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 and Evolutionary Analysis of Salmonella enterica Serovar Kentucky Sequence Type 198 Isolated From Livestock In East Africa.

Since its emergence in the beginning of the 90's, multidrug-resistant (MDR) Salmonella enterica subsp. enterica serovar Kentucky has become a significant public health problem, especially in East Africa. This study aimed to investigate the antimicrobial resistance profile and the genotypic relatedness of Salmonella Kentucky isolated from animal sources in Ethiopia and Kenya (n=19). We also investigated population evolutionary dynamics through phylogenetic and pangenome analyses with additional publicly available Salmonella Kentucky ST198 genomes (n=229). All the 19 sequenced Salmonella Kentucky isolates were identified as ST198. Among these isolates, the predominant genotypic antimicrobial resistance profile observed in ten (59.7%) isolates included the aac(3)-Id, aadA7, strA-strB, blaTEM-1B, sul1, and tet(A) genes, which mediated resistance to gentamicin, streptomycin/spectinomycin, streptomycin, ampicillin, sulfamethoxazole and tetracycline, respectively; and gyrA and parC mutations associated to ciprofloxacin resistance. Four isolates harbored plasmid types Incl1 and/or Col8282; two of them carried both plasmids. Salmonella Pathogenicity islands (SPI-1 to SPI-5) were highly conserved in the 19 sequenced Salmonella Kentucky isolates. Moreover, at least one Pathogenicity Island (SPI 1-4, SPI 9 or C63PI) was identified among the 229 public Salmonella Kentucky genomes. The phylogenetic analysis revealed that almost all Salmonella Kentucky ST198 isolates (17/19) stemmed from a single strain that has accumulated ciprofloxacin resistance-mediating mutations. A total of 8,104 different genes were identified in a heterogenic and still open Salmonella Kentucky ST198 pangenome. Considering the virulence factors and antimicrobial resistance genes detected in Salmonella Kentucky, the implications of this pathogen to public health and the epidemiological drivers for its dissemination must be investigated.

Antimicrobial resistance in the globalized food chain: a One Health perspective applied to the poultry industry.

Antimicrobial resistance (AMR) remains a major global public health crisis. The food animal industry will face escalating challenges to increase productivity while minimizing AMR, since the global demand for animal protein has been continuously increasing and food animals play a key role in the global food supply, particularly broiler chickens. As chicken products are sources of low-cost, high-quality protein, poultry production is an important economic driver for livelihood and survival in developed and developing regions. The globalization of the food supply, markedly in the poultry industry, is aligned to the globalization of the whole modern society, with an unprecedented exchange of goods and services, and transit of human populations among regions and countries. Considering the increasing threat posed by AMR, human civilization is faced with a complex, multifaceted problem compromising its future. Actions to mitigate antimicrobial resistance are needed in all sectors of the society at the human, animal, and environmental levels. This review discusses the problems associated with antimicrobial resistance in the globalized food chain, using the poultry sector as a model. We cover critical aspects of the emergence and dissemination of antimicrobial resistance in the poultry industry and their implications to public health in a global perspective. Finally, we provide current insights using the multidisciplinary One Health approach to mitigate AMR at the human-animal-environment interface.

Staphylococcus sciuri as a Reservoir of mecA to Staphylococcus aureus in Non-Migratory Seabirds from a Remote Oceanic Island.

Aim: Genomic analysis of a methicillin-resistant Staphylococcus aureus (MRSA) strain cultured from a non-migratory seabird at Fernando de Noronha Archipelago (Brazilian oceanic islands) was carried out to investigate the potential origin of MRSA genetic determinants in an ecological setting with minimal or absent antimicrobial selective pressure, and minimal interaction with humans and domestic animals. Results: The study determined mecA gene homology and the phylogenetic relatedness with mecA described in Staphylococcus sciuri, which was the major Staphylococcus spp. cultured from the birds. Our findings corroborate in silico assumptions that the mecA gene in MRSA strains clinically relevant for humans and animals originates from S. sciuri ancestors. Conclusion: Coagulase-negative staphylococci seem to be natural reservoirs of methicillin-resistant genes to S. aureus, even in environments with very low antimicrobial selection pressure.

Occurrence of KPC-Producing Escherichia coli in Psittaciformes Rescued from Trafficking in Paraíba, Brazil.

The emergence and spread of antimicrobial resistance pose a threat to public health globally. Antibiotic-resistant bacteria and genes can disseminate among environments, animals and humans. Therefore, investigation into potential reservoirs of multidrug-resistant bacteria is of great importance to the understanding of putative transmission routes of resistant bacteria and resistance genes. This study aimed to report the occurrence of Escherichia coli harboring the Klebsiella pneumoniae carbapenemase-producing gene (blaKPC) in Psittaciformes rescued from wildlife trafficking in Paraíba State, Brazil. Cloacal swabs were collected from thirty birds and cultured by conventional microbiology using MacConkey and serum tryptone glucose glycerol (STGG) media supplemented with selective antimicrobials. E. coli isolates (n = 43) were identified by phenotypic tests and confirmed by MALDI-TOF. Antimicrobial susceptibility profiles were determined by means of Kirby-Bauer test. All isolates were further screened for extended-spectrum beta-lactamase (ESBL) production, and putative genes encoding ESBL were investigated by PCR. Additionally, blaKPC-harboring strains were genotyped by REP-PCR. A total of 43 E. coli phenotypically resistant isolates were recovered. The highest resistance rate was observed against ciprofloxacin. Among the resistance genes, only blaKPC was found in seven different birds from three species. According to the genotyping, these seven isolates belonged to four different strains. To date, this is the first report on the occurrence of KPC-E. coli in Psittaciformes rescued from trafficking in Northeastern Brazil. Due to the high clinical importance of KPC-E. coli, our findings suggest that wild animals in captivity at wildlife rescue centers can play a role as reservoirs of bacteria that are resistance to Critically Important antimicrobials in human medicine.

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.

Genomic surveillance links livestock production with the emergence and spread of multi-drug resistant non-typhoidal Salmonella in Mexico.

Multi-drug resistant (MDR) non-typhoidal Salmonella (NTS) is increasingly common worldwide. While food animals are thought to contribute to the growing antimicrobial resistance (AMR) problem, limited data is documenting this relationship, especially in low and middle-income countries (LMIC). Herein, we aimed to assess the role of non-clinical NTS of bovine origin as reservoirs of AMR genes of human clinical significance. We evaluated the phenotypic and genotypic AMR profiles in a set of 44 bovine-associated NTS. For comparative purposes, we also included genotypic AMR data of additional isolates from Mexico (n = 1,067) that are publicly available. The most frequent AMR phenotypes in our isolates involved tetracycline (40/44), trimethoprim-sulfamethoxazole (26/44), chloramphenicol (19/44), ampicillin (18/44), streptomycin (16/44), and carbenicillin (13/44), while nearly 70% of the strains were MDR. These phenotypes were correlated with a widespread distribution of AMR genes (i.e. tetA, aadA, dfrA12, dfrA17, sul1, sul2, bla-TEM-1, blaCARB-2) against multiple antibiotic classes, with some of them contributed by plasmids and/or class-1 integrons. We observed different AMR genotypes for betalactams and tetracycline resistance, providing evidence of convergent evolution and adaptive AMR. The probability of MDR genotype occurrence was higher in meat-associated isolates than in those from other sources (odds ratio 11.2, 95% confidence interval 4.5-27.9, P < 0.0001). The study shows that beef cattle are a significant source of MDR NTS in Mexico, highlighting the role of animal production on the emergence and spread of MDR Salmonella in LMIC.

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.

Whole genome sequencing reveals widespread distribution of typhoidal toxin genes and VirB/D4 plasmids in bovine-associated nontyphoidal Salmonella.

Nontyphoidal Salmonella (NTS) is a common pathogen in food-producing animals and a public health concern worldwide. Various NTS serovars may be present in apparently healthy animals. This could result in carcass contamination during the slaughter process leading to human exposure. While most genomic research has focused on Salmonella pathogenesis, little is known on the factors associated with subclinical infections and environmental persistence. We report here the widespread distribution of typhoidal toxin genes (i. e. the cdtB islet, hlyE, taiA), among NTS strains from a beef slaughter operation (n = 39) and from epidemiologically unconnected ground beef (n = 20). These genes were present in 76% of the strains, regardless of serovar, isolation source or geographical location. Moreover, strains that predominated in the slaughterhouse carry plasmid-borne type IV secretion systems (T4SS), which have been linked to persistent infections in numerous pathogens. Population genomics supports clonal dissemination of NTS along the food production chain, highlighting its role as reservoir of genetic variability in the environment. Overall, the study provides a thorough characterization of serovar diversity and genomic features of beef-associated NTS in Mexico. Furthermore, it reveals how common genetic factors could partially explain the emergence and persistence of certain NTS serovars in the beef industry.

Accuracy of PCR targeting different markers for Staphylococcus aureus identification: a comparative study using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry as the gold standard.

Staphylococcus aureus is considered a major pathogen in veterinary and human medicine, and the emergence of multidrug-resistant strains, such as livestock-associated methicillin-resistant S. aureus, means that reliable, inexpensive, and fast methods are required to identify S. aureus obtained from animal sources. We tested the accuracy of a PCR targeting the genes femA, nuc, and coa in identifying S. aureus from animals. A total of 157 Staphylococcus spp. isolates were examined by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry; 18 different Staphylococcus species were identified. Of 68 S. aureus isolates, the genes femA, nuc, and coa were found in 61, 53, and 32 isolates, respectively. Considering MALDI-TOF as the gold standard, the PCR assays targeting all 3 genes showed 100% specificity; the sensitivity values were 89.7, 77.9, and 47.0% for femA, nuc, and coa, respectively. Sensitivity was 100% when femA and nuc markers were targeted simultaneously. These results confirm PCR as an accurate method to identify S. aureus species from animal sources and strongly suggest the simultaneous use of primers targeting femA and nuc genes.

Antimicrobial resistance and genotypic relatedness of environmental staphylococci in semi-extensive dairy farms.

This study aimed to investigate the occurrence, genotypic relatedness and antimicrobial resistance of Staphylococcus aureus and coagulase-negative Staphylococcus from milk and environmental sources in dairy herds. A total of 110 staphylococci recovered from 147 samples collected at 21 semi-extensive dairy farms in Northeastern Brazil were investigated. Staphylococcus aureus isolates were identified and screened for methicillin resistance by means of a duplex-PCR. The highest frequency of contamination by S. aureus was observed for milk samples (38.1%), while contamination by coagulase-negative staphylococci (CoNS) was most commonly detected in milkers' hand swabs (52.4%) and environmental samples (29.5%). Two mecA-positive Staphylococcus aureus (2/40; 5%) were detected, while the same gene was found in fourteen (14/70; 20%) CoNS. Clonally related isolates from milk and environmental sources, such as the surface of gates, were detected by PFGE. This study reports the occurrence of MRSA in dairy farms under semi-extensive production practices and reinforces the importance of environment as a source of Staphylococcus contamination in dairy herds.

Biofilm-forming and antimicrobial resistance traits of staphylococci isolated from goat dairy plants.

INTRODUCTION: Biofilm-associated antimicrobial resistance is of increasing importance to the maintenance and spread of foodborne pathogens in the food industry. This study aimed to investigate the ability to form biofilm and the antimicrobial resistance of staphylococci contaminating small-scale goat milk dairy plants. METHODOLOGY: Sixty isolates were tested for antimicrobial resistance against 20 drugs by the microdilution method. Biofilm-forming traits were assessed by the microtiter plate method (MtP), Congo red agar method (CRA), and icaD gene detection by polymerase chain reaction (PCR). RESULTS: High antimicrobial resistance to ampicillin (60/60; 100%), penicillin G (21/60; 35%), and erythromycin (15/60; 25%) was observed, but all isolates were susceptible to amoxicillin/K-clavulanate, ceftriaxone, ciprofloxacin, gentamicin, levofloxacin, linezolid, and moxifloxacin. No resistance to oxacillin or vancomycin was seen among Staphylococcus aureus. Twenty-seven isolates (27/60; 45%) were considered to form biofilm according to MtP, and similar biofilm-producing frequencies were observed in coagulase-negative staphylococci (CoNS) (20/44; 45.4%) and S. aureus (7/16; 43.7%). The icaD gene was observed only in S. aureus isolates. There was no association between biofilm production and antimicrobial resistance. A higher frequency of biofilm-producing staphylococci was found in isolates from bulk tank milk and hand swabs. On the other hand, isolates from pasteurized milk showed lower frequency of biofilm formation. CONCLUSIONS: Staphylococci contaminating goat dairy plants are potential biofilm producers. The results suggest no association between the ability to form biofilm and antimicrobial resistance.

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.