ANTIMICROBIAL SUSCEPTIBILITY OF STAPHYLOCOCCUS SPP. ISOLATED FROM FELIDS AND CANIDS IN BELO HORIZONTE ZOO, BRAZIL.

The epidemiology of Staphylococcus spp. has become a major concern among humans and animals due to increasing antimicrobial resistance and frequent reports of infection. Despite the importance of animals as reservoirs for staphylococci, little is known about the epidemiology of Staphylococcus spp. in most nondomestic species, including canids and felids. This study evaluated the frequency, distribution, and patterns of antimicrobial resistance of staphylococcal species isolated from captive felids and canids from Belo Horizonte Zoo, Brazil. Rectal, oral, and nasal swabs from apparently healthy maned wolves (Chrysocyon brachyurus, n= 7), a lion (Panthera leo, n = 1), jaguars (Panthera onca, n = 3), and one swab of a cougar (Puma concolor) with an ear infection were streaked onto mannitol salt agar. Colonies identified by matrix-assisted laser desorption/ionization-time of flight mass spectrometry, polymerase chain reaction for the Staphylococcus intermedius group (SIG), and 16S rRNA gene sequencing. Isolates were subjected to antimicrobial susceptibility tests and Staphylococcus pseudintermedius strains were subjected to multilocus sequence typing. Staphylococcus species were isolated from 24 of the 34 samples (70.6%). Among the isolated strains, S. pseudintermedius and Staphylococcus felis were the most frequent species (41.7 and 25%, respectively). Five novel sequence types were identified among the S. pseudintermedius isolates. Resistance to tetracycline (7/24, 29.2%) or penicillin (6/23, 26.1%) was significantly higher than the other antimicrobial agents tested (P < 0.05). One isolate, Staphylococcus nepalensis, was positive for mecA and resistant to five antimicrobials, and was thus classified as multidrug-resistant. The present work suggests that maned wolves are natural hosts of SIG and also reports the isolation of S. felis in sick and healthy, captive, nondomestic carnivores. The isolated staphylococci were susceptible to most classes of antimicrobials tested. However, the multidrug-resistance capability of an S. nepalensis strain reinforces the hypothesis that felids and canids act as reservoirs of pathogens with antimicrobial resistance.

DISTRIBUTION AND ANTIMICROBIAL RESISTANCE OF STAPHYLOCOCCI ISOLATED FROM FREE-LIVING SOUTH AMERICAN COATI (NASUA NASUA) IN AN URBAN PARK IN MINAS GERAIS, BRAZIL.

The scientific information regarding staphylococci in procyonids is scarce. Hence, the aim of this study was to evaluate the frequency, distribution, and pattern of antimicrobial resistance of staphylococcal species isolated from free-roaming coatis (Nasua nasua) in an urban park in Belo Horizonte, Minas Gerais, Brazil. Rectal swabs from 55 free-living coatis were plated onto mannitol salt agar for isolating staphylococci, and species were identified using matrix-assisted laser desorption/ionization-time of flight mass spectrometry, polymerase chain reaction (PCR) of nuc, and sequencing of 16S rRNA and rpoB when needed. Antimicrobial susceptibility was investigated using the disk diffusion method, and the presence of the mecA gene was investigated by PCR. A total of 72.7% of the animals tested positive for staphylococci. Nine different species were identified, and Staphylococcus intermedius (60.4%) and S. delphini (20.9%) were the most frequently isolated species. Most of the isolates were susceptible to most of the antimicrobials evaluated, with a resistance pattern seen for penicillin (13.9%). One isolate was multidrug-resistant (MDR). The present study suggests that coatis are natural hosts of S. intermedius and S. delphini and, despite living in a heavily anthropized environment, the Staphylococcus spp. isolates showed a low incidence of drug resistance.

Antimicrobial susceptibility and phylotyping profile of pathogenic Escherichia coli and Salmonella enterica isolates from calves and pigs in Minas Gerais, Brazil.

The aims of the present study were to determine (i) the profiles of phylogroup and (ii) the antimicrobial susceptibility of pathogenic Escherichia coli strains isolated from calves, and of Salmonella spp. strains isolated from calves and pigs in Minas Gerais State, Brazil. Sixty-one pathogenic E. coli strains and Salmonella spp. (n = 24) strains isolated from fecal samples of calves and Salmonella spp. (n = 39) strains previously isolated from fecal samples of growing/finishing pigs were tested. The minimum inhibitory concentration (MIC) using the agar dilution method was determined for nalidixic acid, amikacin, amoxicillin, ampicillin, cefoxitin, norfloxacin, gentamicin, tetracycline, and trimethoprim-sulfamethoxazole. All E. coli isolates were susceptible to amikacin. Tetracycline was the antimicrobial that presented the higher frequency of resistance among E. coli strains, followed by ampicillin, trimethoprim-sulfamethoxazole, amoxicillin, nalidixic acid, norfloxacin, gentamicin, and cefoxitin. E. coli (n = 61) strains isolated from calves belonged to different phylogroup namely, phylogroup A (n = 26), phylogroup B1 (n = 31), phylogroup E (n = 3), and phylogroup F (n = 1). Phylogroups B2, C, and D were not identified among the E. coli in the present study. All Salmonella spp. (n = 24) strains isolated from fecal samples of calves were susceptible to amikacin, amoxicillin, ampicillin, norfloxacin, gentamicin, tetracycline, and trimethoprim-sulfamethoxazole. Resistance to nalidixic acid and cefoxitin was detected in 16.66 and 8.33 % of the Salmonella spp. strains, respectively. Among the Salmonella spp. (n = 39) strains isolated from fecal samples of pigs, the higher frequency of resistance was observed to tetracycline, followed by amoxicillin, gentamicin, ampicillin, trimethoprim-sulfamethoxazole, nalidixic acid, cefoxitin, and norfloxacin. All strains were susceptible to amikacin. Forty-eight (78.68 %) of the E. coli strains were classified as multidrug-resistant, whereas among Salmonella spp. strains, the percentage of multidrug resistance was 57.14 %, being all multidrug-resistant strains isolated from pigs (92.30 %). The results from the present study indicate a high frequency of antimicrobial resistance among pathogenic E. coli strains isolated from calves and Salmonella spp. strains isolated from pigs and a high rate of susceptibility to most antimicrobials tested among Salmonella spp. strains isolated from calves. Our study highlights the presence of multidrug-resistant strains of E. coli and Salmonella spp. isolated from food-producing animals in Minas Gerais, Brazil.

Genetic stability of Brucella abortus isolates from an outbreak by multiple-locus variable-number tandem repeat analysis (MLVA16).

BACKGROUND: Brucellosis caused by Brucella abortus is one of the most important zoonoses in the world. Multiple-locus variable-number tandem repeat analysis (MLVA16) has been shown be a useful tool to epidemiological traceback studies in B. abortus infection. Thus, the present study aimed (i) to evaluate the genetic diversity of B. abortus isolates from a brucellosis outbreak, and (ii) to investigate the in vivo stability of the MLVA16 markers. RESULTS: Three-hundred and seventy-five clinical samples, including 275 vaginal swabs and 100 milk samples, were cultured from a brucellosis outbreak in a cattle herd, which adopted RB51 vaccination and test-and-slaughter policies. Thirty-seven B. abortus isolates were obtained, eight from milk and twenty-nine from post-partum/abortion vaginal swabs, which were submitted to biotyping and genotyping by MLVA16. Twelve B. abortus isolates obtained from vaginal swabs were identified as RB51. Twenty four isolates, seven obtained from milk samples and seventeen from vaginal swabs, were identified as B. abortus biovar 3, while one isolate from vaginal swabs was identified as B. abortus biovar 1. Three distinct genotypes were observed during the brucellosis outbreak: RB observed in all isolates identified as RB51; W observed in all B. abortus biovar 3 isolates; and Z observed in the single B. abortus biovar 1 isolate. Epidemiological and molecular data show that the B. abortus biovar 1 genotype Z strain is not related to the B. abortus biovar 3 genotype W isolates, and represents a new introduction B. abortus during the outbreak. CONCLUSIONS: The results of the present study on typing of multiple clinical B. abortus isolates from the same outbreak over a sixteen month period indicate the in vivo stability of MLVA16 markers, a low genetic diversity among B. abortus isolates and the usefulness of MLVA16 for epidemiological studies of bovine brucellosis.

Equine Brucellosis: Current Understanding and Challenges.

Brucellosis in equines, including horses, donkeys, and mules, is characterized by abscesses in tendons, bursae, and joints. Reproductive disorders, which are common in other animals, are rare in both males and females. Joint breeding of horses, cattle, and pigs was found as the main risk factor for equine brucellosis, with the transmission from equines to cattle or among equines possible, although unlikely. Hence, evaluation of the disease in equines can be considered an indirect indicator of the effectiveness of brucellosis control measures employed for other domestic species. Generally, the disease in equines reflects disease status in the sympatric domestic species, mainly cattle. It is important to note that in equines, the disease has no validated diagnostic test, which limits the interpretation of available data. Finally, it is important to mention that equines also represent significant Brucella spp. infection sources for humans. Considering the zoonotic aspect of brucellosis, the significant losses due to infection, and the representativeness of horses, mules, and donkeys in the society, as well as the continuous efforts to control and eradicate the disease in livestock, in this review, we covered the various aspects of brucellosis in equines and compile the sparse and diffuse information on the subject.

Risk Factors, Genetic Diversity, and Antimicrobial Resistance of Staphylococcus spp. Isolates in Dogs Admitted to an Intensive Care Unit of a Veterinary Hospital.

Intensive Care Units (ICU) usually provide an excellent environment for the selection of pathogens associated with hospital-acquired infections (HAI), leading to increased mortality and hospitalization costs. Methicillin-resistant Staphylococcus pseudintermedius (MRSP) is a major cause of HAI in dogs worldwide, but the risk factors and dynamics of colonization by MRSP are largely unknown. This study aimed to evaluate the risk factors associated with the acquisition of MRSP in dogs admitted to an ICU, and to report the antimicrobial resistance profiles and genetic relatedness of MRSP isolates. Sterile swabs from the nostril, axilla, and rectum were collected daily during the hospitalization of 54 dogs. Samples were subjected to Mannitol Salt Agar, and colonies were identified by MALDI-ToF, polymerase chain reaction (PCR), and sequencing of the rpoB gene. Antimicrobial susceptibility testing and PCR detection of mecA were performed. Staphylococcus spp. was isolated from 94% of the dogs, and the most frequently isolated species was S. pseudintermedius (88.2%). Carriage of multidrug resistant (MDR) staphylococci was observed in 64.4% of the dogs, and approximately 39% had methicillin-resistant Staphylococcus sp. (MRS), of which 21.6% had MRSP and 1.9% had methicillin-resistant S. aureus (MRSA). The acquisition of MRSP during ICU hospitalization was associated with sex (female), age (>7 years), and dogs that had previously been treated with antimicrobials. Animals colonized by MRSP resistant to ≥9 antimicrobial classes had longer hospital stays than those colonized by other MRS strains. Among the 13 MRSP isolates that were subjected to whole-genome sequencing, ten were classified as ST71. A single nucleotide polymorphism (SNP) analysis revealed three clones, including one that was detected in infected dogs outside the ICU. This study indicates novel risk factors associated with colonization by MRSP. The detection of the same MRSP clone causing HAI outside the ICU reinforces the need for improved infection prevention and control practices at veterinary hospitals in general and at the ICU in particular.

Clostridioides difficile and multi-drug-resistant staphylococci in free-living rodents and marsupials in parks of Belo Horizonte, Brazil.

The global emergence of antimicrobial resistance (AMR) has become a serious threat to human and animal health. Recent studies have shown that synanthropic animals can act as reservoirs and disseminators of pathogens and resistant bacteria. The aim of this study was to evaluate the frequency, distribution, and antimicrobial susceptibility of staphylococcal species and Clostridioides difficile isolated from the feces of free-living rodents and marsupials from two urban parks in Belo Horizonte, Brazil. During a 12-month period, fecal samples from 159 free-living animals, including 136 rodents and 23 marsupials, were collected from two urban parks in Belo Horizonte, Minas Gerais, Brazil. Staphylococcus spp. were more likely to be isolated from rodents than marsupials (p = 0.0164). Eight different staphylococcal species were isolated from 36 (26.5%) rodents and one marsupial (4.3%). S. saprophyticus (48.6%) was the most frequently isolated species, and almost a quarter of the isolates (24.3%) were resistant to at least one antimicrobial agent, four (10.8%) of which were multi-drug resistant (MDR). Two (5.4%) strains were resistant to cefoxitin and were then classified as methicillin-resistant staphylococci, and one also tested positive for the mecA gene. C. difficile was isolated from two rodents (1.5%), and one strain was toxigenic and classified as ribotype 064. One isolate was resistant to rifampicin, but both strains were susceptible to all other antimicrobials tested, including metronidazole and vancomycin. All C. difficile isolates and all staphylococcal strains resistant to antimicrobials were recovered from the same park. The present study suggests that free-living rodents in Belo Horizonte (Brazil) are mainly colonized by S. saprophyticus and may act as reservoirs of antimicrobial-resistant Staphylococcus spp. and C. difficile strains. This is the first study to evaluate the presence of staphylococci and C. difficile from free-living opossums and suggest a low fecal shedding of these organisms by these mammals.

Occurrence and characterization of methicillin-resistant Staphylococcus spp. in diseased dogs in Brazil.

Staphylococcus pseudintermedius is a major commensal bacterium of the skin and mucosae of dogs and an opportunistic agent responsible for several clinical infections, such as pyoderma, otitis, and surgical wound infections. The emergence of methicillin-resistant S. pseudintermedius (MRSP) has become a problem of great concern in veterinary and human medicine because it is multidrug resistant (MDR) and can also infect humans. This study aimed to identify the occurrence of Staphylococcus spp. in infected patients and investigate the antimicrobial resistance profiles and molecular structure of MRSP isolates. Samples were obtained from two different veterinary clinics; suggestive colonies were submitted to matrix-assisted laser desorption ionization-time of flight (MALDI-ToF) mass spectrometry and confirmed at the species level by polymerase chain reaction (PCR). Sequencing of the 16S rRNA and rpoB genes were used in selected samples that were not identified by MALDI-ToF and by the species-specific PCR. Antimicrobial susceptibility and PCR detection of mecA were performed. MRSP isolates were subjected to multilocus sequence typing. Of all the clinical staphylococci (n = 131), 98 (74.8%) were identified as S. pseudintermedius. Multidrug resistance (resistance to ≥3 classes of antimicrobials) was observed in 63.2% of S. pseudintermedius isolates, and 24.5% of S. pseudintermedius isolates were methicillin-resistant. Half of the MRSP isolates were isolated from surgical site infections. Among the ten sequence types (ST) identified, nine were novel. ST71 was the most prevalent and associated with resistance to fluoroquinolones. Prior antimicrobial therapy, hospitalization, and surgical site infections seemed to be risk factors for MRSP acquisition. The present study showed a high rate of MDR staphylococci in infected dogs. MRSP was isolated from different clinical conditions, mainly surgical site infections. Additionally, this is the first study to extensively investigate the population structure of MRSP in Brazil, which revealed the dispersion of CC71 and nine novel ST. These findings raise concerns for both animal and human health due to the zoonotic potential of this species and limited therapeutic options available for MRSP infections.

Fecal Shedding of Multidrug Resistant Escherichia coli Isolates in Dogs Fed with Raw Meat-Based Diets in Brazil.

The practice of feeding dogs raw meat-based diets (RMBDs) is growing in several countries, and the risks associated with the ingestion of pathogenic and antimicrobial-resistant Escherichia coli in dogs fed these diets are largely unknown. We characterized E. coli strains isolated from dogs fed either an RMBD or a conventional dry feed, according to the phylogroup, virulence genes, and antimicrobial susceptibility profiles of the bacteria. Two hundred and sixteen E. coli strains were isolated. Dogs fed RMBDs shed E. coli strains from the phylogroup E more frequently and were positive for the E. coli heat-stable enterotoxin 1-encoding gene. Isolates from RMBD-fed dogs were also frequently positive for multidrug-resistant E. coli isolates including extended-spectrum beta-lactamase (ESBL) producers. Whole-genome sequencing of seven ESBL-producing E. coli strains revealed that they predominantly harbored blaCTX-M-55, and two strains were also positive for the colistin-resistant gene mcr-1. These results suggest that feeding an RMBD can affect the dog's microbiota, change the frequency of certain phylogroups, and increase the shedding of diarrheagenic E. coli. Also, feeding an RMBD seemed to be linked with the fecal shedding of multidrug-resistant E. coli, including the spread of strains harboring mobilizable colistin resistance and ESBL genes. This finding is of concern for both animal and human health.

Identification and Characterization of Escherichia coli, Salmonella Spp., Clostridium perfringens, and C. difficile Isolates from Reptiles in Brazil.

Considering the increasing popularity of reptiles as pets and their possible role as reservoirs of pathogenic microorganisms, the aim of this study was to isolate Escherichia coli, Salmonella spp., Clostridium perfringens, and C. difficile strains from reptiles in Brazil and to characterize the isolated strains. The characterization was based on phylogenetic typing of E. coli, identification of virulence genes of E. coli, C. perfringens, and C. difficile, serotyping of Salmonella spp., ribotyping and MLST of C. difficile and antimicrobial susceptibility test of pathogenic strains. Cloacal swabs were collected from 76 reptiles, of which 15 were lizards, 16 chelonians, and 45 snakes, either living in captivity, in the wild, or as companion animals. E. coli was isolated from 52 (68.4%) reptiles, of which 46 (88.4%) were characterized as phylogroup B1. The virulence factor CNF1 of E. coli was found in seven (9.2%) sampled animals, whereas the gene of EAST1 was found in isolates from two (2.6%) reptiles. Three isolates positive for CNF1 were resistant to cephalothin, one of which was also resistant to ciprofloxacin, trimethoprim/sulfamethoxazole, and chloramphenicol, being then classified as multidrug resistant strain (MDR). Salmonella enterica was identified in 26 (34.2%) reptiles, of which 13 belonged to the subspecies enterica. Serotypes such as S. Mbandaka, S. Panama, S. Infantis, S. Heidelberg, and S. Anatum were identified. One isolate of S. enterica subsp. houtenae was resistant to cephalothin and ciprofloxacin. C. perfringens type A was isolated from six (7.8%) animals. C. difficile was isolated from three (3.9%) reptiles. Two of these isolates were toxigenic and classified into ribotypes/MLST 081/ST9 and 106/ST42, which have been previously reported to infect humans. In conclusion, reptiles in Brazil can harbor toxigenic C. difficile and potentially pathogenic E. coli and Salmonella enterica subsp. enterica, thus representing a risk to human and animal health.

Brucellosis in working equines of cattle farms from Minas Gerais State, Brazil.

The present survey aimed at estimating the seroprevalence of brucellosis in working equines of cattle farms from Minas Gerais State, Brazil, and investigating risk factors associated with the infection. Serum samples from 6439 animals, including 5292 horses, 1037 mules and 110 donkeys, were collected from 1936 herds, between September 2003 and March 2004, in 848 municipalities from the state of Minas Gerais, Brazil. The prevalence of antibodies against smooth Brucella spp. found in equines from Minas Gerais State was 1.37% (95% CI: 0.97-1.78), resulting in a prevalence of herds with infected animals of 4.28% (95% CI: 4.21-4.36). There were differences between regions but these were not of major epidemiological relevance nor were most of them statistically significant, given the considerable overlap of confidence intervals. Nevertheless, the point estimates suggest that the three northeastern regions have slightly higher prevalence than the rest of the state, both at the herd and animal levels. No association of Brucella spp. seropositivity with sex, age or host was observed. In conclusion, the present study showed a low but widespread prevalence of antibodies against smooth Brucella in equines kept in cattle farms in Minas Gerais, a state where bovine brucellosis is also widespread albeit with low prevalence.

Genetic stability of Brucella abortus S19 and RB51 vaccine strains by multiple locus variable number tandem repeat analysis (MLVA16).

The aims of the present study were (i) to assess the in vitro genetic stability of S19 and RB51 Brucella abortus vaccines strains and (ii) to evaluate the ability of multiple-locus variable-number tandem repeat (VNTR) analysis (MLVA) as a tool to be used in the quality control of live vaccines against brucellosis. Sixty-three batches of commercial S19 (n=53) and RB51 (n=10) vaccines, produced between 2006 and 2009, were used in this study. S19 and RB51 vaccines were obtained from, respectively, seven and two different manufacturers. Ten in vitro serial passages were performed on reference strains and on selected batches of commercial vaccines. All batches, reference strains and strains of serial passages were typed by the MLVA16. The results demonstrated that B. abortus S19 and RB51 vaccine strains are genetically stable and very homogeneous in their respective groups. Anyway, batches of S19 from one manufacturer and batches of RB51 from another presented genotypes distincts from the reference vaccine strains. In both cases, differences were found on locus Bruce07, which had addition of one repeat unit in the case of S19 batches and the deletion of one repeat unit in the case of RB51 batches. In summary, MLVA16 proved to be a molecular tool capable of discriminating small genomic variations and should be included in in vitro official tests.

Isolation and genotyping of Clostridium perfringens from goats in Minas Gerais, Brazil / Isolamento e genotipagem de Clostridium perfringens isolados de cabras em Minas Gerais, Brasil

ABSTRACT: Despite the known importance of Clostridium perfringens as an enteropathogen in small ruminants, little is known about the role of its additional virulence factors or the frequency of the various C. perfringens genotypes in healthy goats; this complicates the laboratory diagnosis of the infections caused by this microorganism. In light of this, the aim of the present study was to isolate and genotype C. perfringens from stool samples from healthy goats in Brazil. Stool samples from 250 apparently healthy adult goats from 17 different herds in Minas Gerais, Brazil were collected, and isolation and genotyping of C. perfringens was performed. C. perfringens type A was isolated from 189 (75.6%) goats, whereas C. perfringens types C and D were each detected in one goat (0.4%). All isolates were negative for enterotoxin-, NetB-, NetE-, and NetF-encoding genes. These results confirmed C. perfringens type A as part of the microbiota in these animals, and they suggested that C. perfringens type C and D are rarely isolated from healthy goats.

RESUMO: Apesar da reconhecida importância de Clostridium perfringens como enteropatógeno de pequenos ruminantes, pouco se sabe sobre a frequência dos genótipos ou do papel de fatores de virulência adicionais de C. perfringens em cabras saudáveis, dificultando o diagnóstico laboratorial da infecção causada por esse micro-organismo. Dessa forma, o presente estudo teve como objetivo caracterizar C. perfringens de amostras de fezes de cabras adultas saudáveis. Amostras de fezes de 250 cabras saudáveis de 17 rebanhos diferentes em Minas Gerais, Brasil, foram submetidas ao isolamento e genotipagem de C. perfringens. C. perfringens tipo A foi isolado de 189 (75,6%) cabras, enquanto C. perfringens tipos C e D foram detectados em um animal (0.4%) cada. Todos os isolados foram negativos para os genes codificadores das toxinas NetB, NetE, NetF e enterotoxina. Os resultados apresentados confirmam C. perfringens tipo A como parte da microbiota de cabras saudáveis e sugere que C. perfringens tipos C e D são raramente encontrados em caprinos saudáveis.