Whole genome sequence datasets of Salmonella enterica serovar Saintpaul ST50 and serovar Worthington ST592 strains isolated from raw milk in Brazil.

Herein we report the draft genome sequences of Salmonella enterica subsp. enterica serovars Saintpaul ST50 and Worthington ST592 isolated from raw milk samples in Northeastern Brazil. The 4,696,281 bp S. Saintpaul ST50 genome contained 4,628 genes in 33 contigs, while S. Worthington ST592 genome was 4,890,415 bp in length, comprising 4,951 genes in 46 contigs. S. Worthington ST592 carried a conserved Col(pHAD28) plasmid which contains the antimicrobial resistance determinants tet(C), acc(6')-Iaa, and a nonsynonymous point mutation in ParC (p.T57S). The data could support further evolutionary and epidemiologic studies involving Salmonella organisms.

Genomic Analysis of a Community-Acquired Methicillin-Resistant Staphylococcus aureus Sequence Type 1 Associated with Caprine Mastitis.

This study aimed to investigate the genomic and epidemiological features of a methicillin-resistant Staphylococcus aureus sequence type 1 (MRSA ST1) strain associated with caprine subclinical mastitis. An S. aureus strain was isolated from goat's milk with subclinical mastitis in Paraiba, Northeastern Brazil, by means of aseptic procedures and tested for antimicrobial susceptibility using the disk-diffusion method. Whole genome sequencing was performed using the Illumina MiSeq platform. After genome assembly and annotation, in silico analyses, including multilocus sequence typing (MLST), antimicrobial resistance and stress-response genes, virulence factors, and plasmids detection were performed. A comparative SNP-based phylogenetic analysis was performed using publicly available MRSA genomes. The strain showed phenotypic resistance to cefoxitin, penicillin, and tetracycline and was identified as sequence type 1 (ST1) and spa type 128 (t128). It harbored the SCCmec type IVa (2B), as well as the lukF-PV and lukS-PV genes. The strain was phylogenetically related to six community-acquired MRSA isolates (CA-MRSA) strains associated with human clinical disease in North America, Europe, and Australia. This is the first report of a CA-MRSA strain associated with milk in the Americas. The structural and epidemiologic features reported in the MRSA ST1 carrying a mecA-SCCmec type IVa suggest highly complex mechanisms of horizontal gene transfer in MRSA. The SNP-based phylogenetic analysis suggests a zooanthroponotic transmission, i.e., a strain of human origin.

Antimicrobial susceptibility profiles of Staphylococcus spp. contaminating raw goat milk.

BACKGROUND AND AIM: Antimicrobial resistance poses a major threat to global public health. Foodstuff of animal origin can serve as potential vehicles for the dissemination of antimicrobial-resistant bacteria and resistance genes to consumers. In view of the lack of knowledge about antimicrobial resistance in bacteria associated with goat milk, the aim of this study was to report species-level identification and antimicrobial susceptibility profiles of a large collection of Staphylococcus spp. isolates recovered from raw goat milk in Brazil. MATERIALS AND METHODS: A total of 434 Staphylococcus spp. isolates originated from 510 goat milk samples in Northeast Brazil were investigated. The isolates were obtained by conventional microbiological methods. Species identification and antimicrobial susceptibility testing were performed by means of a semi-automated system using a panel for biochemical tests and broth microdilution method for 19 antimicrobial drugs. RESULTS: Although Staphylococcus aureus (22.6%) accounted for the majority of the isolates, a total of 13 different non-aureus staphylococci spp. were identified. High resistance rates against erythromycin (40.8%), and the beta-lactams ampicillin (45.9%) and penicillin (42.9%) were observed among S. aureus isolates. The most significant findings were related to the resistance against quinupristin-dalfopristin, a drug of last resort used in human medicine to treat infections caused by vancomycin-resistant S. aureus and enterococci. CONCLUSION: The high diversity of Staphylococcus spp. showing phenotypic resistance against different antimicrobial drugs encourages further investigations on the real impact of these bacteria as reservoirs of antimicrobial resistance genes to consumers. Furthermore, the potential impact of technological processes, such as pasteurization, fermentation, and maturation, on the maintenance and dissemination of antimicrobial resistance among the microbial populations in milk and dairy products must also be investigated.

Pathogen-specific changes in composition and quality traits of milk from goats affected by subclinical intramammary infections.

We investigated the effects of pathogens associated with subclinical intramammary infections on yield, composition and quality indicators of goat milk. By means of a longitudinal study, individual half udder milk samples (n = 132) were collected at different lactation periods and assessed for milk yield and physicochemical composition, somatic cell count (SCC), total bacteria count (TBC) and microbiological culture. Staphylococci species accounted for the great majority of the isolates (96.1%). Intramammary infections significantly reduced fat and total solids in goat milk and increased both SCC and TBC. However, these indicators were significantly higher in udder halves affected by S. aureus compared with other staphylococci species.

Chicken embryo development: metabolic and morphological basis for in ovo feeding technology.

Broiler embryonic development depends on the nutrients that are available in the egg, which includes mostly water, lipids, and proteins. Carbohydrates represent less than 1%, and free glucose only 0.3%, of the total nutrients. Considering that energy requirements increase during incubation and metabolism is shifted toward the use of glycogen stores and gluconeogenesis from amino acids, extensive muscle protein degradation in the end of incubation can compromise chick development in the initial days after hatch. Significant prehatch changes occur in embryonic metabolism to parallel the rapid embryonic development. Oral consumption of the amniotic fluid begins around 17 d of incubation and promotes rapid development of the intestinal mucosa, which is characterized by morphological changes and increased expression and activity of enzymes and transporters. Furthermore, ingested substrates are stored as nutritional reserves to be used during hatching and in the first week after hatch. At hatch, this limited-nutrient store is directed to the functional development of the gastrointestinal tract to enable assimilation of exogenous nutrients. In ovo feeding is an alternative to deliver essential nutrients to chick embryos at this critical and challenging phase. The improved nutritional status and physiological changes triggered by in ovo feeding can resonate throughout the entire rearing period with significant health and economic gains. The present review addresses the main changes in metabolism and intestinal development throughout incubation, and also addresses scientific advances, limitations and future perspectives associated with the use of in ovo feeding that has been regarded as an important technology by the poultry industry.

The posthatch prophylactic use of ceftiofur affects the cecal microbiota similar to the dietary sanguinarine supplementation in broilers.

The prophylactic administration of ceftiofur to newly hatched chicks is a common practice in some hatcheries worldwide to mitigate early gastrointestinal infections caused by Enterobacteriaceae. In spite of the crucial role of the gut microbiome for the broiler's health, there is still limited information on how the microbial composition is affected by such procedure. We investigated the effects of posthatch prophylactic application of ceftiofur on the cecal microbiota of 14-day-old broilers fed regular or sanguinarine-supplemented diets. DNA samples were extracted from cecal contents, amplified for the V3-V4 regions of the microbial 16S rRNA gene, and sequenced in a high-throughput sequencing platform (Illumina MiSeq). After downstream bioinformatics and statistical analyses, our results demonstrated that both ceftiofur and sanguinarine treatments similarly increased the proportions of the phylum Bacteroidetes and the genera Bacteroides and Megamonas, whereas reduced the relative abundances of Firmicutes and Lachnospiraceae in the ceca of the birds. Such changes are probably associated with increased carbohydrate fermentation processes favoring the production of short-chain fatty acids. This was also corroborated by the functional prediction findings, which suggest an increase in some metabolic pathways associated with digestibility in broilers receiving ceftiofur. Considering that antimicrobial stewardship in animal production systems is strongly needed to mitigate the threat of antimicrobial resistance, our findings show that supplementation with a phytogenic feed additive can lead to a similar microbial composition in the ceca of commercial broiler chickens, suggesting that the use of alternative products could lead to functional modifications without increasing pressure for antimicrobial resistance.

First reported genome of an mcr-9-mediated colistin-resistant Salmonella Typhimurium isolate from Brazilian livestock.

OBJECTIVES: To investigate the genetic context of colistin resistance in anmcr-9-harbouring Salmonella Typhimurium ST19 strain from swine in Brazil. METHODS: Minimum inhibitory concentrations (MIC) to colistin were determined by broth microdilution. Whole-genome sequencing was performed on an Illumina MiSeq system, followed by de novo genome assembly using SPAdes 1.13.1. The draft genome sequence was annotated in Prokka using KBase online server. Downstream analyses for resistome and plasmid detection were performed using online tools available at the Center for Genomic Epidemiology. The strain was typed in silico using MLST 2.0. Phylogenetic analysis involving 24 other genomes ofSalmonella Typhimurium ST19 and mcr-9-harbouring Salmonella Typhimurium isolated from humans, livestock and foodstuff in different regions was also performed. RESULTS: Assembly of the draft genome resulted in 5245 protein-coding sequences, 14 rRNAs, 83 tRNAs and a GC content of 51.81%. The strain was identified asSalmonella Typhimurium ST19 harbouring a 265.5-kb pN1566-2 plasmid carrying genes encoding resistance to colistin (mcr-9.1), aminoglycosides (aadA1), tetracycline [tet(C)] and sulfonamides (sul1). Our findings indicate that the Salmonella Typhimurium ST19 strain in this study showed low genetic variability compared with Salmonella Typhimurium ST19 isolated from swine and poultry in Brazil, and was less related to those reported in other countries. CONCLUSIONS: This is the first reported genome of a phenotypically colistin-resistantSalmonella Typhimurium harbouring the mcr-9 variant in Brazilian livestock. This genome will aid global investigations on epidemiological and evolutionary aspects of plasmid-mediated colistin resistance and the role of colistin-resistant Salmonella Typhimurium ST19 lineage as a zoonotic pathogen.

Draft genome sequence of mcr-1-mediated colistin-resistant Escherichia coli ST359 from chicken carcasses in Northeastern Brazil.

OBJECTIVES: Considering that polymyxin is a drug of last resort in the treatment of humans infected by multidrug-resistant bacteria, the occurrence of plasmid-mediated colistin resistance mcr gene among Gram-negative bacteria in foods must be investigated. We present herein the draft genome sequence of a phenotypically colistin-resistant Escherichia coli carrying mcr-1 in chicken carcasses from a public market. METHODS: Total genomic DNA from the strain was sequenced by means of the Illumina MiSeq. The assembled contigs were annotated and manually curated. In silico analyses were performed to detect significant epidemiologic (serotyping and MLST) and structural features related plasmids identification, virulence and resistome. RESULTS: The ST359 E. coli strain presented a conserved 747 bp mcr-1 gene within a 9431 kb contig compatible with the IncX4 plasmid, which has been identified as a key vector for the global dissemination of mcr determinants among Enterobacteriacea. Other genes encoding for multidrug resistance such as blaCTX-M-2 and blaTEM-1B, and the virulence factors astA, cma, gad, iroN, ipfA, mchF were also detected. CONCLUSION: We reported a draft genome of a colistin-resistant E. coli ST359 associated with an IncX4 plasmid containing the gene mcr-1. The genomic data can be useful in epidemiological and evolutionary investigations on the spread of colistin-resistance among Enterobacteriacea in the food chain.

Off-label use of ceftiofur in one-day chicks triggers a short-term increase of ESBL-producing E. coli in the gut.

This trial was designed to evaluate the off-label use of ceftiofur with Marek's vaccine in one-day-old broiler chicks, a prophylactic treatment that has been done in some commercial hatcheries, on the emergence of extended-spectrum beta-lactamase producing Escherichia coli (ESBL-E. coli). A total of 168 chicks (Cobb500®) were used in a completely randomized design. Birds were assigned to two treatments (Marek's vaccine plus saline vs Marek's vaccine plus ceftiofur) and six repetitions, with 14 animals each. Cloacal swabs were collected from 1 to 14 days post-hatch. The majority (86%; p<0.0001) of the ESBL-producing isolates harboring blaCTX-M and blaSHV genes originated from animals receiving the antimicrobial. None of the isolates were positive for plasmid-mediated AmpC betalactamase genes (blaACC, blaCMY-2, blaDHA, blaFOX, blaMOX and blaMIR). These findings indicate that the off-label use of ceftiofur with Marek's vaccine is associated with the short-term increase in ESBL-producing Escherichia coli in the gut of chicks.

Intra-Amnionic Threonine Administered to Chicken Embryos Reduces Salmonella Enteritidis Cecal Counts and Improves Posthatch Intestinal Development.

This study assessed the effect of in ovo threonine supplementation on the response of broiler chicks challenged with Salmonella Enteritidis, considering bacterial counts in cecal contents, intestinal morphology, body weight, and weight gain. Fertilized eggs were inoculated in the amniotic fluid with saline (NT) or 3.5% threonine (T) solution at day 17.5 of incubation. At hatch, chicks were individually weighed and cloacal swabs were screened for Salmonella. At 2 days of age, half of the birds from each in ovo treatment were given either 0.5 mL of nutrient broth (sham-inoculated) or nalidixic acid-resistant Salmonella Enteritidis (SE NalR) in nutrient broth (8.3 × 107 colony forming units (CFU) SE NalR/mL). The birds were distributed using a completely randomized design with four treatments after the Salmonella challenge: no in ovo Thr supplementation and sham-inoculated in the posthatch challenge (NT-SHAM), in ovo Thr supplementation and sham-inoculated (T-SHAM), no in ovo Thr supplementation and SE NalR-challenged (NT-SE), and in ovo Thr supplementation and SE NalR-challenged (T-SE). In ovo threonine supplementation reduced Salmonella Enteritidis colonization 168-hour postinoculation and reduced the negative effects associated with Salmonella infection on intestinal morphology and performance, with results similar to those of the sham-inoculated birds. In ovo Thr supplementation increased the expression of MUC2 at hatch and the expression of MUC2 and IgA at 2 days of age and 168-hour postinoculation. Our results suggest that providing in ovo threonine promotes intestinal health in broilers challenged with Salmonella Enteritidis in the first days of life.

Pre-parturition staphylococcal mastitis in primiparous replacement goats: persistence over lactation and sources of infection.

This investigation reported for the first time the occurrence of intramammary infections caused by Staphylococcus in primiparous replacement goats before parturition and the persistence of clinical Staphylococcus aureus infection during the lactation period. Subclinical infections, mainly caused by coagulase negative staphylococci (CoNS), did not persist during lactation. Genotyping analysis indicated that environment seems to play a moderate role as source of intramammary infections to goats before parturition, but causative agents of mastitis in lactating animals are not genotypically related to environmental staphylococci. The occurrence and persistence of intramammary infections in replacement goats demonstrate the need to consider those animals as potential sources of infections in dairy goat herds.

Enterotoxin-encoding genes in Staphylococcus spp. from bulk goat milk.

Although Staphylococcus aureus has been implicated as the main Staphylococcus species causing human food poisoning, recent studies have shown that coagulase-negative Staphylococcus could also harbor enterotoxin-encoding genes. Such organisms are often present in goat milk and are the most important mastitis-causing agents. Therefore, this study aimed to investigate the occurrence of enterotoxin-encoding genes among coagulase-positive (CoPS) and coagulase-negative (CoNS) staphylococci isolated from raw goat milk produced in the semi-arid region of Paraiba, the most important region for goat milk production in Brazil. Enterotoxin-encoding genes were screened in 74 staphylococci isolates (30 CoPS and 44 CoNS) by polymerase chain reaction targeting the genes sea, seb, sec, sed, see, seg, seh, and sei. Enterotoxin-encoding genes were found in nine (12.2%) isolates, and four different genes (sea, sec, seg, and sei) were identified amongst the isolates. The most frequent genes were seg and sei, which were often found simultaneously in 44.5% of the isolates. The gene sec was the most frequent among the classical genes, and sea was found only in one isolate. All CoPS isolates (n=7) harboring enterotoxigenic genes were identified as S. aureus. The two coagulase-negative isolates were S. haemolyticus and S. hominis subsp. hominis and they harbored sei and sec genes, respectively. A higher frequency of enterotoxin-encoding genes was observed amongst CoPS (23.3%) than CoNS (4.5%) isolates (p<0.05), reinforcing the importance of S. aureus as a potential foodborne agent. However, the potential risk posed by CoNS in goat milk should not be ignored because it has a higher occurrence in goat milk and enterotoxin-encoding genes were detected in some isolates.

Nose-to-nose transmission of Salmonella Typhimurium between weaned pigs.

Little attention has been paid to the possibility of transmission of Salmonella in intensive pig production systems through alternate methods, such as airborne or direct nose-to-nose contact. This experimental study tested the hypothesis of nose-to-nose transmission of Salmonella enterica serovars Typhimurium (Trial I) and Agona (Trial II) in weaned pigs using stainless steel/glass isolation cabinets. In each trial, cabinet 1 (control pigs) and cabinet 2 (sentinel pigs) were connected directly to the fan unit. Cabinet 3 (seeded pigs) was not directly linked to the fan, but was arranged to receive a constant unidirectional airflow from cabinet 2 (sentinel pigs) through a 10cm diameter hole, which also allowed nose-to-nose contact between pigs housed in these two cabinets. Air was taken out of the system through ducts connecting cabinets 1 and 3 to the exhauster. Therefore, direct contact among seeded and sentinel pigs was allowed but possible aerial transference of contaminated particles between those cabinets was prevented. The system was opened 21 days post-inoculation and tissue samples were collected for bacteriological analysis. The recovery of nalidixic acid-resistant Salmonella Typhimurium from sentinel pigs corroborates the hypothesis of nose-to-nose transmission of that pathogen in pigs. However, serovar-related differences might exist regarding the nose-to-nose transmissibility of Salmonella in pigs, since Salmonella Agona was not detected in sentinel pigs (Trial II).

Development and application of a Saccharomyces cerevisiae-expressed nucleocapsid protein-based enzyme-linked immunosorbent assay for detection of antibodies against infectious bronchitis virus.

A Saccharomyces cerevisiae-expressed nucleocapsid (N) polypeptide of the M41 strain of infectious bronchitis virus (IBV) was used as antigen in a recombinant yeast-expressed N protein-based enzyme-linked immunosorbent assay (Y-N-ELISA). The Y-N-ELISA was rapid, sensitive, and specific for detecting chicken serum antibodies to IBV, and it compared favorably with a commercial ELISA.