Bacteriological, cytological, and molecular investigation of Corynebacterium pseudotuberculosis, mycobacteria, and other bacteria in caseous lymphadenitis and healthy lymph nodes of slaughtered sheep.

Caseous lymphadenitis (CL) in sheep is a chronic contagious disease caused by Corynebacterium pseudotuberculosis, commonly characterized by abscess formation in peripheral lymph nodes and disseminated infections. Nonetheless, other microorganisms, including with zoonotic relevance, can be isolated from CL-resembling lymph nodes. Currently, mycobacteria have been reported in visceral granulomatous lesions in small ruminants, a fact that poses a public health issue, particularly in slaughtered sheep intended for human consumption. Cytology using fine needle aspiration and microbiological culturing are suitable tests for routine diagnostic, whereas present drawbacks and molecular methods have been confirmatory. Data about the occurrence of mycobacteria in both lymph nodes with aspect of CL and apparently healthy visceral nodes of sheep slaughtered for human consumption are scarce. In this study, 197 visceral lymph nodes of sheep showed lymphadenitis and 202 healthy visceral lymph nodes of slaughtered sheep intended for human consumption were submitted to conventional bacteriological diagnosis, mycobacteria culturing, and cytological evaluation. Compatible Corynebacterium isolates were subjected to multiplex PCR targeting 16S rRNA, rpoB, and pld genes to detect C. pseudotuberculosis. Based on microbiological identification, C. pseudotuberculosis (86/197; 43.7%), streptococci γ-hemolytic (17/197; 8.6%), and Trueperella pyogenes (12/197; 6.1%) were prevalent in lymph nodes with abscesses, as opposed to staphylococci (53/202; 26.2%) in apparently healthy lymph nodes. No mycobacteria were isolated. Cytology identified 49.2% (97/197) Gram-positive pleomorphic organisms (coryneform aspect). Multiplex PCR confirmed genetic material of C. pseudotuberculosis in 74.4% (64/86) of the samples with C. pseudotuberculosis isolation and 66% (64/97) samples with cytological coryneform aspect (κ = 86.78%; 95% CI = 79.87-93.68%). These findings emphasize the prevalence of C. pseudotuberculosis in abscess formation among peripheral lymph nodes of sheep. Other bacteria were also identified in lymph nodes sampled that resembling C. pseudotuberculosis-induced infections that may difficult the diagnosis. Multiplex PCR revealed a valuable assay to detect C. pseudotuberculosis, in addition to routine methods applied to CL-diagnosis. No mycobacteria were identified in lymph nodes sampled, with and without apparent lesions. Nonetheless, due to public health impacts, this pathogen should be considered as a differential diagnosis of C. pseudotuberculosis-induced infections during inspection procedures of slaughtered sheep intended for human consumption.

Prevalência da linfadenite caseosa em ovinos e caprinos de municípios do estado do Amazonas e sensibilidade in vitro de Corynebacterium pseudotuberculosis aos antimicrobianos / Prevalence of caseous lymphadenitis in sheep and goats of the Amazon state municipality and in vitro susceptibility pattern of Corynebacterium pseudotuberculosis to antimicrobials / Prevalencia de linfadenitis caseosa en ovinos y caprinos del municipio del estado de Amazona y en la susceptibilidad in vitrode la Corynebacterium pseudotuberculosis a antimicrobianos

A linfadenite caseosa (LC) é uma enfermidade que acomete pequenos ruminantes, caracterizada clinicamente por lesões abscedantes em linfonodos, pele e órgãos, causada principalmente pela bactéria Corynebacterium pseudotuberculosis. O presente estudo investigou a prevalência da LC em criatórios de pequenos ruminantes do estado do Amazonas, bem como a sensibilidade microbiana in vitro de C. pseudotuberculosis aos antimicrobianos. Foi realizado exame clínico em 1.280 animais, dos quais 4,53% (58/1.280) dos animais apresentavam abscessos e/ou cicatrizes na pele e/ou linfonodos. No perfil de sensibilidade in vitro aos antimicrobianos, 100% dos isolados de C. pseudotuberculosis obtidos de ovinos foram sensíveis à ampicilina (8/8), amoxicilina/ácido clavulânico (8/8), cefotaxima (8/8), ciprofloxacina (8/8), norfloxacino (8/8), sulfazotrim e tetraciclina (8/8). Nos isolados de C. pseudotuberculosis obtidos de caprinos, verificou-se 100% de sensibilidade in vitro diante da amoxicilina/ácido clavulânico (5/5), cefotaxima (5/5), ciprofloxacina (5/5) e norfloxacino (5/5). Ratifica-se a importância do primeiro isolamento do agente em pequenos ruminantes no estado, bem como o primeiro estudo epidemiológico na Região Norte, constatando que a enfermidade possui baixa prevalência em ovinos e caprinos no Amazonas.(AU)

Caseous lymphadenitis (CL) is a disease that affects small ruminants, clinically characterized by abscessing lesions in lymph nodes, skin and organs, caused mainly by the bacterium Corynebacterium pseudotuberculosis. The present study investigated the prevalence of CS in small ruminant farms in the state of Amazonas, as well as the in vitro microbial sensitivity of C. pseudotuberculosis to antimicrobials. A clinical examination was carried out on 1,280 animals evaluated, of which 4.53% (58 / 1,280) of the animals had abscesses and / or scars on the skin and / or lymph nodes. In the in vitro sensitivity profile to antimicrobials, 100% of C. pseudotuberculosis isolates obtained from sheep were sensitive to ampicillin (8/8), amoxicillin / clavulanic acid (8/8),cefotaxime (8/8), ciprofloxacin (8 / 8), norfloxacin (8/8), sulfazotrim and tetracycline (8/8). In isolates of C. pseudotuberculosis obtained from goats, there was 100% sensitivity in vitro to amoxicillin / clavulanic acid (5/5), cefotaxime (5/5), ciprofloxacin (5/5) and norfloxacin (5/5). The importance of the first isolation of the agent in small ruminants in the state is ratified, as well as the first epidemiological study in the North Region, confirming that the disease has a low prevalence in sheep and goats in Amazonas.(AU)

La linfadenitis caseosa (LC) es una enfermedad que afecta a pequeños rumiantes, caracterizada clínicamente por lesiones de absceso en los ganglios linfáticos, la piely los órganos, causada principalmente por la bacteria Corynebacterium pseudotuberculosis. El presente estudio investigó la prevalencia de LC en pequeñas granjas de rumiantes en el estado de Amazona, así como la sensibilidad microbiana in vitro de C. pseudotuberculosis a los antimicrobianos. Se realizó un examen clínico en 1.280 animales evaluados, de los cuales 4.53% (58 / 1,280) de los animales tenían abscesos y / o cicatrices en la piel y / o ganglios linfáticos. En el perfil de sensibilidad in vitro a los antimicrobianos, el 100% de los aislados de C. pseudotuberculosis obtenidos de ovejas eran sensibles a ampicilina (8/8), amoxicilina / ácido clavulánico (8/8), cefotaxima (8/8), ciprofloxacina (8 / 8), norfloxacino (8/8), sulfazotrim y tetraciclina(8/8). En los aislados de C. pseudotuberculosis obtenidos de cabras, 100% de sensibilidad in vitro a amoxicilina / ácido clavulánico (5/5), cefotaxima (5/5), ciprofloxacina (5/5) y norfloxacino (5/5). Se ratifica la importancia del primer aislamiento del agente en pequeños rumiantes en el estado, así como el primer estudio epidemiológico en la Región Norte, lo que confirma que la enfermedad tiene una baja prevalencia en ovejas y cabras en Amazona.(AU)

Prevalência da linfadenite caseosa em ovinos e caprinos de municípios do estado do Amazonas e sensibilidade in vitro de Corynebacterium pseudotuberculosis aos antimicrobianos / Prevalence of caseous lymphadenitis in sheep and goats of the Amazon state municipality and in vitro susceptibility pattern of Corynebacterium pseudotuberculosis to antimicrobials / Prevalencia de linfadenitis caseosa en ovinos y caprinos del municipio del estado de Amazona y en la susceptibilidad in vitrode la Corynebacterium pseudotuberculosis a antimicrobianos

A linfadenite caseosa (LC) é uma enfermidade que acomete pequenos ruminantes, caracterizada clinicamente por lesões abscedantes em linfonodos, pele e órgãos, causada principalmente pela bactéria Corynebacterium pseudotuberculosis. O presente estudo investigou a prevalência da LC em criatórios de pequenos ruminantes do estado do Amazonas, bem como a sensibilidade microbiana in vitro de C. pseudotuberculosis aos antimicrobianos. Foi realizado exame clínico em 1.280 animais, dos quais 4,53% (58/1.280) dos animais apresentavam abscessos e/ou cicatrizes na pele e/ou linfonodos. No perfil de sensibilidade in vitro aos antimicrobianos, 100% dos isolados de C. pseudotuberculosis obtidos de ovinos foram sensíveis à ampicilina (8/8), amoxicilina/ácido clavulânico (8/8), cefotaxima (8/8), ciprofloxacina (8/8), norfloxacino (8/8), sulfazotrim e tetraciclina (8/8). Nos isolados de C. pseudotuberculosis obtidos de caprinos, verificou-se 100% de sensibilidade in vitro diante da amoxicilina/ácido clavulânico (5/5), cefotaxima (5/5), ciprofloxacina (5/5) e norfloxacino (5/5). Ratifica-se a importância do primeiro isolamento do agente em pequenos ruminantes no estado, bem como o primeiro estudo epidemiológico na Região Norte, constatando que a enfermidade possui baixa prevalência em ovinos e caprinos no Amazonas.

Caseous lymphadenitis (CL) is a disease that affects small ruminants, clinically characterized by abscessing lesions in lymph nodes, skin and organs, caused mainly by the bacterium Corynebacterium pseudotuberculosis. The present study investigated the prevalence of CS in small ruminant farms in the state of Amazonas, as well as the in vitro microbial sensitivity of C. pseudotuberculosis to antimicrobials. A clinical examination was carried out on 1,280 animals evaluated, of which 4.53% (58 / 1,280) of the animals had abscesses and / or scars on the skin and / or lymph nodes. In the in vitro sensitivity profile to antimicrobials, 100% of C. pseudotuberculosis isolates obtained from sheep were sensitive to ampicillin (8/8), amoxicillin / clavulanic acid (8/8),cefotaxime (8/8), ciprofloxacin (8 / 8), norfloxacin (8/8), sulfazotrim and tetracycline (8/8). In isolates of C. pseudotuberculosis obtained from goats, there was 100% sensitivity in vitro to amoxicillin / clavulanic acid (5/5), cefotaxime (5/5), ciprofloxacin (5/5) and norfloxacin (5/5). The importance of the first isolation of the agent in small ruminants in the state is ratified, as well as the first epidemiological study in the North Region, confirming that the disease has a low prevalence in sheep and goats in Amazonas.

La linfadenitis caseosa (LC) es una enfermedad que afecta a pequeños rumiantes, caracterizada clínicamente por lesiones de absceso en los ganglios linfáticos, la piely los órganos, causada principalmente por la bacteria Corynebacterium pseudotuberculosis. El presente estudio investigó la prevalencia de LC en pequeñas granjas de rumiantes en el estado de Amazona, así como la sensibilidad microbiana in vitro de C. pseudotuberculosis a los antimicrobianos. Se realizó un examen clínico en 1.280 animales evaluados, de los cuales 4.53% (58 / 1,280) de los animales tenían abscesos y / o cicatrices en la piel y / o ganglios linfáticos. En el perfil de sensibilidad in vitro a los antimicrobianos, el 100% de los aislados de C. pseudotuberculosis obtenidos de ovejas eran sensibles a ampicilina (8/8), amoxicilina / ácido clavulánico (8/8), cefotaxima (8/8), ciprofloxacina (8 / 8), norfloxacino (8/8), sulfazotrim y tetraciclina(8/8). En los aislados de C. pseudotuberculosis obtenidos de cabras, 100% de sensibilidad in vitro a amoxicilina / ácido clavulánico (5/5), cefotaxima (5/5), ciprofloxacina (5/5) y norfloxacino (5/5). Se ratifica la importancia del primer aislamiento del agente en pequeños rumiantes en el estado, así como el primer estudio epidemiológico en la Región Norte, lo que confirma que la enfermedad tiene una baja prevalencia en ovejas y cabras en Amazona.

Comparison between disk diffusion and agar dilution methods to determine in vitro susceptibility of Corynebacterium spp. clinical isolates and update of their susceptibility.

OBJECTIVES: Although Corynebacterium spp. are part of the microbiota of the skin and mucous membranes, human infections caused by Corynebacterium spp. have been reported and the multidrug resistance pattern of the recovered isolates was emphasised. Due to the usefulness of disk diffusion in daily practice, the purpose of this study was to compare disk diffusion with agar dilution to determine disk diffusion breakpoints and to review the antimicrobial susceptibility of the most frequent Corynebacterium spp. isolated in clinical samples. METHODS: Susceptibility to 20 antimicrobial agents of 143 Corynebacterium spp. isolates recovered from relevant clinical samples was determined. Comparison between the disk diffusion and agar dilution methods for eight antimicrobial agents was performed to establish new breakpoints using simple linear regression analysis. RESULTS: All of the isolates tested were susceptible to vancomycin, minocycline and linezolid. A typical susceptibility profile to ß-lactam antibiotics among the different species included was not observed. Almost all isolates showed resistance to macrolides and lincosamides. Using a simple linear regression method, it was possible to establish breakpoints for penicillin, erythromycin, clindamycin, gentamicin and ciprofloxacin. However, the low correlation coefficient obtained for vancomycin, minocycline and trimethoprim/sulfamethoxazole did not allow establishment of breakpoints for the disk diffusion method. CONCLUSION: The disk diffusion method could only be used to evaluate susceptibility to penicillin, erythromycin, clindamycin, gentamicin and ciprofloxacin. These data show that the presence of a Corynebacterium spp. isolate in a clinical sample demands the performance of antimicrobial susceptibility testing since the susceptibility profile is not predictable.

Searching whole genome sequences for biochemical identification features of emerging and reemerging pathogenic Corynebacterium species.

Biochemical tests are traditionally used for bacterial identification at the species level in clinical microbiology laboratories. While biochemical profiles are generally efficient for the identification of the most important corynebacterial pathogen Corynebacterium diphtheriae, their ability to differentiate between biovars of this bacterium is still controversial. Besides, the unambiguous identification of emerging human pathogenic species of the genus Corynebacterium may be hampered by highly variable biochemical profiles commonly reported for these species, including Corynebacterium striatum, Corynebacterium amycolatum, Corynebacterium minutissimum, and Corynebacterium xerosis. In order to identify the genomic basis contributing for the biochemical variabilities observed in phenotypic identification methods of these bacteria, we combined a comprehensive literature review with a bioinformatics approach based on reconstruction of six specific biochemical reactions/pathways in 33 recently released whole genome sequences. We used data retrieved from curated databases (MetaCyc, PathoSystems Resource Integration Center (PATRIC), The SEED, TransportDB, UniProtKB) associated with homology searches by BLAST and profile Hidden Markov Models (HMMs) to detect enzymes participating in the various pathways and performed ab initio protein structure modeling and molecular docking to confirm specific results. We found a differential distribution among the various strains of genes that code for some important enzymes, such as beta-phosphoglucomutase and fructokinase, and also for individual components of carbohydrate transport systems, including the fructose-specific phosphoenolpyruvate-dependent sugar phosphotransferase (PTS) and the ribose-specific ATP-binging cassette (ABC) transporter. Horizontal gene transfer plays a role in the biochemical variability of the isolates, as some genes needed for sucrose fermentation were seen to be present in genomic islands. Noteworthy, using profile HMMs, we identified an enzyme with putative alpha-1,6-glycosidase activity only in some specific strains of C. diphtheriae and this may aid to understanding of the differential abilities to utilize glycogen and starch between the biovars.

Etiology And In Vitro Antimicrobial Susceptibility Profile Of Strains Isolated From Bovine Mastitis In Dairy Herds From The Midwest Region Of Santa Catarina State, Brazil

Dairy farming is an important social and economic activity in the Midwest region of Santa Catarina State, Brazil. However, despite technological advances in the productive chain, mastitis is still considered the main disease of dairy herds, due its high economic impact. Bacterial pathogens are the main cause of this disease and failures in the treatment of clinical cases are attributed in part to the antimicrobial resistance of these pathogens. As an alternative, in vitro antimicrobial susceptibility tests of strains isolated from bovine mastitis cases have been carried out; the information generated contributes to strategic treatment measures, and also to the control of microorganism resistance. Therefore, the aims of the present study were to evaluate the etiology and in vitro antimicrobial susceptibility profiles of bacterial strains isolated from cases of bovine mastitis in dairy herds from the Midwest region of Santa Catarina State, Brazil. For this purpose, 345 milk samples were collected from 70 herds located in 16 different municipalities. The main pathogens isolated were: staphylococci (55.9%), streptococci (34.8%), Corynebacterium spp. (7.8%) and Escherichia coli (1.4%). The resistance rates of staphylococcus strains to ampicillin and to gentamicin were lower (24.9%) and higher (33%), respectively, than those obtained in other Brazilian studies. The resistance profiles of streptococcus strains were similar to those described in the literature, while Corynebacterium spp., showed high resistance rates to tetracycline (85.2%). Moreover, a considerable prevalence of staphylococcus (27.5%) and streptococcus (11.7%) multiresistant strains was verified. These data reinforce the need for regional characterization of microorganisms.

Etiology And In Vitro Antimicrobial Susceptibility Profile Of Strains Isolated From Bovine Mastitis In Dairy Herds From The Midwest Region Of Santa Catarina State, Brazil

Dairy farming is an important social and economic activity in the Midwest region of Santa Catarina State, Brazil. However, despite technological advances in the productive chain, mastitis is still considered the main disease of dairy herds, due its high economic impact. Bacterial pathogens are the main cause of this disease and failures in the treatment of clinical cases are attributed in part to the antimicrobial resistance of these pathogens. As an alternative, in vitro antimicrobial susceptibility tests of strains isolated from bovine mastitis cases have been carried out; the information generated contributes to strategic treatment measures, and also to the control of microorganism resistance. Therefore, the aims of the present study were to evaluate the etiology and in vitro antimicrobial susceptibility profiles of bacterial strains isolated from cases of bovine mastitis in dairy herds from the Midwest region of Santa Catarina State, Brazil. For this purpose, 345 milk samples were collected from 70 herds located in 16 different municipalities. The main pathogens isolated were: staphylococci (55.9%), streptococci (34.8%), Corynebacterium spp. (7.8%) and Escherichia coli (1.4%). The resistance rates of staphylococcus strains to ampicillin and to gentamicin were lower (24.9%) and higher (33%), respectively, than those obtained in other Brazilian studies. The resistance profiles of streptococcus strains were similar to those described in the literature, while Corynebacterium spp., showed high resistance rates to tetracycline (85.2%). Moreover, a considerable prevalence of staphylococcus (27.5%) and streptococcus (11.7%) multiresistant strains was verified. These data reinforce the need for regional characterization of microorganisms.(AU)

Efficient differentiation of Corynebacterium striatum, Corynebacterium amycolatum and Corynebacterium xerosis clinical isolates by multiplex PCR using novel species-specific primers.

A multiplex-PCR (mPCR) assay was designed with species-specific primers which generate amplicons of 226bp, 434bp and 106bp for differentiating the species C. striatum, C. amycolatum, and C. xerosis, respectively. mPCR results were 100% in agreement with identifications achieved by 16S rRNA and rpoB gene sequencing and by VITEK-MS.

Draft Genome Sequences of Two Pathogenic Corynebacterial Species Isolated from Cows.

The species Corynebacterium renale, Corynebacterium pilosum, and Corynebacterium cystitidis were initially thought to be the same species C. renale, but with different immunological types. These bacteria are the causative agent of cystitis, urethritis and pyelonephritis and are found usually as constituents of the normal flora in the lower urogenital tract of cattle. Therefore, we present the draft genome sequences of two pathogenic Corynebacterium species: C. renale CIP 52.96 and C. pilosum CIP 103422. The genome sequences of these species have 2,322,762 bp with 2,218 protein encoding genes and 2,548,014 bp with 2,428 protein encoding genes, respectively. These genomes can help clarify the virulence mechanisms of these unknown bacteria and enable the development of more effective methods for control.

Effects of bovine subclinical mastitis caused by Corynebacterium spp. on somatic cell count, milk yield and composition by comparing contralateral quarters.

Subclinical mastitis caused by Corynebacterium spp. (as a group and at the species level) was investigated by evaluating contralateral (healthy and infected) mammary quarters for somatic cell count (SCC), milk yield and composition. Selection of cows with subclinical mastitis caused by Corynebacterium spp. was performed by microbiological culture of composite samples collected from 1242 dairy cows from 21 dairy herds. For each of the selected cows, milk yield was measured and milk samples were collected at the mammary quarter level (i.e., 1140 mammary samples collected from 285 cows) for analysis of milk composition and SCC. The identification of Corynebacterium spp. isolates was performed by 16S rRNA gene sequencing. One hundred and eighty Corynebacterium spp. isolates were identified, of which 167 (92.77%) were C.bovis and eight (4.44%) non-C.bovis; for five of the Corynebacterium spp. isolates (2.77%), sequencing of 16S rRNA genes did not allow identification at the species level. Mammary quarters infected with Corynebacterium spp. as a group had a higher geometric mean SCC (197,900 cells/mL) than healthy contralateral mammary quarters (85,800 cells/mL). Species of Corynebacterium non-C.bovis were infrequently isolated and did not change SCC, milk yield or milk solid contents when evaluated at the contralateral quarter level. Although C.bovis infection showed no effect on milk yield, fat, protein, casein or total solids in milk, it increased SCC and decreased lactose and milk solids non-fat content.

Draft Genome Sequences of Two Species of "Difficult-to-Identify" Human-Pathogenic Corynebacteria: Implications for Better Identification Tests.

Non-diphtheriae Corynebacterium species have been increasingly recognized as the causative agents of infections in humans. Differential identification of these bacteria in the clinical microbiology laboratory by the most commonly used biochemical tests is challenging, and normally requires additional molecular methods. Herein, we present the annotated draft genome sequences of two isolates of "difficult-to-identify" human-pathogenic corynebacterial species: C. xerosis and C. minutissimum. The genome sequences of ca. 2.7 Mbp, with a mean number of 2,580 protein encoding genes, were also compared with the publicly available genome sequences of strains of C. amycolatum and C. striatum. These results will aid the exploration of novel biochemical reactions to improve existing identification tests as well as the development of more accurate molecular identification methods through detection of species-specific target genes for isolate's identification or drug susceptibility profiling.

Identification of Corynebacterium spp. isolated from bovine intramammary infections by matrix-assisted laser desorption ionization-time of flight mass spectrometry.

Corynebacterium species (spp.) are among the most frequently isolated pathogens associated with subclinical mastitis in dairy cows. However, simple, fast, and reliable methods for the identification of species of the genus Corynebacterium are not currently available. This study aimed to evaluate the usefulness of matrix-assisted laser desorption ionization/mass spectrometry (MALDI-TOF MS) for identifying Corynebacterium spp. isolated from the mammary glands of dairy cows. Corynebacterium spp. were isolated from milk samples via microbiological culture (n=180) and were analyzed by MALDI-TOF MS and 16S rRNA gene sequencing. Using MALDI-TOF MS methodology, 161 Corynebacterium spp. isolates (89.4%) were correctly identified at the species level, whereas 12 isolates (6.7%) were identified at the genus level. Most isolates that were identified at the species level with 16 S rRNA gene sequencing were identified as Corynebacterium bovis (n=156; 86.7%) were also identified as C. bovis with MALDI-TOF MS. Five Corynebacterium spp. isolates (2.8%) were not correctly identified at the species level with MALDI-TOF MS and 2 isolates (1.1%) were considered unidentified because despite having MALDI-TOF MS scores >2, only the genus level was correctly identified. Therefore, MALDI-TOF MS could serve as an alternative method for species-level diagnoses of bovine intramammary infections caused by Corynebacterium spp.

Produção e composição do leite de vacas com mastite causada por Corynebacterium spp / Milk yield and composition of cows with subclinical mastitis caused by Corynebacterium spp

A mastite é a inflamação da glândula mamária cuja causa mais comum são as infecções bacterianas. É considerada a doença de maiores prejuízos e prevalência em vacas leiteiras. A forma subclínica da mastite é o tipo mais predominante de infecção intramamária (IIM), sendo Corynebacterium spp. um dos agentes mais frequentemente isolados. Portanto, os objetivos gerais deste estudo foram: 1) avaliar o efeito de IIMs subclínicas causadas por Corynebacterium spp. sobre a composição e a produção de leite de quartos mamários; 2) determinar o efeito da IIM causada por Corynebacterium spp. sobre o teor de gordura, proteína bruta, lactose, caseína, extrato seco total, extrato seco desengordurado do leite e contagem de células somáticas (CCS); 3) avaliar a técnica de Espectrometria de massas por Ionização e Dessorção à Laser Assistida por Matriz Tempo de Voo (MALDI/TOF-MS) para a identificação das espécies de Corynebacterium isoladas de quartos mamários. Foram utilizados 21 rebanhos leiteiros (n=1.242 animais), nos quais foram selecionadas vacas em lactação previamente identificadas com IIM causada por Corynebacterium spp. (n=285). [...] Houve equivalência de resultados de 95% entre as duas técnicas de identificação utilizadas. Em conclusão, IIMs causadas por Corynebacterium spp. não apresentam efeito sobre a produção e composição de leite, porém apresentam aumento significativo da CCS. Adicionalmente, a técnica de MALDI/TOF-MS com o uso da extração em tubo por ácido fórmico/acetonitrila foi aplicada com 91,57% de sensibilidade de identificação das espécies de Corynebacterium nos casos de mastite bovina subclínica, quando comparado aos resultados do sequenciamento de genes 16S rRNA

Mastitis is an inflammation of the mammary glands which bacteria are the most common cause of infections. It is considered a disease with higher prevalence and losses in dairy cows. The subclinical form of mastitis is the most prevalent type of intramammary infection (IMI), and Corynebacterium spp., one of the agents most frequently isolated. Therefore, the main objectives of this study were: 1) to evaluate the effect of subclinical IMI caused by Corynebacterium spp. on milk composition and yield of mammary quarters; 2) to determine the effect of IMI caused by Corynebacterium spp. on the concentration of fat, protein, lactose, casein, total solids, non fat dry milk and somatic cell count (SCC); 3) to evaluate the technique of Matrix-Assisted Laser Desorption Ionization - Time-of-Flight - Mass Spectrometry (MALDI/TOF-MS) for species identification of Corynebacterium isolated from mammary glands. Twenty-one dairy herds were used (n = 1242 animals) on which lactating cows were selected previously identified with IMI caused by Corynebacterium spp. (n = 285). [...]There were equivalence results of 95% between the two identification techniques used. In conclusion, IMI caused by Corynebacterium spp. have no effect on milk yield and composition, but exhibit a significant increase in CCS. Additionally, the technique of MALDI/TOF-MS using the tube-extraction by formic acid/acetonitrile was applied with 91.57% sensitivity for identifying the species of Corynebacterium on cases of bovine mastitis subclinical when compared to the results of genes sequencing 16S rRNA

Desenvolvimento e implementação de métodos laboratoriais aplicados ao diagnóstico fenotípico e genotípico do Corynebacterium diptheriae / Development and implementation of laboratory methods applied to phenotypic and genotypic diagnosis of Corynebacterium diphtheriae

A emergência de cepas de Corynebacterium diphtheriae atoxinogênicas como agentes de endocardite e outras infecções sistêmicas aliada ao aumento do número de adultos susceptíveis à difteria enfatizam a necessidade de métodos alternativos para o diagnóstico laboratorial desta doença, especialmente para laboratórios de rotina clínica. Neste estudo avaliou-se a atividade de DNase de 91 amostras de C. diphtheriae (37 toxinogênicas e 54 atoxinogênicas) e de 564 cepas clínicas de bacilo Gram positivo não diftérico. A atividade de DNase foi detectada em todas as amostras de C. diphtheriae examinadas, previamente identificadas por métodos bioquímicos e pelo sistema API Coryne System. Diferentemente, os resultados do teste de DNase foram negativos em 93.9 porcento das cepas clínicas de bacilo Gram positivo não diftérico. Também foi documentado o valor de uma PCR espécie-específica que tem como alvo o gene dtxR como um método para diferenciação entre C. diphtheriae e colônias similares ao gênero Corynebacterium. Os resultados da PCR-dtxR foram positivos para todas as amostras de C. diphtheriae estudadas e foram concordantes com os obtidos através de metodologia bioquímica padrão. Diferentemente, os resultados da PCR-dtxR foram negativos para 100 porcento das 111 amostras de bacilos Gram positivos não diftéricos estudadas. A partir destes resultados, uma PCR multiplex utilizando três pares de oligonucleotídeos iniciadores foi desenvolvida para a detecção do C. diphtheriae e diferenciação em amostras toxinogênicas ou atoxinogênicas. Dois pares de oligonucleotídeos iniciadores têm como alvo as regiões do gene tox relativas aos domínios A e B da toxina diftérica e um terceiro par direcionado para o gene dtxR. Todas as amostras de C. diphtheriae foram identificadas pela reação de PCR multiplex em concordância com os testes bioquímicos padrão e os ensaios de citotoxicidade celular...

The emergence of non-toxigenic Corynebaterium diphtheriae strains as the causative agent of endocarditis and other systemic infections and the significant rise in the percentage of adults susceptible to diphtheria emphasize the need for new laboratory diagnostic procedures. In this study, we examine techniques as alternative procedures for differentiating C. diphtheriae from Corynebacterium-like colonies for the presumptive identification of this pathogen, especially in the diagnosis laboratory. This study evaluated the DNase activity of 91 C. diphtheriae (37 toxigenic and 54 non-toxigenic) and 564 non-diphtherial Gram-positive rod clinical strains. The DNase activity was detected in all C. diphtheriae strains examined, previously identified by both conventional biochemical methods and API Coryne System. Conversely, DNase test results were negative in 93.9 percent of the 564 non-diphtherial Gram-positive rod clinical strains. We also documented the value of a species-specific PCR assay that targets the dtxR gene as a procedure for differentiating C. diphtheriae from Corynebacterium-like colonies. The results of the PCR-dtxR were all positive for 91 C. diphtheriae strains and completely correlated with the standard biochemical methods and commercial identification system for all strains tested. In other hand, the PCR-dtxR results were negative in 100 percent of the 111 non-diphtherial Gram-positive rod strains. Considering these results, a multiplex PCR using three primers pairs was developed for detection of C. diphtheriae infection and differentiation between toxigenic and non-toxigenic strains. Two primer pairs targeted to domains A and B of tox gene and a third primer pair targeted to a region of dtxR gene. All C. diphtheriae strains were diagnosed by the multiplex PCR in agreement with standard biochemical tests and citotoxicity assay in Vero cells. Thus, these tecniques emerged as viable, cost-effective screening methods for C. diphtheriae laboratory...