Global analysis of sRNA target genes in Mycoplasma hyopneumoniae

Background: Small RNAs (sRNAs) are noncoding molecules that regulate different cellular activities in several bacteria. The role of sRNAs in gene expression regulation is poorly characterized in the etiological agent of porcine enzootic pneumonia Mycoplasma hyopneumoniae. We performed a global analysis of the sRNAs, sRNA target genes and regulatory elements previously identified in their genome and analyzed the expression of some sRNAs and their target genes by quantitative RT-PCR (qPCR) in three different culture conditions. Results: Seven of the 145 sRNA target genes are organized as monocistronic genes (mCs) while the other 138 sRNA target genes are organized into transcriptional units (TU). The identification of transcriptional regulatory elements (promoter motif, DNA repeat sequence or intrinsic terminator) was verified in 116 of the 145 sRNA target genes. Moreover, the 29 sRNA target genes without regulatory elements revealed the presence of at least one regulatory element in the boundaries of the TU or in other internal genes of the TU. We verified that 16 sRNAs showed differential expression, seven in heat shock condition and 14 in oxidative stress condition. Analysis of the differential expression of the sRNA target genes showed that the tested sRNAs possibly regulate gene expression. The sRNA target genes were up- or down-regulated possibly in response to sRNA only under oxidative stress condition. Moreover, the sRNA target genes are involved in diverse processes of the cell, some of which could be linked to transcription processes and cell homeostasis. Conclusion: Our results indicate that bacterial sRNAs could regulate a number of targets with various outcomes, and different correlations between the levels of sRNA transcripts and their target gene mRNAs were found, which suggest that the regulation of gene expression via sRNAs may play an important role in mycoplasma

Microbial contamination and antimicrobial resistance profiles indicate potential risks of infection at the veterinary medical teaching hospital - UFRGS, Porto Alegre, Brazil

Background: This study aimed to assess the level of bacterial contamination in the Small Animals Sector of the Veterinary Medical Teaching Hospital (HCV) of the Universidade Federal do Rio Grande do Sul (UFRGS). Firstly, a committee was invited to complete a questionnaire and to list critical sample sites for collection. With the identification of the places to be sampled, collections were made with sterile swabs on different surfaces of environments of the Veterinary Hospital. The identification of important bacteria in the veterinary area, in the different sampled environments, raises the concern for hygiene procedures in the veterinary hospital environment. Materials, Methods & Results: Sixteen samples were collected from these different areas, and microbiological analyses were performed. Standard counts of viable and strictly aerobic mesophilic microorganisms were realized. Collections were made to assess ambient air quality. With the microbiological analysis performed, bacteria of clinical importance were identified. To assess the resistance profile of the bacteria, the susceptibility test to antimicrobials was performed. MALDI-TOF/MS measurement identified 29 bacteria at the genus level and 10 bacteria at the species level and the antimicrobial susceptibility test was realized. Most of the isolates identified (60%) were bacteria of the genus Staphylococcus spp. Regarding antimicrobial susceptibility analysis the 10 bacteria identified at the species level were assessed. Test results showed that the isolates S. aureus, S. epidermidis and S. haemolyticus - collected from treatment room 2 - and S. haemolyticus, which had been isolated from samples from treatment room 2 of the cattery, presented multiresistance. Pantoea ananatis isolates from room 5 also showed a multiresistant profile for erythromycin, cephalothin, vancomycin and ampicillin. Micrococcus luteus isolates from the x-ray room and the kennel showed resistance to ceftazidime. Staphylococcus equorum isolates from room 4 were sensitive to all tested antimicrobials. Discussion: In Brazilian legislation there are no official microbiological parameters for surfaces in a veterinary hospital environment. The microorganisms present in the air are transient and variable, and the number and types of airborne agents is determined by the various sources of contamination in the environment. These microorganisms can be found in suspension, particulate matter and water droplets. Veterinary medical care tables are potentially contaminated by the animals handling, including those that sometimes defecate or urinate during their medical visit. Frequent handwashing is also known to be an important means of personal protection and disease prevention, although it is estimated that only 40% of practitioners do so routinely. Based on these results, we recommend a plan of bacterial control and disinfection that should be implemented to ensure more effective sanitary conditions. Microorganism counts were high in some of the veterinary hospital environments tested, indicating that current disinfection and hygiene practices are not sufficient to control the establishment of these microorganisms at the study sites. In view of this, it is reasonable to conclude that permanent monitoring and assessment of the effectiveness of hygiene protocols is needed in different sectors of the hospital. This may be an essential tool in a preventive approach to stop the spread of selectively resistant microorganisms, as well as cases of hospital infections. In addition, continuous staff training and awareness of the importance of personal and environmental hygiene is vital for minimizing the presence of these microorganisms in hospitals and avoid their transmission to patients. Finally, a more systematic hygiene guideline should be implemented in areas that showed higher counts

Mannheimia varigena como agente etiológico de claudicação e lesão na banda coronária em bovinos

Mannheimia varigena was identified as the etiologic agent of lameness and coronary band lesion in 30% of cattle in a farm located in Rio Grande do Sul, Brazil. Swab samples from the lesions were cultured in McConkey Agar and Blood Agar for microbiological identification. Culture growth was submitted to Gram staining and Matrix Assisted Laser Desorption Ionization Time of Flight Mass Spectrometry (MALDI-TOF MS) identification. Antimicrobial susceptibility test based on disc diffusion was performed for three antibiotics: ceftiofur, gentamicin and florfenicol. Furthermore, molecular characterization of 16S rDNA gene sequencing was performed and the data was used in a phylogenetic analysis. For that purpose, total DNA was extracted by thermo extraction directly from the bacterial colonies and the polymerase chain reaction (PCR) was performed. Gram-negative Mannheimia varigena strain LBV010/22 was identified as the causative of the lesions. The strain was susceptible to all antibiotics tested. The phylogenetic analysis demonstrated that the analyzed strain is closely related to M. varigena strains from pyelonephritis and respiratory tract. Overall, this is the first report of M. varigena as the causative agent of coronary band injury in bovine. Therefore, our findings show the importance of an accurate microbiological identification of infectious agent in lameness cases in order to prevent the occurrence and perform an appropriate treatment in the future.Mannheimia varigena foi identificada como agente etiológico de claudicação e lesão de banda coronária em 30% dos bovinos de uma fazenda localizada no Rio Grande do Sul, Brasil. Amostras de swab das lesões foram cultivadas em Ágar McConkey e Ágar Sangue para identificação microbiológica. O crescimento da cultura foi submetido à coloração de Gram e identificação por Espectrometria de Massa de Ionização por Dessorção a Laser Assistida por Matriz (MALDI-TOF MS). O teste de suscetibilidade antimicrobiana baseado na difusão em disco foi realizado para três antibióticos: ceftiofur, gentamicina e florfenicol. Além disso, foi realizada a caracterização molecular do sequenciamento do gene 16S rDNA e o resultado utilizado para análise filogenética. Para tanto, o DNA total foi extraído por termoextração diretamente das colônias bacterianas e uma reação em cadeia da polimerase (PCR) foi realizada. Foi identificada como causadora das lesões a cepa gram-negativa de Mannheimia varigena_LBV010/22. Ela foi suscetível a todos os antibióticos testados. A análise filogenética demonstrou que a cepa analisada está intimamente relacionada às M. varigena presentes em pielonefrite e no trato respiratório. No geral, este é o primeiro relato de M. varigena como agente causador de lesão de banda coronária em bovinos. Portanto, nossos achados mostram a importância de uma identificação microbiológica precisa do agente infeccioso nos casos de claudicação, a fim de prevenir a ocorrência e realizar um tratamento adequado no futuro

Streptococcus spp. in Equines - Infection and Antimicrobial Susceptibility Profiles

Background: Empirical antimicrobial prescribing is commonly used in equine veterinary. Therefore, professionals can obtain information about antimicrobial susceptibility profile of the bacterial strains based on veterinary literature. Considering equine infections, Streptococcus spp. are important pathogens that can cause serious damage in horses. Therefore, the aim of this study was to describe the antimicrobial susceptibility testing (AST) and infection profiles of Streptococcus spp. strains isolated from equines with infectious diseases subjected to microbiological analysis.   Materials, Methods & Results: Veterinarians sent 13 samples and culture in Blood and MacConkey Agar were performed. After the incubation period, suspected colonies, which showed significative growth, were analyzed by Gram-staining, biochemical tests, and subjected to confirmatory identification in Matrix Assisted Laser Desorption Ionization Time of Flight Mass Spectrometry. In vitro AST analysis were performed by disc diffusion method, in accordance with the veterinarians' request. The antimicrobials tested in this study were: ceftiofur, gentamicin, ampicillin, enrofloxacin, amikacin, penicillin, trimethoprim-sulfamethoxazole, ciprofloxacin, doxycycline, vancomycin and metronidazole. The samples included uterine exudate, hock fistula, osteosynthesis exudate, exudate from the guttural pouch, and were originated from animals located in different and distant geographical regions in the cities of Porto Alegre, Pelotas, and Bagé, Rio Grande do Sul, Brazil. Streptococcus dysgalactiae, Streptococcus equi and Streptococcus thoraltensis were the Streptococcus species identified in the samples. S. dysgalactiae was the mainly species found in the uterus samples, while S. thoraltensis, an unusual Streptococcus species, was identified as etiological agent of endometritis in 2 of the analyzed animals. On the other hand, S. equi was found in both the guttural pouch, representing the etiological agent of the strangle case, and in the osteosynthesis exudate, as infectious agent of post-osteosynthesis surgery. The majority of streptococci strains were susceptible to ceftiofur drug. Amikacin and ciprofloxacin, however, were the drugs for which the strains were mainly resistant according to the results. Discussion: The present study provided the AST and infection profile of Streptococcus species related to equine infectious diseases. S. dysgalactiae is considered an unusual bacterium isolated from horses that can be related to endometritis, S. equi is the causative agent of strangles, and S. thoraltensis is unusual in equines. Generally, the observed susceptibility to ceftiofur of the strains analyzed was in agreement with previous results reported in the literature. However, ceftiofur is a third-generation cephalosporin and is considered a critically important antibiotic for human health and its use in veterinary medicine should be cautious. Considering the resistance profile found, Streptococcus spp. can be intrinsically resistant to low drug concentrations of aminoglycosides. Moreover, the emergence and spread of fluoroquinolones resistance may also be due to the acquisition of resistance via horizontal gene transfer. Therefore, the present study described both infection and antimicrobial susceptibility patterns of Streptococcus strains related to equine infectious diseases. Considering the findings, the results found in this study might contribute to the decision-making by veterinarians to further equine treatments. Keywords: antimicrobial susceptibility, pattern, AST, drug, resistant bacteria, horses, veterinarians

Streptococcus spp. in equines : infection and antimicrobial susceptibility profiles

Background: Empirical antimicrobial prescribing is commonly used in equine veterinary. Therefore, professionals can obtain information about antimicrobial susceptibility profile of the bacterial strains based on veterinary literature. Considering equine infections, Streptococcus spp. are important pathogens that can cause serious damage in horses. Therefore, the aim of this study was to describe the antimicrobial susceptibility testing (AST) and infection profiles of Streptococcus spp. strains isolated from equines with infectious diseases subjected to microbiological analysis. Materials, Methods & Results: Veterinarians sent 13 samples and culture in Blood and MacConkey Agar were performed. After the incubation period, suspected colonies, which showed significative growth, were analyzed by Gram-staining, biochemical tests, and subjected to confirmatory identification in Matrix Assisted Laser Desorption Ionization Time of Flight Mass Spectrometry. In vitro AST analysis were performed by disc diffusion method, in accordance with the veterinarians’ request. The antimicrobials tested in this study were: ceftiofur, gentamicin, ampicillin, enrofloxacin, amikacin, penicillin, trimethoprim-sulfamethoxazole, ciprofloxacin, doxycycline, van¬comycin and metronidazole. The samples included uterine exudate, hock fistula, osteosynthesis exudate, exudate from the guttural pouch, and were originated from animals located in different and distant geographical regions in the cities of Porto Alegre, Pelotas, and Bagé, Rio Grande do Sul, Brazil. Streptococcus dysgalactiae, Streptococ¬cus equi and Streptococcus thoraltensis were the Streptococcus species identified in the samples. S. dysgalactiae was the mainly species found in the uterus samples, while S. thoraltensis, an unusual Streptococcus species, was identified as etiological agent of endometritis in 2 of the analyzed animals. On the other hand, S. equi was found in both the guttural pouch, representing the etiological agent of the strangle case, and in the osteosynthesis exu¬date, as infectious agent of post-osteosynthesis surgery. The majority of streptococci strains were susceptible to ceftiofur drug. Amikacin and ciprofloxacin, however, were the drugs for which the strains were mainly resistant according to the results. Discussion: The present study provided the AST and infection profile of Streptococcus species related to equine infectious diseases. S. dysgalactiae is considered an unusual bacterium isolated from horses that can be related to endometritis, S. equi is the causative agent of strangles, and S. thoraltensis is unusual in equines. Generally, the observed susceptibility to ceftiofur of the strains analyzed was in agreement with previous results reported in the literature. However, ceftiofur is a third-generation cephalosporin and is considered a critically important antibiotic for human health and its use in veterinary medicine should be cautious. Considering the resistance profile found, Streptococcus spp. can be intrinsically resistant to low drug concentrations of aminoglycosides. Moreover, the emergence and spread of fluoroquinolones resistance may also be due to the acquisition of resistance via horizontal gene transfer. Therefore, the present study described both infection and antimicrobial susceptibility patterns of Streptococcus strains related to equine infectious diseases. Considering the findings, the results found in this study might contribute to the decision-making by veterinarians to further equine treatments

Perception of poultry veterinarians on the use of antimicrobials and antimicrobial resistance in egg production

This study aimed to describe the perception of veterinarians who work with commercial laying hens in the state of Rio Grande do Sul, Brazil, regarding the use of antibiotics and their possible impacts on animal, human, and environmental health. A descriptive epidemiological study was carried out through face-to-face or web conferencing interviews with the veterinarians that provide technical assistance at commercial laying hen operations. A standardized and structured questionnaire was developed based on the literature and expert opinion, which contained 1 opened and 40 closed questions. Conventional non-probabilistic sampling was used, based on an initial list of 15 veterinarians registered in the Poultry Production Association of Rio Grande do Sul, followed by the snowball technique. The acquisition of 26 contacts of veterinarians was accomplished, and 16 were interviewed. Through the answers obtained it was possible to verify that the interviewees' understanding regarding both the antibiotic resistance impact and the decision-making about the use of antibiotics seem to be linked to their practical experiences. Besides that, according to the veterinarians, farmers can acquire and administer the antimicrobials on their farms. Moreover, both farm storage and administration of lower doses of antibiotics than the recommended one could be contributing factors to resistant bacteria selection. Furthermore, controversially, the professionals believed that resistant bacteria can be transmitted to humans from eggs, but they said that there are no bacteria in eggs. Therefore, the veterinarians practices can be improved considering national and international guidelines on antimicrobial resistance to minimize the development of resistance. Finally, it is expected that the present results will contribute to a more complex discussion about antimicrobial resistance, helping to formulate public policies in the egg production industry

Aborto bovino associado à infecção por Staphylococcus aureus : caracterização da cepa de S. aureus isolada a partir de tecidos fetais

Staphylococcus aureus is a gram-positive bacterium, commonly found colonizing the skin and mucous membranes of humans and animals. This report describes a case of fetal loss associated with S. aureus infection in a cow. A six-month old, crossbred male bovine fetus from a beef farm was submitted for necropsy. At gross examination fibrinous pleuropneumonia was observed. Histologically, lesions were restricted to the lungs and consisted of marked multifocal to coalescing areas of inflammatory infiltrate of neutrophils, abundant fibrin exudation, necrosis of bronchiolar epithelium and numerous aggregates of coccoid bacteria. Lung and abomasal fluid bacterial culture yielded pure culture of S. aureus, which was characterized as a multidrug resistant strain. Molecular analysis indicated that the studied strain presented several genes of virulence factors including toxic shock syndrome toxin-1 (tst), staphylococcal enterotoxin type A (sea), Panton–Valentine leukocidin (pvl), alpha-hemolysin (hla) and delta-hemolysin (hld). This report documents an infrequent case of fetal loss in cattle due to infection with a highly virulent S. aureus strain.Staphylococcus aureus é uma bactéria gram-positiva, comumente encontrada colonizando a pele e as membranas mucosas de humanos e animais. O presente relato descreve um caso de aborto bovino associado à infecção por S. aureus. Um feto bovino, macho, cruzado, com seis meses de idade gestacional proveniente de uma fazenda de gado de corte foi submetido para a necropsia. Pleuropneumonia supurativa foi observada na avaliação macroscópica. Histologicamente as lesões encontravam-se restritas aos pulmões e eram representadas por infiltrado inflamatório acentuado, multifocal a coalescente de neutrófilos, acentuada exsudação de fibrina, necrose do epitélio bronquiolar e numerosos agregados bacterianos cocoides. A cultura bacteriana de fragmento de pulmão e líquido do abomaso revelou o crescimento puro de S. aureus, que foi caracterizado como uma cepa multirresistente a drogas. Análises moleculares indicaram que a cepa estudada apresentava vários fatores de virulência, incluindo toxina 1 da síndrome do choque tóxico (TSST-1), enterotoxina estafilocócica tipo A (sea), leucocidina Panton-Valentine (pvl), hemolisina alfa (hla) e hemolisina delta (hld). O presente relato documenta um caso infrequente de aborto bovino devido à infecção por uma cepa altamente virulenta de S. aureus

Pet pyometra : correlating bacteria pathogenicity to endometrial histological changes

Pyometra is a life-threatening infectious disease that frequently affects bitches and queens. Although histopathological patterns of pyometra have been extensively explored, the microbiological aspects, such as bacteria pathogenicity, have not been correlated to microscopy endometrial lesions so far. In this study, these two pathological aspects of pyometra were analysed and correlated. Uterus fragments and intrauterine content samples were collected from pets diagnosed with pyometra (30) and submitted to histopathology analysis and bacterial culture, respectively. The degree of endometrial histopathological lesions in pyometra cases were classified as mild, moderate and severe. Thirty different bacteria isolates were identified from intrauterine content culture. Escherichia coli(E. coli) was pure isolated in 57.7% and highly related to severe endometrial lesions. Immunohistochemistry assay revealed the adhesion and invasion of this bacteria agent to the injured endometrium. Virulence aspects of these E. coli strains were explored, demonstrating biofilm formation ability and a set of virulence genes in most isolates. These results support the adaptive genetic and phenotypic advantages of E. coli for uterus infection, and justify the high frequency of this agent involved in pyometra cases

Genes encoding Shiga toxin and the intimin receptor detected in faecal samples collected from wild canids

Background: Enteropathogenic Escherichia coli (EPEC) and Shiga toxin-producing E. coli (STEC) are diarrheagenic E. coli that can cause disease in humans. The pathotype EPEC leads to the attaching and effacing lesion, causing damage to the microvilli following to diarrhea. STEC pathotypes produces cytotoxins, which in humans are responsible for hemorrhagic colitis or hemolytic uremic syndrome. Animals are the reservoirs of these pathotypes, especially ruminants. However, other animal’s species can be associated as carriers of EPEC and STEC strains. The aim of this study was to analyze wild canid crab-eating fox (Cerdocyon thous) as potential natural carriers of STEC and EPEC E. coli. Materials, Methods & Results: Seven fecal samples were analyzed from the crab-eating fox of free-living, captured in a peri-urban area. Samples were collected from the rectal ampulla, and the animals were clinic evaluated, being considered healthy at the captured moment. The feces were inoculated on medium MacConkey agar, and then the plates were incubated at 37°C for 24 h. All colony forming units (CFU) were collected by plate washing with ultrapure water (2 mL) and posterior freezing at -20°C. The total bacterial DNA from the CFU collected was extracted, followed by PCR assay to search for three genes: stx1, stx2 (responsible for the synthesis of the Shiga toxin) and tir, which encodes the translocated intimin receptor, related to the A/E lesion formation. Three samples were detected as positive, being one animal detected as carrier of the stx2 gene (STEC strain), while two animals were identified as carrier of the tir gene (EPEC strains).The stx1 gene was not identified on the samples. Also, in the samples, only the presence of one gene studied at a time was observed. Therefore, we have found out that the crab-eating fox can act as reservoirs of STEC and EPEC strains. Discussion: The carrier’s animals of STEC and EPEC strains do not have receptors for the Shiga toxin, serving as asymptomatic vehicle. The wild canids collected and positives to STEC and EPEC E. coli strains analyzed here, did not shown any clinical signs, wherefore they have the potential of being a source of pathogens to other animals and even humans. The observation of this additional wild species, the crab-eating fox, as carriers of STEC and EPEC reinforces the importance of monitoring wild species and the need for caution because of the potential zoonosis. Nevertheless, there are other species of wild animals that were described as rarely-carriers of these pathogens, like deer, wild ruminants and birds. These E. coli pathotypes, potentially foodborne zoonotic pathogens, are acquired by humans from food and water contaminated, by oral-fecal route, or by direct contact with carries animals and their feces. The human contact with wild animals is getting ordinary; because of that, wild animals, like wild canids, may constitute a considerable risk to animal and humans health, mostly by the transmission of these bacteria strains to the environment. In conclusion, the identification of genes encoding Shiga toxin and the intimin receptor in wild canids feces highlight that STEC and EPEC pathogens could be spread by these wild animals. We reinforce that STEC and EPEC pathogens could be naturally carried by wild animals, such as the crab-eating fox, and therefore, is needed further research for better understanding their potential effect, and also the interaction between the pathogen and the host