potent antimicrobial agent against Erysipelothrix rhusiopathiae.

KEY MESSAGE: We produced a biologically active phage-encoded endolysin, LysP11, in N. benthamiana. Plant-produced LysP11 exhibited robust antimicrobial activity against E. rhusiopathiae, and C-terminal domain of LysP11 bound specifically to E. rhusiopathiae. Bacterial resistance to antibiotics, a serious issue in terms of global public health, is one of the leading causes of death today. Thus, new antimicrobial agents are needed to combat pathogens. Recent research suggests that bacteriophages and endolysins derived from bacteriophages are potential alternatives to traditional antibiotics. Here, we examined the antimicrobial activity of LysP11, which is encoded by Propionibacterium phage P1.1 and comprises an N-terminal amidase-2 domain and a C-terminal domain with no homology to other bacteriophage endolysins. LysP11 was produced in Nicotiana benthamiana (N. benthamiana) using an Agrobacterium-mediated transient expression strategy. LysP11 was purified on microcrystalline cellulose-binding resin after attachment of the Clostridium thermocellum-derived family 3 cellulose-binding domain as an affinity tag. The affinity tag was removed using the small ubiquitin-related modifier (SUMO) domain and SUMO-specific protease. Plant-produced LysP11 showed strong antimicrobial activity toward Erysipelothrix rhusiopathiae (E. rhusiopathiae), mediated via lysis of the cell wall. Lytic activity was optimal at pH 8.0-9.0 (37 °C) and increased at higher concentrations of NaCl up to 400 mM. Furthermore, the C-terminal domain of LysP11 bound specifically to the E. rhusiopathiae cell wall. Based on these results, we propose that LysP11 is a potential candidate antimicrobial agent against E. rhusiopathiae.

Presence and new genetic environment of pleuromutilin-lincosamide-streptogramin A resistance gene lsa(E) in Erysipelothrix rhusiopathiae of swine origin.

Erysipelothrix rhusiopathiae is a Gram-positive bacillus that causes erysipelas in swine. In recent years, erysipelas infection among swine in China has been increasing. A combined resistance phenotype to pleuromutilins, lincosamides, and streptogramin A (PLSA phenotype) was found in some E. rhusiopathiae isolates. The aim of this study was to identify the resistance genes responsible for the PLSA phenotype in E. rhusiopathiae strains and to map the genetic environment of the identified resistance gene. A total of 46 E. rhusiopathiae isolates from 31 pig farms in China were studied. Minimum inhibitory concentrations (MICs) of 11 antimicrobial agents were determined by broth microdilution method. Seven were highly resistant to tiamulin (MICs 32 µg/ml) and clindamycin (MICs 64 µg/ml). Resistance genes responsible for the PLSA phenotype were screened by PCR. The lsa(E), spw, lnu(B), aadE and aphA3 genes were detected in strains had the PLSA phenotype, whereas none was detected in susceptible strains. The genetic environment of lsa(E) gene was determined by whole-genome sequencing and overlapping PCR assays. A novel multiresistance gene cluster, orf1-aadE-apt-spw-lsa(E)-lnu(B)-rec-orf2-orf1-aadE-sat4-aphA3, was found. Horizontal gene transfer experiments and whole-genome sequencing suggested that the lsa(E)-carrying multiresistance gene cluster was located in the chromosome. This is the first molecular characterization of PLSA resistance in E. rhusiopathiae. The lsa(E), spw and lnu(B) genes were found in E. rhusiopathiae for the first time. A novel lsa(E)-carrying multiresistance gene cluster was found. The location of lsa(E) in different gene cluster facilitates its persistence and dissemination.

Virulence determinants, antimicrobial susceptibility, and molecular profiles of Erysipelothrix rhusiopathiae strains isolated from China.

The aim of this study was to understand the epidemiology, serotype, antibiotic sensitivity, and clonal structure of Erysipelothrix rhusiopathiae strains in China. Forty-eight strains were collected from seven provinces during the period from 2012 to 2013. Pulse-field electrophoresis identified 32 different patterns which were classified into clonal groups A­D. Most pulsed-field gel electrophoresis (PFGE) patterns were observed in clonal complex A and B, suggesting high diversity of genetic characterization in these two predominant clonal complexes. Antibiotic sensitivity test shows that all the stains were susceptible to ampicillin, erythromycin, and cefotaxime, and resistant to kanamycin, cefazolin, sulfadiazine, and amikacin. Erythromycin and ampicillin are recommended as first-line antibiotics for treatment of E. rhusiopathiae in China. The high variation in PFGE pattern among the main clonal groups shows that the E. rhusiopathiae in China may originate from different lineages and sources instead of from expansion of a single clonal lineage across different regions.

Native valve endocarditis caused by Erysipelothrix rhusiopathiae in an immunocompetent individual.

Infective endocarditis is a very rare clinical form caused by Erysipelothrix rhusiopathiae. It is rarely seen in immunocompetent individuals. Even after surgery it may entail mortality rates as high as 30-40 %. This report describes a case of native valve endocarditis caused by E. rhusiopathiae and cured with crystallized penicillin G and surgery.

Active infective endocarditis due to Erysipelothrix rhusiopathiae: zoonosis caused by vancomycin-resistant gram-positive rod.

A 42-year-old female who was a voluntary worker in a school for handicapped children was referred to us for surgery for active infective endocarditis. Trans-esophageal echocardiography showed 2 large mobile vegetations on the aortic valve and severe aortic regurgitation. Aortic valve replacement was performed to prevent septic embolism and deterioration of congestive heart failure. The empiric therapy with vancomycin, ampicillin, and gentamycin was initiated because a pathogen was not identified. But Erysipelothrix rhusiopathiae (gram-positive rod) was isolated on the 4th day after surgery. The target therapy with penicillin G and clindamycin was started and continued for 4 weeks after surgery. The inflammatory parameters improved steadily and the patient was discharged on the 36th day after surgery. Infective endocarditis due to gram-positive rods can be easily mistaken for streptococci or dismissed as a skin contamination. But, E. rhusiopathiae endocarditis should be considered in the differential diagnosis.

Bile acid is a host factor that regulates the composition of the cecal microbiota in rats.

BACKGROUND & AIMS: Alterations in the gastrointestinal microbiota have been associated with metabolic diseases. However, little is known about host factors that induce changes in gastrointestinal bacterial populations. We investigated the role of bile acids in this process because of their strong antimicrobial activities, specifically the effects of cholic acid administration on the composition of the gut microbiota in a rat model. METHODS: Rats were fed diets supplemented with different concentrations of cholic acid for 10 days. We used 16S ribosomal RNA gene clone library sequencing and fluorescence in situ hybridization to characterize the composition of the cecal microbiota of the different diet groups. Bile acids in feces, organic acids in cecal contents, and some blood parameters were also analyzed. RESULTS: Administration of cholic acid induced phylum-level alterations in the composition of the gut microbiota; Firmicutes predominated at the expense of Bacteroidetes. Cholic acid feeding simplified the composition of the microbiota, with outgrowth of several bacteria in the classes Clostridia and Erysipelotrichi. Externally administered cholic acid was efficiently transformed into deoxycholic acid by a bacterial 7α-dehydroxylation reaction. Serum levels of adiponectin decreased significantly in rats given the cholic acid diet. CONCLUSIONS: Cholic acid regulates the composition of gut microbiota in rats, inducing similar changes to those induced by high-fat diets. These findings improve our understanding of the relationship between metabolic diseases and the composition of the gastrointestinal microbiota.

Phenotypic and molecular characterization of recent and archived Erysipelothrix spp. isolated from Brazilian swine.

One hundred fifty-one Erysipelothrix spp. isolates from Brazilian swine were characterized by serotyping, determination of antimicrobial susceptibility, amplified fragment length polymorphism (AFLP), and pulsed-field gel electrophoresis (PFGE). Among all isolates, 139 were classified in 18 different serotypes and serotype 2b was the most frequent. The susceptibility profiles of the isolates were very similar among each other, which did not permit subtyping Erysipelothrix spp. isolates by the antimicrobial susceptibility testing. Despite the fact that AFLP and PFGE provided the same discriminatory index (0.98), PFGE was more discriminatory than AFLP, given the types of groups it generates. Regardless the technique employed (AFLP or PFGE), no discrimination between recent and historical isolates was established, neither a fixed epidemiologic pattern for their grouping was observed. Nevertheless, AFLP could be an interesting alternative for discriminating the Erysipelothrix species, while PFGE could be an indication for discerning this bacterium according to the serotypes.

Comparison of bacterial diversity in wheat bran and in the gut of larvae and newly emerged adult of Musca domestica (Diptera: Muscidae) by use of ethidium monoazide reveals bacterial colonization.

The objective of the current study is to investigate the bacterial colonization within the gut of the house fly, Musca domestica L. (Diptera: Muscidae), at the larval stage and the bacterial community of the gut of the house fly at the newly emerged adult stage. After using ethidium monoazide to inhibit recovery of nucleic acids from dead bacteria, three polymerase chain reaction (PCR)-amplified 16S rDNA libraries from wheat bran, larvae, and newly emerged adults was constructed, analyzed, and compared. In total, 24, 11, and four phylotypes in the 16S rDNA libraries of wheat bran and the gut of larvae and adults, respectively, were found and assigned to three phylogenetic phyla of the domain Bacteria: Firmicutes, Proteobacteria, and Bacteroidetes. In the wheat bran library, 76% of the total number of sequences were affiliated to the genera Pseudomonas, Halomonas, Providencia, and Ignatzschineria. The three genera Morganella (79.05%), Providencia (8.78%), and Ignatzschineria (9.46%) dominated the library of the larval gut. Compared with the wheat bran library, the relative abundance of Morganella morganii (Winslow) was significantly higher (79.05 versus 0.8%), whereas that of Ignatzschineria larvae and of Providencia spp. was similar. These results demonstrate that M. morganii, Providencia spp., and I. larvae colonized the gut of the house fly larvae. Live bacteria of M. morganii, Providencia spp., and Proteus spp. were found in the gut of newly emerged adults. Therefore, the bacteria M. morganii and Providencia spp. colonized the larval gut could survive in the gut from larval metamorphosis to adult eclosion of the house fly.

Antimicrobial susceptibility of Erysipelothrix rhusiopathiae isolated from pigs in Southern Japan with a modified agar dilution method.

The determination of antimicrobial minimum inhibitory concentration (MIC) in Erysipelothrix rhusiopathiae by using the agar dilution method has not been covered by the Clinical and Laboratory Institute (CLSI). Only the broth microdilution method has been outlined. This report describes a modification of the agar dilution procedure for E. rhusiopathiae using Trypto-soy agar supplemented with 0.1% Tween 80 and incubation in ambient air at 37 degrees C for 24 hr. The MICs of the assay were in agreement with those of the broth microdilution method recommended by the CLSI. Antimicrobial susceptibility test was performed using this method for 149 E. rhusiopathiae isolates from 2 meat processing plants in Kagoshima Prefecture during the period of April 2004 to March 2005. The number of strains resistant to oxytetracycline, erythromycin, lincomycin, ofloxacin and enrofloxacin were 56 (37.6%), 4 (2.7%), 18 (12.1%), 21 (14.1%) and 19 (12.8%), respectively. All strains were susceptible to ampicillin.

Development of quantitative real-time polymerase chain reaction for detection of and discrimination between Erysipelothrix rhusiopathiae and other Erysipelothrix species.

Quantitative real-time polymerase chain reaction (qPCR) assays were developed and validated in combination with enrichment culture for the detection and discrimination of Erysipelothrix rhusiopathiae and other Erysipelothrix species from tissue samples. The targets for SYBR green qPCR assays were the 16S ribosomal RNA gene for Erysipelothrix species and a gene involved in capsular formation for E. rhusiopathiae. The specificity of the assays was assessed with Erysipelothrix species and other related bacterial species. The limit of detection was found to be 5 colony-forming units per reaction. Amplification of DNA extracted from spleen and joint samples spiked with increasing quantities of Erysipelothrix cells was shown to be equally sensitive to DNA extracted from a pure bacterial culture. The assays were evaluated with 88 tissue samples from 3 experimentally infected pigs and 50 mice and with 36 tissue samples from 3 naturally infected pigs and 11 noninfected pigs. Results were compared with those of direct qPCR and conventional culture. The qPCR after enrichment increased the diagnostic sensitivity over that of culture and qPCR, thereby significantly reducing the total time taken for the detection of E. rhusiopathiae and other Erysipelothrix species. Therefore, this technique could be used for practical applications.

The evaluation of trifloxystrobin in protection of Calendula officinalis (Pot marigold) against Erysiphe cichoracearum DC.

The aim of the two-years field's examinations was the evaluation of the fungicide Zato 50 WG (biologically active substances BAS--trifloxystrobin 50%) in protection of Calendula officinalis (Pot marigold) against Erysiphe cichoracearum. Mentioned fungicide was applied at three concentrations: 0.1, 0.15 and 0.2%. As the standard fungicide Amistar 250 SC (biologically active substances BAS - azoxystrobin 250 g/dm3) was used. In every year of research work the four protective treatments were carried out. The estimation of infestation degree of Calendula officinalis leafs by the Erysiphe cichoracearum was made 5 times. Before each treatment four analysis were done, whereas the last analysis--the fifth one was executed after 10 days from the last protective spraying. According to the results, it was found that investigated preparations significant reduced the mean infestation degree of Calendula officinalis leafs by the Erysiphe cichoracearum compared to the control. The results pointed, that in protection of the mentioned plant by the powdery mildew the 0.2% dose of Zato 50WG showed the best suitability.

[Fatal mitral valve endocarditis by Erysipelothrix rhusiopathiae]. / Endocarditis fatal con localizaci'on mitral producida por Erysipelothrix rhusiopathiae.

A fatal case of Erysipelothrix rhusiopathiae mitral valve endocarditis is described in a 45 years old male, with a history of chronic alcohol abuse and without animals contact. He presented intermittent fever, polyarthralgia, weight loss, and low back pain. In blood cultures (2 bottles), gram-positive pleomorphic rods grew after 48 hours of incubation. The subculture on blood agar media showed a small, alpha-hemolytic colony, catalase and oxidase negative, PYR and LAP positive and the production of H2S in triple sugar iron agar, was demonstrated. The isolate was initially identified as E. rhusiopathiae, and confirmed by API Coryne (BioMérieux). On the basis of these findings and a transthoracic echocardiogram, an endocarditis was confirmed. Intravenous ampicillin and gentamicin treatment was initiated. The patient became afever, nevertheless he died on day 19 after admission as a consequence of acute pulmonary edema. Susceptibility testing by E-test showed that the microorganism was resistant to vancomycin and gentamicin, and susceptible to penicillin and cefotaxime. We emphasize the importance to consider the isolates of gram-positive pleomorphic rods, catalase and oxidase negative, and the addition of H2S production test in TSI medium. Vancomycin-resistance helps in the identification, and to establish the correct antimicrobial therapy. Although E. rhusiopathiae is usually reported as an occupational pathogen, the contact with pigs and other farm animals may be underestimated.

Susceptibility of Erysipelothrix rhusiopathiae to antimicrobial agents and home disinfectants.

AIM: Erysipelothrix rhusiopathiae causes the occupationally-related infection erysipeloid in humans, and may be responsible for infections in lobster fishermen in Western Australia. There are little recent data pertaining to antimicrobial susceptibility, or susceptibility to disinfectants that might be used in the environment. The aim of this study was to determine the susceptibility of E. rhusiopathiae from human, animal and environmental sources to various antimicrobial agents and disinfectants. METHODS: The susceptibility of 60 E rhusiopathiae isolates was determined using a recommended agar dilution procedure. Susceptibility to disinfectants was achieved using a broth microdilution method. RESULTS: Penicillin and ceftriaxone, with low minimum inhibitory concentrations (MICs) (MIC90 0.03 mg/l and 0.125 mg/l, respectively), remained active against E. rhusiopathiae and should continue to be recommended for treatment. Ciprofloxacin MICs were particularly low (MIC90 0.06 mg/l), offering an alternative agent for the penicillin allergic patient. Erysipelothrix rhusiopathiae is still resistant to vancomycin (MIC90 64 mg/l), highlighting the importance of early diagnosis of E. rhusiopathiae infection in cases of endocarditis. In addition, 31 E. rhusiopathiae isolates were tested against several commercially available home disinfectants. Most were effective in killing E. rhusiopathiae with minimum bactericidal concentrations of 0.001% for Pine O Cleen, and 0.03% for Domestos, Linely and the Wheelie Bin Phenyl Cleanser. CONCLUSIONS: There appeared to be no new emergence of antibiotic resistance in E. rhusiopathiae. Various disinfectants could be used following mechanical cleaning of work environments, such as fishing boats, and equipment, to reduce the risk of infection with E. rhusiopathiae.

Antimicrobial susceptibilities of Erysipelothrix rhusiopathiae isolated from pigs with swine erysipelas in Japan, 1988-1998.

The susceptibility to 21 antimicrobial agents of 214 strains of Erysipelothrix rhusiopathiae isolated from pigs affected with swine erysipelas in Japan between 1988 and 1998 was determined. Ampicillin, cloxacillin, benzylpenicillin, ceftiofur, tylosin, enrofloxacin and danofloxacin were the most active agents [minimum inhibitory concentrations (MICs); < or = 0.025-0.78 microgram/ml], followed by cefazolin, virginiamycin, tiamulin, chloramphenicol, florphenicol and oxolinic acid (MICs; 0.1-25 micrograms/ml). Activity was poor or absent with kanamycin and sulfadimethoxine. Strains resistant to dihydrostreptomycin, erythromycin, clindamycin, lincomycin, oxytetracycline and doxycycline were detected. The susceptibilities to dihydrostreptomycin and oxytetracycline tended to decrease. Investigation of the differences in antimicrobial susceptibility of the 214 strains according to their serotypes, sources, isolation years and regions, showed that the strains resistant to dihydrostreptomycin were most frequently found in the strains of serotype 1a and in strains from septicaemic cases. Strains resistant to oxytetracycline were detected in all serotypes and all sources, and most of the strains resistant to erythromycin were detected in the strains of serotype 2. The frequency of strains resistant to dihydrostreptomycin gradually increased from 1988 to 1996, but then decreased between 1997 and 1998. The frequency of strains resistant to oxytetracycline was remained more than 38% from 1988 to 1998. It was suggested that the strains resistant to dihydrostreptomycin and oxytetracycline were distributed over almost all districts of Japan.

Identification of the tetracycline resistance gene, tet(M), in Erysipelothrix rhusiopathiae.

This is the first report to demonstrate the presence of tet(M) in naturally occurring isolates of tetracycline-resistant Erysipelothrix rbusiopathiae, which causes swine erysipelas. The tet(M) gene was isolated from E. rhusiopathiae strain KY5-42. The nucleotide and the deduced amino acid sequence were 99% identical to the tet(M) gene from Enterococcus faecalis. The gene was necessary and sufficient for the expression of tetracycline resistance in Escherichia coli. The presence of the tet(M) gene in the 114 tetracycline-resistant E. rhusiopathiae isolates from diseased pigs was detected by the polymerase chain reaction assay. The specific amplified DNA fragment was obtained from all 114 tetracycline-resistant strains. It was suggested that the tet(M) gene was widely present in the field isolates of E. rhusiopathiae resistant to tetracycline.

In vitro susceptibility of porcine respiratory pathogens to tilmicosin.

Bacterial isolates obtained from swine with various clinical diseases were tested for susceptibility to tilmicosin by minimum inhibitory concentration (MIC) and Kirby-Bauer disk diffusion tests using National Committee on Clinical Laboratory Standards methodology. The tilmicosin MIC90 was < or =0.125 microg/ml for Erysiopelothrix rhusiopathiae, < or = 1 microg/ml for Haemophilus parasuis isolates, 8 microg/ml for Actinobacillus suis and Pasteurella multocida type A, 16 microg/ml for toxigenic and nontoxigenic P. multocida type D, 64 microg/ml for Bordetella bronchiseptica, and >128 microg/ml for Staphylococcus hyicus and Streptococcus suis. The results of disk diffusion testing matched well with the MIC results for each pathogen. This in vitro survey of tilmicosin activity against various swine isolates suggests that further clinical evaluation of tilmicosin in swine may be warranted for disease associated with E. rhusiopathiae, H. parasuis, and A. suis but not B. bronchiseptica, S. suis, or S. hyicus.

Fatal outcome of Erysipelothrix rhusiopathiae bacteremia in a patient with oropharyngeal cancer.

Bacteremia due to Erysipelothrix rhusiopathiae is rare; the most common presentation reported in the literature is endocarditis. We report a 32-year-old man with oropharyngeal cancer who developed aspiration pneumonia and E. rhusiopathiae bacteremia, and presented with fever, chills, dyspnea, and productive cough with purulent sputum. Despite treatment with amoxicillin/clavulanate and nutritional support for 9 days, he died of respiratory failure. He had no clinical evidence of endocarditis. He had no history of animal or occupational exposure, and might have been colonized with E. rhusiopathiae in the oral cavity, followed by aspiration pneumonia and bacteremia. A fatal outcome in a patient with bacteremia due to E. rhusiopathiae without endocarditis is rare.

Serovar, pathogenicity and antimicrobial susceptibility of erysipelothrix rhusiopathiae isolates from farmed wild boars (Sus scrofa) affected with septicemic erysipelas in Japan.

Six strains of Erysipelothrix rhusiopathiae were isolated from farmed wild boars with acute septicemic erysipelas during the period from 1983 to 1998 in Japan. All isolates belonged to serovar 1a or 2 (predominant serovars in swine). The 50 per cent lethal dose values of those isolates ranged from 10(1.3)to 10(6.2)colony forming units in mice. In swine, all isolates were virulent, capable of inducing localized or generalized urticarial lesions after intradermal inoculation. All of the isolates were resistant to oxytetracycline and/or dihydrostreptomycin. These observations suggest that E. rhusiopathiae strains isolated from wild boars may have aetiological significance in swine erysipelas.