Detection of Escherichia coli and other Enterobacteriales members in seabirds sampled along the Brazilian coast.

Escherichia coli, an Enterobacterales member, is a normal representative of the microbiota of homeothermic animals. Most strains are commensal, but several pathotypes can cause disease, and numerous antimicrobial resistance factors have been identified. These bacteria have spread rapidly in recent years, highlighting the importance of screening the environment and non-human reservoirs for virulent strains and/or those presenting resistance factors, in addition to other microorganisms of public health importance. In this context, this study aimed to survey Enterobacteriales present in seabirds sampled along the Brazilian coast, comparing findings between migratory and resident birds, as well as between wrecked and non-wrecked animals. Escherichia coli pathotypes were also characterized through rapid seroagglutination and polymerase chain reaction techniques and antimicrobial resistance profiles were investigated through the disc agar diffusion method. Cloacal, ocular, oral, tracheal, and skin lesion swabs, as well as fresh feces, were collected from 122 seabirds. The findings indicate these animals as important hosts for opportunistic human pathogens. Escherichia coli strains were identified in 70 % of the analyzed seabirds, 62 % of which displaying resistant or intermediate profiles to at least one antimicrobial, while 7% were multiresistant. Resistance to tetracycline (22 %), nalidixic acid (15 %), trimethoprim-sulfamethozaxol (14 %) and ampicillin (12 %) were the most prevalent. Resistance to cefoxitin, a critically important antimicrobial for human medicine, was also detected. Virulence genes for one of the EAEC, ETEC or EPEC pathotypes were detected in 30 % of the identified strains, the first two described in seabirds for the first time. The EAEC gene was detected in 25 % of the sampled seabirds, all resident, 8 % of which exhibited a multidrug-resistant profile. Thus, seabirds comprise important reservoirs for this pathotype. Escherichia coli was proven an ubiquitous and well-distributed bacterium, present in all evaluated bird species and sampling sites (except Marajó Island). According to the chi-square test, no significant differences between E. coli prevalences or antimicrobial resistance profiles between migratory and resident and between wrecked and non-wrecked seabirds were observed. Thus, migratory birds do not seem to contribute significantly to E. coli frequencies, pathotypes or antimicrobial resistance rates on the Brazilian coast.

Genomic analyses of drug-resistant Salmonella enterica serovar Heidelberg strains isolated from meat and related sources between 2013 and 2017 in the south region of Brazil.

Salmonella enterica serovar Heidelberg (S. Heidelberg) is a zoonotic, ubiquitous, and worldwide-distributed pathogen, responsible for gastroenteritis in humans caused by the consumption of contaminated food. In this study, 11 S. Heidelberg strains isolated from chicken and bovine meat, drag swab, and animal feed between 2013 and 2017 in states of the southern region of Brazil were characterized by whole-genome sequencing (WGS) analyses. Antimicrobial resistance against 18 antimicrobials was determined by disk-diffusion and ciprofloxacin's minimum inhibitory concentration by Etest®. The search for resistance and virulence genes, plasmids, Salmonella Pathogenicity Islands (SPIs) plus multi-locus sequence typing (MLST), and single-nucleotide polymorphisms (SNPs) analyses was conducted using WGS data. All strains harbored resistance genes fosA7, aac(6')-Iaa, sul2, tet(A), blaCMY-2, mdsA, and mdsB, and point mutations in gyrA and parC. All strains showed a phenotypic multidrug-resistant profile, with resistant or intermediate resistant profiles against 14 antimicrobials tested. Plasmids ColpVC, IncC, IncX1, and IncI1-I(Alpha) were detected. Virulence genes related to adherence, macrophage induction, magnesium uptake, regulation, and type III secretion systems plus 10 SPIs were detected. All strains were assigned to ST15 and belonged to two SNP clusters showing high similarity to isolates from the United Kingdom, Chile, Germany, the Netherlands, China, South Africa, and South Korea. In conclusion, the presence of multidrug-resistant S. Heidelberg strains in Brazil showing a global genomic relationship may alert for the necessity of stronger surveillance measures by food safety and public health authorities to limit its spread to humans and animals through foods.

Whole genome sequencing analyses revealed that Salmonella enterica serovar Dublin strains from Brazil belonged to two predominant clades.

Salmonella Dublin is a cattle-associated serovar sporadically causing disease in humans. S. Dublin strains isolated in Brazil and in other countries were analyzed to determine their phylogenetic relationships, the presence of genes, plasmids, genomic regions related to virulence and antimicrobial resistance genes repertoire, using WGS analyses. Illumina was used to sequence the genome of 112 S. Dublin strains isolated in Brazil from humans (n = 82) and animals (n = 30) between 1983 and 2016. Furthermore, 87 strains from other countries were analyzed. WGSNP analysis revealed three different clades, in which the strains from Brazil belonged to two clades, A and C. Most of the genes and genomic regions searched varied among the strains studied. The siderophore genes iroB and iroC were exclusively found in strains from Brazil and pegD gene, related to fimbrial adherence determinants, were positive in 124 strains from clades A and B but absent in all the strains from clade C (n = 71). Eleven plasmid replicons were found in the strains from Brazil, and nine were exclusively found in strains from other countries. The antimicrobial resistance genes mdsA and mdsB, that encode an efflux pump, were found in all the strains studied. The strains from Brazil carried other resistance genes, such as tet(A) (n = 11), tet(B) (n = 4) and tet(C) (n = 4), blaTEM-1 (n = 4), catA1 (n = 1), aadA1 (n = 1), and sul1 (n = 1). In conclusion, S. Dublin strains isolated in Brazil presented some few unique genes not found in strains from other countries and were allocated into two distinct clades with strains of human and animal origin epidemiologically related. This fact stresses the zoonotic potential of S. Dublin circulating in Brazil for more than 30 years.

High Genetic Diversity and Antimicrobial Resistance in Escherichia coli Highlight Arapaima gigas (Pisces: Arapaimidae) as a Reservoir of Quinolone-Resistant Strains in Brazilian Amazon Rivers.

The increasing prevalence of multi-drug resistant (MDR) Escherichia coli in distinct ecological niches, comprising water sources and food-producing animals, such as fish species, has been widely reported. In the present study, quinolone-resistant E. coli isolates from Arapirama gigas, a major fish species in the Brazilian Amazon rivers and fish farms, were characterized regarding their antimicrobial susceptibility, virulence, and genetic diversity. A total of forty (40) specimens of A. gigas, including 20 farmed and 20 wild fish, were included. Thirty-four quinolone-resistant E. coli isolates were phenotypically tested by broth microdilution, while resistance and virulence genes were detected by PCR. Molecular epidemiology and genetic relatedness were analyzed by MLST and PFGE typing. The majority of isolates were classified as MDR and detected harboring blaCTX-M, qnrA and qnrB genes. Enterotoxigenic E. coli pathotype (ETEC) isolates were presented in low prevalence among farmed animals. MLST and PFGE genotyping revealed a wide genetic background, including the detection of internationally spread clones. The obtained data point out A. gigas as a reservoir in Brazilian Amazon aquatic ecosystems and warns of the interference of AMR strains in wildlife and environmental matrices.

Salmonella in the processing line of farmed Tambatinga (Colossoma macropomum x Piaractus brachypomus) in Mato Grosso, Brazil: serotypes of occurrence and antimicrobial profile.

The objective of this study was to evaluate the dispersion dynamics and antimicrobial resistance profiles of Salmonella in the processing of Tambatinga (Colossoma macropomum x Piaractus brachypomus). Thirty fish were monitored during four processing stages (reception, first wash, evisceration, and prepackage area) in a fish slaughterhouse. One hundred and twenty fish surface samples were collected and tested through bacteriological analysis, PCR, serotyping, and antimicrobial resistance profile (disk-diffusion). Of these samples, 7.5% (9/120) were positive for Salmonella, with 0.83% being observed in the pre-packaging phase, indicating a low occurrence at this stage. All the analyzed stages were positive for Salmonella, with the prevalent serovars being Ndolo, Mbandaka, Typhimurium, Rough, and O:16. All strains were sensitive to various antimicrobials. Improvements in microbiological control during all processing stages should be implemented to ensure a Salmonella-free product.

A high number of multidrug-resistant and predominant genetically related cluster of Shigella flexneri strains isolated over 34 years in Brazil.

Shigella flexneri has been a major public health problem in developing countries. This work analyzed the frequency of 16 virulence genes, the genotypic diversity, and the antimicrobial resistance profiles of 130 S. flexneri strains isolated in Brazil. The ipaH gene was found in all the 130 strains. The frequencies of the other genes were variable ial (88.5%), sigA (82.3%), iuc (74.6%), virA (73%), pic (72.3%), virF (57.7%), sat (48.5%), ipaBCD (37%), sen (36%), set1A (35.4%), sepA (30%), set1B (30%), virB (14%), icsA (10%), and ipgD (5.4%). A total of 57 (43.8%) strains were multidrug-resistant. ERIC-PCR grouped 96 of the strains into a single cluster with ≥ 70.4% of similarity, 75 of these strains presented a similarity ≥ 80.9%. PFGE grouped 120 of the strains into a single cluster with 57.4% of similarity and 82 of these strains presented a similarity ≥ 70.6%. In conclusion, the high frequency of some virulence genes reinforces the pathogenic potential of the strains studied. The high rates of MDR strains are alarming once it may lead to failure when antimicrobial treatment is necessary. Genotype techniques reveled a major cluster with high genetic similarity including S. flexneri strains from the different Brazilian states and distinct years of isolation, showing that they probably emerged from a common ancestor.

Phylogenetic analysis revealed that Salmonella Typhimurium ST313 isolated from humans and food in Brazil presented a high genomic similarity.

Salmonella Typhimurium sequence type 313 (S. Typhimurium ST313) has caused invasive disease mainly in sub-Saharan Africa. In Brazil, ST313 strains have been recently described, and there is a lack of studies that assessed by whole genome sequencing (WGS)-the relationship of these strains. The aims of this work were to study the phylogenetic relationship of 70 S. Typhimurium genomes comparing strains of ST313 (n = 9) isolated from humans and food in Brazil among themselves, with other STs isolated in this country (n = 31) and in other parts of the globe (n = 30) by 16S rRNA sequences, the Gegenees software, whole genome multilocus sequence typing (wgMLST), and average nucleotide identity (ANI) for the genomes of ST313. Additionally, pangenome analysis was performed to verify the heterogeneity of these genomes. The phylogenetic analyses showed that the ST313 genomes were very similar among themselves. However, the ST313 genomes were usually clustered more distantly to other STs of strains isolated in Brazil and in other parts of the world. By pangenome calculation, the core genome was 2,880 CDSs and 4,171 CDSs singletons for all the 70 S. Typhimurium genomes studied. Considering the 10 ST313 genomes analyzed the core genome was 4,112 CDSs and 76 CDSs singletons. In conclusion, the ST313 genomes from Brazil showed a high similarity among them which information might eventually help in the development of vaccines and antibiotics. The pangenome analysis showed that the S. Typhimurium genomes studied presented an open pangenome, but specifically tending to become close for the ST313 strains.

High similarity and high frequency of virulence genes among Salmonella Dublin strains isolated over a 33-year period in Brazil.

Salmonella Dublin is a strongly adapted serovar that causes enteritis and/or systemic disease with high rates of mortality in cattle and occasionally infects humans. Despite the importance of this serovar, there is a lack of studies in Brazil. The aim of this study was to characterize the genetic diversity of 112 S. Dublin strains isolated from humans and animals in Brazil by CRISPR and CRISPR-MVLST and the relatedness among strains by MLST. In addition, the frequency of some important virulence genes was verified. The strains studied belonged to nine different sequence types, being all of them single- or double-locus variants of the ST10. CRISPR discriminated the strains into 69 subtypes with a similarity ≥ 84.4% and CRISPR-MVLST into 72 subtypes with a similarity ≥ 84.7%. The virulence genes ratB, lpfA, mgtC, avrA, sopB, sopE2, sifA, sseA, ssrA, csgA, fliC, and sinH were found in all the strains studied, while spvB, spvC, sodCl, rpoS, sipA, sipD, invA, and hilA were detected in ≥ 93.7% of the strains. In conclusion, the high similarity among the strains reinforces the clonal nature of the strains of this serovar that may have descended from a common ancestor that little differed over 33 years in Brazil. CRISPR and CRISPR-MVLST showed to be good alternatives to type S. Dublin strains. MLST suggested that S. Dublin strains from Brazil were phylogenetically related to strains from other parts of the globe. Moreover, the high frequency of virulence genes among the strains studied reinforces the capacity of S. Dublin to cause invasive diseases.

Salmonella isolated from chicken carcasses from a slaughterhouse in the state of Mato Grosso, Brazil: antibiotic resistance profile, serotyping, and characterization by repetitive sequence-based PCR system.

Salmonella is one of the major causative agents of foodborne infections. Salmonellosis becomes more dangerous when strains resistant to several antibiotics are found in food, especially in chicken, one of the primary transmission vehicles of this pathogen for humans. The present study aimed to estimate the occurrence of Salmonella in chicken carcasses from the state of Mato Grosso, Brazil, as well as determine the antibiotic resistance profile and genotypic characteristic of multi-drug resistant (MDR) isolates. During a 15-month period, from 01/2014 to 05/2015, 850 samples of chilled fresh chicken carcasses were sampled from a slaughterhouse and submitted to Salmonella determinations according to the ISO-6579/2002 method, serotyping and multiplex PCR. The disc diffusion test was applied for 17 antibiotics, according to CLSI (2014). Five isolates were genotyped by repetitive sequence-based PCR using the semi-automated DiversiLab (bioMérieux®) system. The occurrence of Salmonella in chicken carcasses was of 3.7% (31/850), with only 4 strains (12.9%) presenting as MDR, and 6 strains (19.35%) displaying ESBL. The predominant serovars were Salmonella Infantis (35.4%, 11/31), and S. Abony (25.8%, 8/31), followed by serovars S. Agona (12.9%, 4/31), S. Schwarzengrund (9.7%, 3/31), S. Anatum and Salmonella enterica O:4,5 (6.5%, 2/31), and only one Salmonella enterica O:6,7 strain (3.2%, 1/31). All isolates were resistant to one to 5 classes of antibiotics in decreasing order: folate pathway inhibitors, ß-lactams (cephalosporins, penicillin, monobactams), tetracyclines, chloramphenicol, and gentamicin. However, strains sensitive to florfenicol, streptomycin, nalidixic acid, ciprofloxacin, enrofloxacin, and nitrofurantoin were also found in this study. Genotyping revealed 98 to 99% homology between 3 Salmonella strains, which displayed high phenotypic resistance similarity to ß-lactams and folate pathway inhibitors. Detection of MDR non-typhoid Salmonella in chicken slaughterhouses with quality assurance systems such as Hazard Analysis and Critical Points and Implemented Good Manufacturing Practices is a concern, reinforcing the need for constant monitoring of these pathogens, with the purpose of safeguarding the safety of their products.

Elimination of coliforms and Salmonella spp. in sheep meat by gamma irradiation treatment.

This study aimed at evaluating the bacteriological effects of the treatment of sheep meat contaminated with total coliforms, coliforms at 45 °C and Salmonella spp. by using irradiation at doses of 3 kGy and 5 kGy. Thirty sheep meat samples were collected from animals located in Rio de Janeiro State, Brazil, and then grouped in three lots including 10 samples: non-irradiated (control); irradiated with 3 kGy; and irradiated with 5 kGy. Exposure to gamma radiation in a (137)Cs source-driven irradiating facility was performed at the Nuclear Defense Section of the Brazilian Army Technological Center (CTEx) in Rio de Janeiro. The samples were kept under freezing temperature (-18 °C) until the analyses, which occurred in two and four months after irradiation. The results were interpreted by comparison with the standards of the current legislation and demonstrated that non-irradiated samples were outside the parameters established by law for all groups of bacteria studied. Gamma irradiation was effective in inactivating those microorganisms at both doses tested and the optimal dose was achieved at 3 kGy. The results have shown not only the need for sanitary conditions improvements in slaughter and processing of sheep meat but also the irradiation effectiveness to eliminate coliform bacteria and Salmonella spp.

Colistin resistance in Escherichia coli and Salmonella enterica strains isolated from swine in Brazil.

Reports about acquired resistance to colistin in different bacteria species are increasing, including E. coli of animal origin, but reports of resistance in wild S. enterica of different serotypes from swine are not found in the literature. Results obtained with one hundred and twenty-six E. coli strains from diseased swine and one hundred and twenty-four S. enterica strains from diseased and carrier swine showed a frequency of 6.3% and 21% of colistin-resistant strains, respectively. When comparing the disk diffusion test with the agar dilution test to evaluate the strains, it was confirmed that the disk diffusion test is not recommended to evaluate colistin resistance as described previously. The colistin MIC 90 and MIC 50 values obtained to E. coli were 0.25 µg/mL and 0.5 µg/mL, the MIC 90 and MIC 50 to S. enterica were 1 µg/mL and 8 µg/mL. Considering the importance of colistin in control of nosocomial human infections with Gram-negative multiresistant bacteria, and the large use of this drug in animal production, the colistin resistance prevalence in enterobacteriaceae of animal origin must be monitored more closely.

Occurrence of integrons and antimicrobial resistance genes among Salmonella enterica from Brazil.

OBJECTIVES: To determine the occurrence of antimicrobial resistance genes and role of integrons among 135 antimicrobial-resistant Salmonella enterica from Brazil. METHODS: The presence of antimicrobial resistance genes, class 1 and 2 integrons and gene cassettes was analysed by PCR and sequencing. The genetic location of class 1 integrons was determined in 25 isolates by hybridization and plasmid transfer experiments. RESULTS: Fifty-five of the isolates were positive for class 1 integrons. Integron-positive isolates represented 17 different serovars and were mainly from human (n=28) and animal (n=13) sources. The gene cassette arrangements could be determined in 51 of the positive isolates, which harboured one [dfrA22, aadA1 or orf3 (putative trimethoprim resistance)], two [aadA1-dfrA1, aac(6')-Ib-orf1 (unknown function) or aacA4-aadA1], three [dfrA15b-cmlA4-aadA2, orf2 (unknown function)-dfrA5-orfD] or four [orf4-aacA4-blaOXA-30 (interrupted by an IS1 element)-aadA1] cassettes in their variable region. Only one isolate harboured a class 2 integron with the gene cassette array dfrA1-sat-aadA1. Several integron unrelated resistance genes were also detected in the isolates. Sulphonamide resistance was primarily mediated by sul2 and sul3, tetracycline resistance by tet(B) and tet(A), chloramphenicol resistance by catA1, streptomycin resistance by strA and ampicillin resistance by blaTEM. blaCTX and blaCMY-2 were found in cephalosporin-resistant isolates. Mating and hybridization experiments demonstrated that a high-molecular-weight plasmid mediated the gene transfer of integrons and additional resistance determinants. CONCLUSIONS: The present study revealed that integron-mediated resistance genes contributed to the multiresistance phenotype observed in the isolates, but most resistance genes were located outside the integron structure, as independent genes. However, they might be located on the same conjugative plasmid.

Occurrence of integrons and resistance genes among sulphonamide-resistant Shigella spp. from Brazil.

OBJECTIVES: To determine the occurrence of class 1 and 2 integrons and antimicrobial resistance genes among sulphonamide-resistant Shigella strains isolated in Brazil during 1999-2003. METHODS: Sixty-two Shigella (Shigella flexneri, n = 47 and Shigella sonnei, n = 15) were tested against 21 antimicrobial agents. The presence of integrons classes 1 and 2 and antimicrobial resistance genes was investigated by PCR using specific primers. RESULTS: A total of eight antimicrobial resistance profiles were identified, with the profile of resistance to sulfamethoxazole, trimethoprim, spectinomycin, streptomycin and tetracycline being the most common among S. sonnei, and additionally to ampicillin and chloramphenicol among S. flexneri. Class 1 integrons were found in only two strains, whereas class 2 integrons were found in 56 (90.3%) of the strains. All class 2-positive strains had a similar fragment of 2214 bp harbouring a gene cassette array conferring resistance to trimethoprim, streptothricin and spectinomycin/streptomycin. The genes coding for resistance to chloramphenicol (catA1), tetracycline [tet(A) and tet(B)] and ampicillin (bla(OXA) and bla(TEM)), were detected in resistant strains. CONCLUSIONS: The detection of class 1 and 2 integrons and additional antimicrobial resistance genes allowed us to identify the most frequent antimicrobial resistance patterns of Shigella spp. isolated in Brazil.

Escherichia coli isolated from seafood: toxicity and plasmid profiles.

Thirty-two Escherichia coli strains were isolated from red snapper (Lutjanus purpureus) and from seabob shrimp (Xiphopenaeus kroyeri). The strains were numbered S1-S16, and F1-F16, which corresponds to the isolation origin from shrimp (S) and fish (F). The isolates were biologically and antigenically characterized by agglutination tests with enteropathogenic E. coli (EPEC)-, enteroinvasive E. coli (EIEC)- and enterohemorrhagic E. coli (EHEC) O157-specific antisera. The ETEC enterotoxins were characterized by GMI-ELISA for enterotoxin LT-1 (thermolabile) and by inoculation of supernatants prepared from newly born mice for enterotoxin Sta. A total of 14 strains produced exotoxins, of which seven were thermolabile (LT) and seven were thermostable (ST). Antimicrobial susceptibility profiles were determined by disc diffusion in agar using ampicillin, cephalothin, cefoxitin, ceftriaxone, imipenem, nalidixic acid, ciprofloxacin, chloramphenicol, gentamicin, nitrofurantoin, sulfamethoxazole-trimethoprim, and tetracycline. Four isolates showed lower susceptibility to some antibiotics, two strains were resistant to ampicillin, tetracycline, and sulfamethoxazole-trimethoprim, and two were resistant to tetracycline and nitrofurantoin. Plasmids were extracted in the four resistant isolates; two of them contained plasmids whose molecular weight varied from low to high. The characterization of LT- and ST-toxin-producing E. coli strains displaying multiresistance and containing plasmids suggests the need for tightening current control measures for the use of antimicrobials.