Search

The study was conducted in the Biology research laboratory in Garmian University during the period of summer to autumn 2016, which aimed to the isolation and biochemically identification of Lactobacillus sp. and comparing their antioxidant activity with Lactobacillus reuteri ATCC 23272, as well as both of the isolates and reference strain with ascorbic acid which used as a control. Out of 25 samples were collected from different sources such as vaginal swabs, infant stool, sheep and cow’s milk, six strains of Lactobacillus sp were Identified according to their colony morphology on MRS agar along with microscopic examination. Further identification strategies are carried out by biochemical analysis including catalase, oxidase, and API 50 CHL kit system through which strains as L. pentosus, L.acidophilus, L. collinoides, L. fermentum, L. salivarius and L. brevis were identified. The results of antioxidant activity which carried out by 2,2-DiPhenyl-2-Picryl hydrazyl hydrate ( DPPH) assay for all six isolated strains revealed that the best antioxidant activity was recorded by L. pentosus strain IC50=0.0464 which showed no-significantly differences with the ascorbic acid antioxidant activity IC50 = 0.0186, on the other hand, Lactobacillus reuteri ATCC 23272 exhibited antioxidant activity about IC50=1.998, in addition, other isolated strains L. acidophilus, L. fermentum, L. salivarius, L. collinoides and L. brevis demonstrates good antioxidant activity with IC50= 0.2311, 0.2792, 0.3707, 3.744 and 5.766 respectively.

In addition to Staphylococcus aureus nowadays other coagulase-positive staphylococci (CoPS) and coagulase-negative staphylococci (CoNS), earlier considered of minor importance, are now accepted as relevant pathogens for humans and animals. The involvement of these microorganisms in bovine mastitis etiology and the possibility their transmission through milk to humans justify the requirement of developing reliable methods for identification of the most frequent species among them. The purpose of this study was to compare the phenotypic techniques with the genotypic method carried out by sequencing of the rpoB gene in identification of several species of the genus Staphylococcus isolated from bovine mastitis. A total of 300 staphylococci isolates of bovine mastitis cases from several Brazilian dairy herds were studied by phenotypic and genotypic techniques, respectively: 150 CoPS and 150 CoNS strains. A total of 18 CoNS different species and 4 CoPS species were identified. Among the CoNS the following species were recognized: 48 (32%) Staphylococcus warneri, 22(15%) S. epidermidis, 20(13%) S. hyicus, 10(7%) S. xylosus, 7(5%) S. haemolyticus, 6(4%) S. simulans, 6(4%) S. schleiferi subsp schleiferi, 6(4%) S. hominis, 5(3%) S. pasteuri, 4(2.7%) S. cohnii, 3(2%) S. saprophyticus subsp. saprophyticus 3(2%) S. chromogenes 3(2%) S. sciuri, 2(1%) S. saccharolyticus, 2(1%) S. lugdunensi, 1(0,7%) S. auricularis, 1(70%) S. saprophyticus subsp. bovis, 1(0.7%) S. capitis. And among the 150 CoPS were identified respectively: 105 (70%) S. aureus, 21(14%), S. hyicus, 19(13%) S. intermedius e 5(3%) S. schleiferi subsp coagulans. Considering the 150 CoNS isolates, the identifications performed by phenotypic and genotypic tests presented 96.7% of concordance, kappa coefficient of agreement = 0.933, SE (standard error) of kappa=0.021 (95% confidence interval: 0.893 to 0.974), Pearson’s correlation coefficient (r) = 0.9977, (confidence interval 95%: 0.9938 a 0.9992) and in relation to 150 CPS isolates it was detected an agreement of 98.7%, kappa = 0.960, SE of kappa = 0.016, (95% confidence interval: 0.929 to 0.992) Pearson’s correlation coefficient (r) = 0.9994 (95% confidence interval: 0.9681 to 1.0000). The verified agreement strength between the identification methods can be considered as excellent. These results assure that according to laboratory resources any of them will be suitable to perform the staphylococci identification. RESUMO: Além de Staphylococcus aureus atualmente outros estafilococos coagulase positiva (SCP) e estafilococos coagulase-negativos (SCN), anteriormente considerados de menor relevância, são reconhecidos como importantes patógenos para humanos e animais. O envolvimento desses micro-organismos na etiologia da mastite bovina e a possibilidade da sua transmissão através do leite aos humanos justifica a utilização de métodos confiáveis para a identificação das espécies mais frequentes. O objetivo deste estudo foi comparar as técnicas fenotípicas com o método genotípico realizada por sequenciamento do gene rpoB na identificação de espécies do gênero Staphylococcus spp. isolados de mastite bovina. Um total de 300 estafilococos isolados de casos de mastite bovina em diferentes rebanhos leiteiros brasileiros foram estudados por técnicas fenotípicas e genotípicas, respectivamente: 150 linhagens de SCP e 150 linhagens de SCN. Foram identificados um total de 18 espécies de SCN e 4 espécies SCP. Entre os SCN as seguintes espécies identificadas: 48 (32%) Staphylococcus warneri, 22 (15%) S. epidermidis, 20 (13%) S. hyicus, 10 (7%) S. xylosus, 7 (5%) S. haemolyticus, 6 (4%) S. simulans, 6 (4%) S. schleiferi subsp schleiferi, 6 (4%) S. hominis, 5 (3%) S. pasteuri, 4 (2,7%) S. cohnii, 3 (2%) S. saprophyticus subsp. saprophyticus, 3 (2%) S. chromogenes, 3 (2%) S. sciuri, 2 (1%) S. saccharolyticus, 2 (1%) S. lugdunensi, 1 (0,7%) S. auricularis, 1 (70 %) S. saprophyticus subsp. bovis, 1 (0,7%) S. capitis. E entre as 150 SCP foram identificados, 105 (70%) S. aureus, 21 (14%), S. hyicus, 19 (13%) S. intermedius e 5 (3%) S. schleiferi subsp coagulans. Considerando-se os 150 SCN isolados, as identificações realizadas por testes fenotípicos e genotípicos apresentaram 96,7% de concordância, coeficiente de concordância kappa = 0,933, SE (erro padrão) de kappa = 0,021 (95% intervalo de confiança: 0,893-0,974), coeficiente de correlação de Pearson (r) = 0,9977, (intervalo de confiança de 95%: 0,9938 a 0,9992) e em relação a 150 SCP isolados foi observado uma concordância de 98,7%, kappa = 0,960, sE de kappa = 0,016, (95% de intervalo de confiança: 0,929 a 0,992) coeficiente de correlação de Pearson (r) = 0,9994 (95% intervalo de confiança: 0,9681-1,0000). A correlação entre os métodos de identificação pode ser considerada como excelente. Esses resultados demonstraram que de acordo com os recursos disponíveis no laboratório, poderia ser utilizada qualquer uma das metodologias.

BACKGROUND : Tuberculosis is considered to be a major cause of death among the infectious diseases prevalent in India. Differentiation of M. tuberculosis from Nontuberculous Mycobacteria (NTM) is very important in the management of the disease. Recently, Standard Diagnostics (SD Yongin Korea) developed a simple and rapid assay using a mouse monoclonal anti - MPT64 antibody to discriminate between M. tuberculosis and NTM by Immu no - chromatography. These tests were found to have good sensitivity and specificity. METHOD : 200 sputum samples were processed and inoculated on conventional Lowenstein Jensen culture medium. The Mycobacterial isolates were subjected to biochemical tests (Ni acin test and Nitrate reduction test) and Immunochromatographic assay. The isolates were also tested by Polymerase chain reaction. H37 RV strain was used as a positive control. RESULT : The culture isolates showed positive in 47 (23.5%) cases. 44 isolates w ere positive and 3 were negative by both biochemical tests and immunochromatographic assay. Similar results were obtained by PCR. The sensitivity and specificity of Immunochromatography test when compared to biochemical tests and PCR was 100%. Positive pre dictive value and Negative predictive values were 100%. CONCLUSION : Immunochromatographic assay showed comparable results with biochemical tests and this assay can be used as an alternative to biochemical tests for the confirmation of Mycobacterium tubercu losis complex, which avoids use of hazardous chemicals (Cyanogen bromide).

Aeromonas hydrophila is an opportunistic microorganism. It is a secondary biological agent that contributes to the occurrence of fish diseases and its deterioration. This research was undertaken to determine the prevalence of A. hydrophila in some freshwater fishes collected from three different fish markets of Dhaka City and to test their antibiotic susceptibility. Total bacterial count and total aeromonas on different aeromonas selective media were enumerated using serial dilution technique. Bacterial isolates were characterized to identify A. hydrophila using biochemical tests and with comparison to reference strain (ATCC 7966). The lowest Aeromonas count was detected to be 2.83±0.40×10 2 cfu/g in Anabas testudineus and the highest was 1.03±0.153×10 3 cfu/g in Oreochromis mossambicus . On market basis highest aeromonas count was found in Anando Bazar (8.10±1.09×10 2 cfu/g) and lowest in Hatirpool Bazar (5.63±0.90×10 2 cfu/g) with no significant difference. Maximum susceptibility to amikacin and gentamicin was observed whereas all of the isolates were found resistant to a commonly used antibiotic amoxycillin. The obtained results point that antimicrobial susceptibility was more or less similar regardless of the origin of the samples collected. All the fishes investigated in this study contained A. hydrophila in their different organs.

Clostridium perfringens is an important pathogen that provokes numerous different diseases. This bacterium is classified into five different types, each of which capable of causing a different disease. There are various methods for the bacterial identification, many are labor-intensive, time-consuming, expensive and also present low sensitivity and specificity. The aim of this research was to identify the different types of C. perfringens using PCR molecular method. In this study, 130 sheep-dung samples were randomly collected from areas around the city of Kerman, southeastern Iran. After processing and culturing of samples, the produced colonies were morphologically studied, gram stain test was also carried out and the genera of these bacteria were identified through biochemical tests. DNA extracted from isolated bacteria for genotyping was tested by multiplex PCR with specific primers. Based on length of synthesized fragments by PCR, toxin types and bacterial strains were detected. C. perfringens isolated types were divided as follows: 17.39% type A, 21.74% type B, 34.78% type C and 26.09% type D. It should be emphasized that, up to the present moment, C. perfringens type A has not been reported in Iran.

The present research aimed at the biochemical characterization and molecular identification of pathogenic strains of Aeromonas sp, isolated from juvenile rainbow trout with clinical signs compatible with aeromoniasis in six fish farms in Junín (2), Pasco (2), Cajamarca (1) and Ancash (1), Peru. In total, 60 juvenile trout of 4-5 months were selected, with suggestive signs of outbreaks of aeromoniasis between 2017 and 2018. Euthanasia and necropsy were performed and the external and internal lesions present were recorded. Bacterial isolation of spleen and anterior kidney samples was carried out on TSA and GSP agar for 24-48 hours at 25 °C, bacterial colonies were characterized by biochemical tests and identified by conventional PCR using specific primers of the 16S rRNA gene (Aeromonas spp.), fstA (A. salmonicida) and gyrB (A. hydrophila), and sequencing was performed and confirmed by BLAST analysis (NCBI). The external lesions were melanosis, necrotic and haemorrhagic fins, skin ulcers and abdominal distention. Internal lesions were hepatomegaly, splenomegaly, and petechial haemorrhage in the liver, pyloric cecum, and peritoneal fat. Twelve strains were isolated in GSP (7 from Junín and 5 from Cajamarca), characterized as Aeromonas spp, being negative in the Pasco and Ancash samples. The biochemical tests determined four strains as Aeromonas salmonicida and eight as mobile Aeromonas sp, differing by motility and indole tests. Molecular identification by PCR determined four A. salmonicida strains from Junín, one A. hydrophila strain from Cajamarca and seven Aeromonas spp mobile strains (3 from Junín and 4 from Cajamarca) that were negative to the PCR of A. salmonicida and A. hydrophila. BLAST analysis (NCBI) confirmed the strains A. salmonicida and Aeromonas spp (98-100% identity). Furthermore, the Cajamarca strain (identified as A. hydrophila by biochemistry) presented 95.65% identity with A. hydrophila subspecies dhakensis. In conclusion, Aeromonas salmonicida, A. hydrophila subspecies dhakensis and other pathogenic mobile Aeromonas produce symptoms associated with aeromoniasis in juvenile rainbow trout in the Junín and Cajamarca fish farms. It is the first time that A. hydrophila subsp. dhakensis is detected in the country., La investigación tuvo como objetivo la caracterización bioquímica e identificación molecular de cepas patógenas de Aeromonas sp aisladas de truchas arcoíris juveniles con signología compatible a aeromoniasis, cultivadas en seis piscigranjas de los departamentos de Junín (2), Pasco (2), Cajamarca (1) y Ancash (1), Perú. Se seleccionaron 60 truchas juveniles de 4-5 meses, con signología sugerente de brotes de aeromoniasis entre 2017 y 2018. Se realizó la eutanasia y necropsia y se registraron las lesiones externas e internas presentes. Se efectuó el aislamiento bacteriano de muestras de bazo y riñón anterior en agar TSA y GSP por 24-48 horas a 25 °C, se caracterizaron las colonias bacterianas mediante pruebas bioquímicas y se identificaron por PCR convencional utilizando cebadores específicos del gen ARNr 16S (Aeromonas spp.), fstA (A. salmonicida) y gyrB (A. hydrophila). Además, se realizó la secuenciación y se confirmó por análisis BLAST (NCBI). Las lesiones externas registradas fueron melanosis, aletas necróticas y hemorrágicas, úlceras en la piel y distensión abdominal. Las lesiones internas fueron hepatomegalia, esplenomegalia y hemorragia petequial en hígado, ciegos pilóricos y grasa peritoneal. Se aislaron 12 cepas en GSP (7 de Junín y 5 de Cajamarca), caracterizadas como Aeromonas spp, siendo negativas en las muestras de Pasco y Ancash. Las pruebas bioquímicas determinaron cuatro cepas como Aeromonas salmonicida y ocho como Aeromonas sp móviles, diferenciándose por pruebas de motilidad e indol. La identificación molecular por PCR determinó cuatro cepas A. salmonicida procedentes de Junín, una cepa A. hydrophila de Cajamarca y siete cepas móviles Aeromonas spp (3 de Junín y 4 de Cajamarca) que fueron negativas a la PCR de A. salmonicida y A. hydrophila. El análisis BLAST (NCBI) confirmó las cepas A. salmonicida y Aeromonas spp (identidad de 98-100%); además, la cepa de Cajamarca (identificada como A. hydrophila por bioquímica) presentó identidad de 95.65% con A. hydrophila subespecie dhakensis. En conclusión, Aeromonas salmonicida, A. hydrophila subespecie dhakensis y otras Aeromonas móviles patógenas causan signología asociada a aeromoniasis en truchas arcoiris juveniles en las piscigranjas de Junín y Cajamarca. Es la primera vez que se detecta A. hydrophila subsp. dhakensis en el país.