RESUMEN
BACKGROUND: Mycobacterioses in animals cause economical losses and certain Mycobacterium avium subspecies are regarded as potential zoonotic agents. The evaluation of the zoonotic risk caused by M. avium subspecies requires information about the quantities of Mycobacterium strains in infected animals. Because M. avium subspecies in pig tissues are difficult or even impossible to quantify by culturing, we tested the suitability of a culture-independent real-time quantitative PCR (qPCR) assay for this purpose. METHODS: Mycobacterial DNA was extracted from porcine tissues by a novel method and quantified by Mycobacterium genus specific qPCR assay targeting the 16S rRNA gene. RESULTS: The response of the qPCR assay to the amount of M. avium subspecies avium mixed with porcine liver was linear in the range of approximately log105 to log107 Mycobacterium cells per 1 g of liver. The assay was validated with three other M. avium subspecies strains. When the assay was applied to porcine lymph nodes with or without visible lesions related to Mycobacterium avium subspecies infections, around 104-107 mycobacterial genomes per gram of lymph nodes were detected. CONCLUSIONS: The qPCR assay was found to be suitable for the quantification of Mycobacterium avium subspecies in porcine lymph nodes and liver.
Asunto(s)
Mycobacterium avium/clasificación , Mycobacterium avium/aislamiento & purificación , Reacción en Cadena en Tiempo Real de la Polimerasa/veterinaria , Porcinos , Animales , Hígado/microbiología , MicroscopíaRESUMEN
BACKGROUND: Animal mycobacterioses are regarded as a potential zoonotic risk and cause economical losses world wide. M. avium subsp. hominissuis is a slow-growing subspecies found in mycobacterial infected humans and pigs and therefore rapid and discriminatory typing methods are needed for epidemiological studies. The genetic similarity of M. avium subsp. hominissuis from human and porcine origins using two different typing methods have not been studied earlier. The objective of this study was to compare the IS1245 RFLP pattern and MIRU-VNTR typing to study the genetic relatedness of M. avium strains isolated from slaughter pigs and humans in Finland with regard to public health aspects. METHODS: A novel PCR-based genotyping method, variable number tandem repeat (VNTR) typing of eight mycobacterial interspersed repetitive units (MIRUs), was evaluated for its ability to characterize Finnish Mycobacterium avium subsp. hominissuis strains isolated from pigs (n = 16) and humans (n = 13) and the results were compared with those obtained by the conventional IS1245 RFLP method. RESULTS: The MIRU-VNTR results showed a discriminatory index (DI) of 0,92 and the IS1245 RFLP resulted in DI 0,98. The combined DI for both methods was 0,98. The MIRU-VNTR test has the advantages of being simple, reproducible, non-subjective, which makes it suitable for large-scale screening of M. avium strains. CONCLUSIONS: Both typing methods demonstrated a high degree of similarity between the strains of human and porcine origin. The parallel application of the methods adds epidemiological value to the comparison of the strains and their origins. The present approach and results support the hypothesis that there is a common source of M. avium subsp. hominissuis infection for pigs and humans or alternatively one species may be the infective source to the other.
Asunto(s)
Mycobacterium avium/genética , Polimorfismo de Longitud del Fragmento de Restricción , Enfermedades de los Porcinos/microbiología , Secuencias Repetidas en Tándem/genética , Tuberculosis/microbiología , Animales , Finlandia , Humanos , Mycobacterium avium/clasificación , Filogenia , ARN Ribosómico 16S/genética , PorcinosRESUMEN
Boreal soils have been suspected reservoirs of infectious environmental mycobacteria. Detection of these bacteria in the environment is hampered by their slow growth. We applied a quantitative sandwich hybridization approach for direct detection of mycobacterial 16S rRNA in soil without a nucleic acid amplification step. The numbers of mycobacterial 16S rRNA molecules found in the soil indicated the presence of up to 10(7) to 10(8) mycobacterial cells per gram of soil. These numbers exceed by factor of 10 to 100 x the previous estimates of mycobacteria in soil based on culture methods. When real-time PCR with mycobacteria targeting primers was used to estimate the number of 16S rDNA copies in soil, one copy of 16S rDNA was detected per 10(4) copies of 16S rRNA. This is close to the number of 16S rRNA molecules detected per cell by the same method in laboratory pure cultures of M. chlorophenolicum. Therefore a major part of the mycobacterial DNA in the studied soils may thus have represented metabolically active cells. The 16S rRNA sandwich hybridization method described in this paper offers a culture independent solution for tracking environmental reservoirs of viable and potentially infectious mycobacteria.
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Mycobacterium/aislamiento & purificación , Hibridación de Ácido Nucleico/métodos , ARN Ribosómico 16S/análisis , Microbiología del Suelo , ADN Ribosómico/análisis , ADN Ribosómico/genética , Mycobacterium/genética , ARN Ribosómico 16S/genética , Sensibilidad y EspecificidadRESUMEN
Automated ribotyping as a tool for identifying of nontuberculous mycobacteria was evaluated. We created a database comprising of riboprints of 60 strains, representing 32 species of nontuberculous mycobacteria. It was shown that combined ribopatterns generated after digestion with EcoRI and PvuII were distinguishable between species of both slow-growing and rapid-growing mycobacteria. The findings were in good agreement with the 16S rRNA gene sequencing results, allowing correct identification of Mycobacterium lentiflavum isolated from clinical specimens and from biofilms growing in public water distribution system. The automated ribotyping was powerful in discriminating between M. lentiflavum and closely related species M. simiae and M. palustre. Mycobacterium lentiflavum strains from drinking water biofilms were resistant to two to four antimycobacterial drugs. The drinking water distribution system may, thus, be a source of nontuberculous mycobacteria resistant to multiple drugs.
Asunto(s)
Infecciones por Mycobacterium/microbiología , Mycobacterium/clasificación , Mycobacterium/aislamiento & purificación , Reconocimiento de Normas Patrones Automatizadas/métodos , Ribotipificación/métodos , Microbiología del Agua , Abastecimiento de Agua , Biopelículas , Análisis por Conglomerados , Dermatoglifia del ADN , ADN Bacteriano/química , ADN Bacteriano/genética , ADN Bacteriano/metabolismo , ADN Ribosómico/química , ADN Ribosómico/genética , Desoxirribonucleasa EcoRI/metabolismo , Desoxirribonucleasas de Localización Especificada Tipo II/metabolismo , Farmacorresistencia Bacteriana Múltiple , Humanos , Pruebas de Sensibilidad Microbiana , Datos de Secuencia Molecular , ARN Ribosómico 16S/genética , Análisis de Secuencia de ADN , Estadística como AsuntoRESUMEN
PURPOSE: Microbial biofilm has become difficult to control by antibiotic and biocide regimes that are effective against suspended bacteria. Their colonization of surfaces can be a problem and is generally controlled through cleaning and disinfection. This study was undertaken to examine the efficacy of the disinfectants including Bio-Ow, Econase CE, Gamanase GC 140, IndiAge 44L, Mannanase AMB, Multifect P-3000, Neutrase, Pandion, Paradigm, Pectinex Ultra SP-L, Promozyme, Resinase A2X, Spezyme AA300, Spezyme GA300 and Vinozym EC, and the proteinase against bacterial biofilms. METHODS: The effectiveness of 20 commercial disinfectants against Pseudomonas aeruginosa (P. aeruginosa) biofilms using a fluorometric technique was examined. Additionally the disinfectants were also tested against Lactobacillus bulgaricus (L. bulgaricus), Lactobacillus lactis (L. lactis) and Streptococcus thermophilus (S. thermophilus) isolates using microtitration tray based turbidimetric techniques. Escherichia coli (E. coli) was used as the test bacteria in the fluorometric control method. RESULTS: Among the first group of the enzymatic cleaning agents tested, four disinfectants (Pandion, Resinase A2X, Spezyme GA300 and Paradigm) were the most potent against bacterial biofilms after 30 min incubation time (residual bacterial count less than 10(3) CFU (colony forming units)/ml). However, only Resinase A2X and Paradigm showed a good effect on bacterial biofilms after 15 min incubation time. Proteinase disinfectants (alkalase, chymotrypsin, cryotin and krilltrypsin) from the second group of the disinfectants showed a good effect against P. aeruginosa biofilm when tested in the absence of milk. The performance of the disinfectants was reduced in the presence of milk. The minimum inhibitory concentration (MIC) of the cleaning agents was determined as the lowest concentration inhibiting bacterial growth. The MIC was tested on Lactobacillus bulgaricus (L. bulgaricus), Lactobacillus lactis (L. lactis) and Streptococcus thermophilus (S. thermophilus) isolates. The minimum inhibitory concentrations (MIC) for Paradigm against S. thermophilus and L. Lactis were lower than L. Bulgaricus. Whereas, the MIC of Pandion against L. bulgaricus was lower than MIC against L. lactis. Resinase A2X had no inhibitory effect on bacterial growth when the concentration was less than or equal to 2.4 mg/ml and Spezyme GA 300 concentration less than or equal to 7.3 mg/ml. Minimum inhibitory concentration of Pandion against L. bulgaricus was 2.7 microg/ml and against L. lactis 5.3 microg/ml. Growth of S. thermophilus was inhibited in all concentration of Pandion tested. CONCLUSIONS: The choice of disinfectant or cleaning agent along with the optimum concentration and the time of action is very important when destroying microbes. It is also important that the resistances of microbes to different disinfectants and cleaning agents be taken into account when planning the cleaning process
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Biopelículas/efectos de los fármacos , Detergentes/farmacología , Desinfectantes/farmacología , Pseudomonas aeruginosa/efectos de los fármacos , Endopeptidasas/farmacología , Lactobacillus/efectos de los fármacos , Tiempo de Reacción , Streptococcus/efectos de los fármacosRESUMEN
Mycotoxins are fungal secondary metabolites that are toxic to vertebrates produced by organisms that occur as plant pathogens, soilborne fungi, airborne fungi and aeroallergens. They are distributed worldwide and may be recovered from a wide range of substrates. Their presence in food and feeds, as the result of fungal diseases in crops, can present a danger to animal and human health. Many mycotoxins have also been shown to be phytotoxic and in some cases, such as with trichothecenes produced by the wheat head blight fungus Fusarium graminearum, mycotoxins may act as virulence factors. Several natural (vitamin, provitamins, carotenoids, chlorophyll and its derivatives, phenolics, and selenium) and synthetic (butylated hydroxyanisole and butylated hydroxytoluene) compounds with antioxidant properties seem to be potentially very efficacious in protecting against the toxic effects of mycotoxins. The protective properties of antioxidants are probably due to their ability to act as superoxide anion scavengers, thereby protecting cell membranes from mycotoxin-induced damage and in some cases, antioxidant vitamins may play a role in preventing mycotoxicosis. However, much less information is available from studies carried out on antioxidants and mycotoxins, such as OTA, FB(1), T-2 toxin, ZEN, and citrinin. No such studies have been performed on recently discovered toxins such as beauvericin, fusaproliferin, moniliformin, and fusaric acid. However, supplementation with antioxidant nutrients to prevent mycotoxicosis has been controversial. The case for the use of supplemental antioxidant vitamins at the present time needs further research.