Genome sequencing of Mycobacterium pinnipedii strains: genetic characterization and evidence of superinfection in a South American sea lion (Otaria flavescens).

BACKGROUND: Mycobacterium pinnipedii, a member of the Mycobacterium tuberculosis Complex (MTBC), is capable of infecting several host species, including humans. Recently, ancient DNA from this organism was recovered from pre-Columbian mummies of Peru, sparking debate over the origin and frequency of tuberculosis in the Americas prior to European colonization. RESULTS: We present the first comparative genomic study of this bacterial species, starting from the genome sequencing of two M. pinnipedii isolates (MP1 and MP2) obtained from different organs of a stranded South American sea lion. Our results indicate that MP1 and MP2 differ by 113 SNPs (single nucleotide polymorphisms) and 46 indels, constituting the first report of a mixed-strain infection in a sea lion. SNP annotation analyses indicate that genes of the VapBC family, a toxin-antitoxin system, and genes related to cell wall remodeling are under evolutionary pressure for protein sequence change in these strains. OrthoMCL analysis with seven modern isolates of M. pinnipedii shows that these strains have highly similar proteomes. Gene variations were only marginally associated with hypothetical proteins and PE/PPE (proline-glutamate and proline-proline-glutamate, respectively) gene families. We also detected large deletions in ancient and modern M. pinnipedii strains, including a few occurring only in modern strains, indicating a process of genome reduction occurring over the past one thousand years. Our phylogenomic analyses suggest the existence of two modern clusters of M. pinnipedii associated with geographic location, and possibly host species, and one basal node associated with the ancient M. pinnipedii strains. Previously described MiD3 and MiD4 deletions may have occurred independently, twice, over the evolutionary course of the MTBC. CONCLUSION: The presence of superinfection (i.e. mixed-strain infection) in this sea lion suggests that M. pinnipedii is highly endemic in this population. Mycobacterium pinnipedii proteomes of the studied isolates showed a high degree of conservation, despite being under genomic decay when compared to M. tuberculosis. This finding indicates that further genomes need to be sequenced and analyzed to increase the chances of finding variably present genes among strains or that M. pinnipedii genome remodeling occurred prior to bacterial speciation.

Virulence factors and antimicrobial resistance in Staphylococcus aureus isolated from bovine mastitis in Brazil.

This study aimed to evaluate virulence factors and genetic markers of antimicrobial resistance in 400 Staphylococcus aureus strains isolated from bovine mastitis in four Brazilian states, as well as to assess the association between these characteristics and field information. Virulence factors and drug resistance genes were identified by PCR screening. Biofilm-forming and hemolytic phenotype were detected using Congo red Tryptic Soy Broth and defibrinated sheep blood agar, respectively. Of all isolates, 83.5% were biofilm-forming and 98.5% strains exhibited biofilm gene icaAD, and a significant association between phenotype and genotype for biofilm was observed (P = 0.0005). Hemolysin genes were observed in 82.85% (hla+hlb+), 16.5% (hla+) and 0.75% (hlb+) isolates, whereas the hemolytic phenotype exhibited was complete and incomplete hemolysis in 64.25%, complete in 28.25%, incomplete in 4.75%, and negative in 2.75% of the strains. Virulence factors genes luk, seb, sec, sed, and tst were observed in 3.5%, 0.5%, 1%, 0.25%, and 0.74% isolates, respectively. The gene blaZ was detected in 82.03% of penicillin-resistant isolates, whereas tetK and aac(6')-Ie-aph(2')-Ia were observed in 33.87% and 45.15% of the tetracycline and aminoglycosides-resistant isolates, respectively. Fluoroquinolone resistance gene mepA was detected for the first time in S. aureus from bovine mastitis. Resistance genes tetM (3.22%), tetL (1.61%), ermA (14.29%), ermB (14.29%), ermC (33.3%), ermT (9.52%), ermY (4.76%), msrA (9.52%), and mphC (9.52%) were also detected among resistant isolates. No association between virulence factors or antimicrobial-resistant genes and year of isolation, geographic origin, or antimicrobial resistance profile was observed. Our results showed that S. aureus strains isolated from bovine mastitis in the four Brazilian states sampled are mainly biofilm-forming and hemolytic, whereas virulence genes associated with enterotoxins, luk and tst, were less frequently observed. Moreover, a wide variety of resistance genes that confer resistance to almost all classes of antimicrobial agents approved for use in animals and humans were found. Overall, the data point to a great pathogenic potential of S. aureus associated with bovine mastitis and to the non-negligible risks to public health of staphylococcal infections from animal origin.

Prevalence of Leptospira serogroups in buffaloes from the Brazilian Amazon.

Although Brazil has one of the largest buffalo populations in the Americas, buffalo leptospirosis is still poorly explored when compared to that in bovines; thus, the aim of this research was to carry out a large serological study for leptospirosis in this species in the Brazilian Amazon. For this, we collected 1,405 serum samples from buffaloes raised in the Amazon delta region, which is considered a major area of buffalo production in Brazil. The test used was a microscopic agglutination test (MAT) adopting 34 Leptospira antigens, some of which have never been tested for buffaloes in Brazil, including autochthonous strains; in total, 20 serogroups were evaluated. From the total of 1,405 serum samples, 894 (63.6%) reacted in the MAT to at least one of the 20 serogroups, and 511 (36.4%) did not react. The serogroups Sejroe, Autumnalis and Pomona were the most prevalent, with titres ranging from 100 to 12,800, and the autochthonous strains used were not significant in relation to the reference serovars. Leptospirosis in buffaloes seems to have a serological profile similar to leptospirosis in cattle, mainly due to the prevalence of the Sejroe serogroup; however, the results of this study suggested that in the Brazilian Amazon, Leptospira strains that are serologically distinct from the autochthonous strains isolated in the southeastern region of Brazil may be circulating in these animals. Other serovars could also be inserted into the panel of antigens used in MAT for serological studies on buffaloes.

Molecular Typing and Antimicrobial Susceptibility Profile of Staphylococcus aureus Isolates Recovered from Bovine Mastitis and Nasal Samples.

In the present study, we aimed to determine the antimicrobial resistance and molecular typing of Staphylococcus aureus recovered from transient and persistent intramammary infections and nares/muzzles in dairy cows. We investigated the antimicrobial resistance of 189 S. aureus strains using a broad antimicrobial susceptibility profile. Furthermore, 107 S. aureus isolates were strain-typed using staphylococcal protein-A (spa) typing. A large proportion of strains exhibited multidrug resistance to antimicrobials, including resistance to critically important antimicrobials, although no methicillin-resistant S. aureus strains were found. Our study did not strengthen the idea that extramammary niches (i.e., nares/muzzles) are an important source of S. aureus for bovine mastitis. A discrepancy in the antimicrobial resistance between S. aureus strains isolated from nares/muzzles and milk samples was observed. Furthermore, S. aureus isolates from transient and persistent intramammary infections (IMIs) did not differ by spa typing, suggesting that the persistence of bovine IMIs was determined by cow factors. Thus, the high level of multidrug-resistant S. aureus found in the two herds, considered together with the predominance of a well udder-adapted S. aureus strain, may contribute to our knowledge of the history of the high prevalence of mastitis caused by S. aureus, which is of great concern for animal and public health.

Global Distribution and Evolution of Mycobacterium bovis Lineages.

Mycobacterium bovis is the main causative agent of zoonotic tuberculosis in humans and frequently devastates livestock and wildlife worldwide. Previous studies suggested the existence of genetic groups of M. bovis strains based on limited DNA markers (a.k.a. clonal complexes), and the evolution and ecology of this pathogen has been only marginally explored at the global level. We have screened over 2,600 publicly available M. bovis genomes and newly sequenced four wildlife M. bovis strains, gathering 1,969 genomes from 23 countries and at least 24 host species, including humans, to complete a phylogenomic analyses. We propose the existence of four distinct global lineages of M. bovis (Lb1, Lb2, Lb3, and Lb4) underlying the current disease distribution. These lineages are not fully represented by clonal complexes and are dispersed based on geographic location rather than host species. Our data divergence analysis agreed with previous studies reporting independent archeological data of ancient M. bovis (South Siberian infected skeletons at ∼2,000 years before present) and indicates that extant M. bovis originated between 715 and 3,556 years BP, with later emergence in the New World and Oceania, likely influenced by trades among countries.

Complete Genome Sequencing of Mycobacterium bovis SP38 and Comparative Genomics of Mycobacterium bovis and M. tuberculosis Strains.

Mycobacterium bovis causes bovine tuberculosis and is the main organism responsible for zoonotic tuberculosis in humans. We performed the sequencing, assembly and annotation of a Brazilian strain of M. bovis named SP38, and performed comparative genomics of M. bovis genomes deposited in GenBank. M. bovis SP38 has a traditional tuberculous mycobacterium genome of 4,347,648 bp, with 65.5% GC, and 4,216 genes. The majority of CDSs (2,805, 69.3%) have predictive function, while 1,206 (30.07%) are hypothetical. For comparative analysis, 31 M. bovis, 32 M. bovis BCG, and 23 Mycobacterium tuberculosis genomes available in GenBank were selected. M. bovis RDs (regions of difference) and Clonal Complexes (CC) were identified in silico. Genome dynamics of bacterial groups were analyzed by gene orthology and polymorphic sites identification. M. bovis polymorphic sites were used to construct a phylogenetic tree. Our RD analyses resulted in the exclusion of three genomes, mistakenly annotated as virulent M. bovis. M. bovis SP38 along with strain 35 represent the first report of CC European 2 in Brazil, whereas two other M. bovis strains failed to be classified within current CC. Results of M. bovis orthologous genes analysis suggest a process of genome remodeling through genomic decay and gene duplication. Quantification, pairwise comparisons and distribution analyses of polymorphic sites demonstrate greater genetic variability of M. tuberculosis when compared to M. bovis and M. bovis BCG (p ≤ 0.05), indicating that currently defined M. tuberculosis lineages are more genetically diverse than M. bovis CC and animal-adapted MTC (M. tuberculosis Complex) species. As expected, polymorphic sites annotation shows that M. bovis BCG are subjected to different evolutionary pressures when compared to virulent mycobacteria. Lastly, M. bovis phylogeny indicates that polymorphic sites may be used as markers of M. bovis lineages in association with CC. Our findings highlight the need to better understand host-pathogen co-evolution in genetically homogeneous and/or diverse host populations, considering the fact that M. bovis has a broader host range when compared to M. tuberculosis. Also, the identification of M. bovis genomes not classified within CC indicates that the diversity of M. bovis lineages may be larger than previously thought or that current classification should be reviewed.

Direct detection of Mycobacterium tuberculosis complex in bovine and bubaline tissues through nested-PCR.

Post-mortem bacterial culture and specific biochemical tests are currently performed to characterize the etiologic agent of bovine tuberculosis. Cultures take up to 90 days to develop. A diagnosis by molecular tests such as PCR can provide fast and reliable results while significantly decreasing the time of confirmation. In the present study, a nested-PCR system, targeting rv2807, with conventional PCR followed by real-time PCR, was developed to detect Mycobacterium tuberculosis complex (MTC) organisms directly from bovine and bubaline tissue homogenates. The sensitivity and specificity of the reactions were assessed with DNA samples extracted from tuberculous and non-tuberculous mycobacteria, as well as other Actinomycetales species and DNA samples extracted directly from bovine and bubaline tissue homogenates. Regarding the analytical sensitivity, DNA of the M. bovis AN5 strain was detected up to 1.5 pg by nested-PCR, whereas DNA of M. tuberculosis H37Rv strain was detected up to 6.1 pg. The nested-PCR system showed 100% analytical specificity for MTC when tested with DNA of reference strains of non-tuberculous mycobacteria and closely-related Actinomycetales. A clinical sensitivity level of 76.7% was detected with tissues samples positive for MTC by means of the culture and conventional PCR. A clinical specificity of 100% was detected with DNA from tissue samples of cattle with negative results in the comparative intradermal tuberculin test. These cattle exhibited no visible lesions and were negative in the culture for MTC. The use of the nested-PCR assay to detect M. tuberculosis complex in tissue homogenates provided a rapid diagnosis of bovine and bubaline tuberculosis.

Identificação e genotipagem de mycobacterium bovis em bovinos positivos no teste intradérmico para tuberculose em Mato Grosso do Sul / Identification and genotyping of mycobacterium bovis from positive cattle in skin test for tuberculosis in the state of Mato Grosso do Sul, Brazil

Neste estudo, realizou-se genotipagem de isolados de Mycobacterium bovis, provenientes de amostras de tecidos de bovinos positivos no teste cervical comparativo (TCC) para tuberculose em Mato Grosso do Sul, por meio da técnica de spoligotyping. Tecidos de 13 bovinos positivos, oriundos de diferentes municípios do estado, foram cultivados em meio de Stonebrink. As colônias resultantes foram submetidas à coloração de Ziehl-Neelsen e todos os isolados apresentaram características tintoriais de BAAR. Os 13 isolados de BAAR foram identificados por PCR multiplex (mPCR). O gene hsp65 foi alvo para identificação de Mycobacterium spp, a sequência de inserção IS6110 foi alvo para identificação de complexo Mycobacterium tuberculosis (CMT) e a região rvd1rv2031c foi explorada para detecção de M. bovis. Os isolados micobacterianos foram genotipados pela técnica de spoligotyping. Dos 13 bovinos, sete tinham pelo menos uma lesão sugestiva de tuberculose em linfonodos retrofaríngeos, parotídeos e pulmonares ou no pulmão, e em seis não foram encontradas lesões visíveis sugestivas da doença. Na mPCR, 11/13 (84,6%) isolados foram positivos para Mycobacterium spp, 8/13 (61,5%) positivos para CMT e 7/13 (53,8%) positivos para M. bovis. Com base no spoligotyping, oito isolados de BAAR foram agrupados dentro de três diferentes agrupamentos de genótipos e uma amostra remanescente apresentou perfil único, sendo quatro isolados com padrão de espoligotipo SB0121, dois SB1145, dois SB0881 e um SB0140. A técnica de spoligotyping demonstrou que há diversidade genética entre os espoligotipos presentes no estado de Mato Grosso do Sul, embora predomine o perfil SB0121.

Spoligotyping was performed in the present study to genotype Mycobacterium bovis isolates obtained from tissues of cattle that were positive in the comparative intradermal tuberculin test (CITT) in the state of Mato Grosso do Sul (Brazil). Tissue samples from 13 positive cattle from different municipalities of the state were cultured using a Stonebrink medium. The resulting colonies were subjected to Ziehl-Neelsen staining and all isolates exhibited the staining characteristics of AFB. The 13 isolates of AFB were identified by means of a multiplex PCR (mPCR) assay. The hsp65 gene was targeted for the identification of Mycobacterium spp., whereas the IS6110 insertion sequence was targeted for the identification of the Mycobacterium tuberculosis complex (MTC) and the rvd1rv2031c region was explored for the detection of Mycobacterium bovis. The spoligotyping assay was performed to genotype mycobacterial isolates. Of the 13 cattle, seven had at least one lesion suggestive of tuberculosis in the retropharyngeal, parotid and lung lymph nodes or lung. The remaining six exhibited no lesions suggestive of the disease. In the mPCR, 11 of the 13 isolates (84.6%) were positive for Mycobacterium spp., 8/13 (61.5%) were positive for the MTC and 7/13 (53.8%) were positive for M. bovis. Based on the spoligotyping, eight isolates were grouped into three different groups of genotypes and one isolate exhibited an orphan type. Four isolates exhibited spoligotype pattern SB0121, while two isolates were associated with the pattern SB1145, another two were associated with pattern SB0881 and one was associated with pattern SB0140. Spoligotyping confirmed the genetic diversity present among isolates found in the state of Mato Grosso do Sul. In addition, SB0121 was confirmed as the predominant profile.

Direct detection of Mycobacterium tuberculosis complex in bovine and bubaline tissues through nested-PCR

Post-mortem bacterial culture and specific biochemical tests are currently performed to characterize the etiologic agent of bovine tuberculosis. Cultures take up to 90 days to develop. A diagnosis by molecular tests such as PCR can provide fast and reliable results while significantly decreasing the time of confirmation. In the present study, a nested-PCR system, targeting rv2807, with conventional PCR followed by real-time PCR, was developed to detect Mycobacterium tuberculosis complex (MTC) organisms directly from bovine and bubaline tissue homogenates. The sensitivity and specificity of the reactions were assessed with DNA samples extracted from tuberculous and non-tuberculous mycobacteria, as well as other Actinomycetales species and DNA samples extracted directly from bovine and bubaline tissue homogenates. Regarding the analytical sensitivity, DNA of the M. bovis AN5 strain was detected up to 1.5 pg by nested-PCR, whereas DNA of M. tuberculosis H37Rv strain was detected up to 6.1 pg. The nested-PCR system showed 100% analytical specificity for MTC when tested with DNA of reference strains of non-tuberculous mycobacteria and closely-related Actinomycetales. A clinical sensitivity level of 76.7% was detected with tissues samples positive for MTC by means of the culture and conventional PCR. A clinical specificity of 100% was detected with DNA from tissue samples of cattle with negative results in the comparative intradermal tuberculin test. These cattle exhibited no visible lesions and were negative in the culture for MTC. The use of the nested-PCR assay to detect M. tuberculosis complex in tissue homogenates provided a rapid diagnosis of bovine and bubaline tuberculosis.