Salmonella detection with LAMP and qPCR and identification of serovars of interest by multiplex qPCR in poultry carcasses.

Salmonella is present in the poultry production chain and is a major challenge in terms of food safety and animal health. The early Salmonella detection is one of the main tools to control and prevent the transmission of this pathogen. Microbiological isolation and serotyping to identify and differentiate Salmonella serovars are laborious processes, time-consuming, and expensive. Therefore, molecular diagnostic methods can be rapid and efficient alternatives to the detection of this pathogen. Thus, the aim herein was to standardize and evaluate the use of loop-mediated isothermal amplification (LAMP) in comparison with real-time PCR (qPCR) for detection of Salmonella associated with a multiplex qPCR for simultaneous identification and differentiation of S. Enteritidis, S. Typhimurium, S. Pullorum, and S. Gallinarum. The LAMP, qPCR, and multiplex qPCR assays were comparable in specificity. The three techniques were evaluated for specificity for 16 different serovars of Salmonella and for 37 strains of the serovars of interest. The limit of detection and the efficiency of the LAMP, qPCR, and multiplex qPCR reactions were determined. The techniques were applied to 33 samples of chicken carcasses and compared to the results of conventional microbiology for validation. As results, LAMP was specific in the detection of different Salmonella serovars but presented lower limit of detection ranging from 101 to 104 CFU/reaction. In comparison, qPCR could detect less cells (100 to 102 CFU/reaction), reaching equal specificity and better repeatability in the assays. The qPCR multiplexing for identification of the different serovars also showed good specificity, with the detection threshold between entre 101 and 102 CFU/reaction. The results obtained in the analyses on poultry carcasses suggested a correspondence between the results obtained in molecular methods and in conventional microbiology. Thus, the proposed assays are promising for the diagnosis of Salmonella in poultry carcasses, already proved to be faster and more efficient than conventional diagnostics techniques, being of great interest for poultry production, animal, and public health.

Whole genome sequence analysis of the first reported isolate of Salmonella Agona carrying blaCTX-M-55 gene in Brazil.

This study analyzes the genomic findings of the first report of Salmonella isolate carrying the blaCTX-M-55 gene, recovered from a bacteremic patient from Brazil. A bacterial isolate positive for the blaCTX-M-55 gene was submitted to antimicrobial susceptibility testing by disk diffusion and epsilometric test. Whole genome sequencing was performed using Illumina technology. Conjugation assay was performed; plasmid sizes determined by S1-PFGE and plasmid content were investigated by hybrid assembly after MinION long reads sequencing. Isolate 288_18 was identified as sequence type ST13, resistant to ampicillin, cefotaxime, ceftazidime, cefepime, ceftriaxone, and aztreonam. A transferable IncFII plasmid sized approximately 67 kb was found to carry the blaTEM-1 and blaCTX-M-55 in a module consisting of IS26-blaTEM-1B-WbuC-blaCTX-M-55-IS26. In addition, an 117 kb IncI1plasmid was also identified in the 288_18 isolate, but without additional resistance genes. To the best of our knowledge, this is the first report of blaCTX-M-55 in Salmonella isolated from human infection in Brazil. The occurrence of blaCTX-M-55 in the IncFII epidemic plasmid in a relevant clinical human isolate of Salmonella Agona underscores the urgent need for enhanced and effective continuous surveillance for controlling its dissemination.

Salmonella Heidelberg and Salmonella Minnesota in Brazilian broilers: Genomic characterization of third-generation cephalosporin and fluoroquinolone-resistant strains.

Salmonella serovars Heidelberg and Minnesota encoding antimicrobial resistance to third-generation cephalosporins and fluoroquinolones are often detected in poultry/poultry meat. We analysed the genomes of 10 Salmonella Heidelberg (SH) and 4 Salmonella Minnesota (SM) from faecal isolates of Brazilian poultry. These featured virulent and multidrug-resistant characteristics, with AmpC beta-lactamase (blaCMY-2 ) predominance (9/14), for all SM (4/4) and some SH (3/10) located on IncC plasmid replicons. IncC carrying blaCTX-M-2 was only detected among SH (3/10). Mutation in the gyrA/parC genes was present in all SH, whereas SM harboured parC mutation plus qnrB19 on ColRNAI plasmids (3/4). In silico resistance overall corroborated with phenotypic results. Core genome phylogenies showed close clustering and high similarities between the Brazilian and poultry meat/food isolates from Europe, and to human isolates from European countries with documented import of Brazilian poultry meat. Conjugation assays with SM successfully transferred blaCMY-2 , and qnrB19 to an Escherichia coli recipient. The findings reinforce the ongoing antimicrobial resistance acquisition of SH and Minnesota and the risks for disseminating resistant strains and/or mobile elements which may increasingly affect importing countries and the need for controlling AMR in major poultry-exporting countries like Brazil.

Salmonella Heidelberg and Salmonella Minnesota in Brazilian broilers: Genomic characterization of third-generation cephalosporin and fluoroquinolone-resistant strains

Salmonella serovars Heidelberg and Minnesota encoding antimicrobial resistance to third-generation cephalosporins and fluoroquinolones are often detected in poultry/poultry meat. We analysed the genomes of 10 Salmonella Heidelberg (SH) and 4 Salmonella Minnesota (SM) from faecal isolates of Brazilian poultry. These featured virulent and multidrug-resistant characteristics, with AmpC beta-lactamase (blaCMY-2 ) predominance (9/14), for all SM (4/4) and some SH (3/10) located on IncC plasmid replicons. IncC carrying blaCTX-M-2 was only detected among SH (3/10). Mutation in the gyrA/parC genes was present in all SH, whereas SM harboured parC mutation plus qnrB19 on ColRNAI plasmids (3/4). In silico resistance overall corroborated with phenotypic results. Core genome phylogenies showed close clustering and high similarities between the Brazilian and poultry meat/food isolates from Europe, and to human isolates from European countries with documented import of Brazilian poultry meat. Conjugation assays with SM successfully transferred blaCMY-2 , and qnrB19 to an Escherichia coli recipient. The findings reinforce the ongoing antimicrobial resistance acquisition of SH and Minnesota and the risks for disseminating resistant strains and/or mobile elements which may increasingly affect importing countries and the need for controlling AMR in major poultry-exporting countries like Brazil.

Whole genome sequence analysis of the first reported isolate of Salmonella Agona carrying blaCTX-M-55 gene in Brazil

This study analyzes the genomic findings of the first report of Salmonella isolate carrying the blaCTX-M-55 gene, recovered from a bacteremic patient from Brazil. A bacterial isolate positive for the blaCTX-M-55 gene was submitted to antimicrobial susceptibility testing by disk diffusion and epsilometric test. Whole genome sequencing was performed using Illumina technology. Conjugation assay was performed; plasmid sizes determined by S1-PFGE and plasmid content were investigated by hybrid assembly after MinION long reads sequencing. Isolate 288_18 was identified as sequence type ST13, resistant to ampicillin, cefotaxime, ceftazidime, cefepime, ceftriaxone, and aztreonam. A transferable IncFII plasmid sized approximately 67 kb was found to carry the blaTEM-1 and blaCTX-M-55 in a module consisting of IS26-blaTEM-1B-WbuC-blaCTX-M-55-IS26. In addition, an 117 kb IncI1plasmid was also identified in the 288_18 isolate, but without additional resistance genes. To the best of our knowledge, this is the first report of blaCTX-M-55 in Salmonella isolated from human infection in Brazil. The occurrence of blaCTX-M-55 in the IncFII epidemic plasmid in a relevant clinical human isolate of Salmonella Agona underscores the urgent need for enhanced and effective continuous surveillance for controlling its dissemination.

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.

Prevalence of Extended-Spectrum ß-Lactamases CTX-M-8 and CTX-M-2-Producing Salmonella Serotypes from Clinical and Nonhuman Isolates in Brazil.

We characterized extended-spectrum ß-lactamases (ESBL) enzymes among Salmonella strains isolated in Brazil from 2009 to 2014. Salmonella recovered from both clinical and nonhuman (food, poultry, and environment) sources were subjected to antimicrobial susceptibility testing. ß-lactamases genes were detected by polymerase chain reaction/sequencing; plasmid profiles and transferability were assessed by S1-pulsed field gel electrophoresis (PFGE). Genetic diversity was evaluated by XbaI-PFGE. Out of 630 Salmonella strains screened, 46 displayed ESBL phenotype, distributed across 11 different serotypes. blaCTX-M-8 and blaCTX-M-2 genes were detected at frequencies of 47% and 41%, respectively. blaSHV-5 and blaSHV-2 were also detected but in lower frequencies (4%, 2%). blaTEM-1 gene was detected in 22% of the strains. Most of the ESBL genes were transferable by conjugation, and the respective blaESBL gene was detected in the recipient strain, indicating the location of ESBL determinants on transferable plasmids. XbaI-PFGE revealed genomic diversity of Salmonella Typhimurium bearing blaCTX-M-2, blaCTX-M-8, blaTEM-1, and blaSHV-2 genes. Salmonella Muenchen (harboring blaCTX-M-2) and Salmonella Corvallis (blaCTX-M-8 and blaSHV-5) showed clonal relatedness within respective serotypes. Our findings underscore the occurrence of diverse ESBL genes in several Salmonella serotypes, reinforcing the need for continuous surveillance of resistance genes circulating in human and nonhuman sources.

Presence of plasmid-mediated quinolone resistance determinants and mutations in gyrase and topoisomerase in Salmonella enterica isolates with resistance and reduced susceptibility to ciprofloxacin.

In recent decades, the emergence and spread of resistance to nalidixic acid are usually associated with reduced susceptibility to ciprofloxacin among Salmonella serotypes. The aims of this study were to investigate the mechanisms associated with resistance to fluoroquinolone and the clonal relatedness of Salmonella strains isolated from human and nonhuman origins, in a 5-year period in São Paulo, Brazil. Antimicrobial susceptibility testing for Salmonella isolates was performed. PCR and DNA sequencing were accomplished to identify mutations in the quinolone resistance-determining regions of the topoisomerase genes and to determine the fluoroquinolone determinants. The strains presented MIC to ciprofloxacin ranging from 0.125 to 8.0 mg/L (all nonsusceptible). From these, 16 strains (17.5%) were resistant to ciprofloxacin (MIC ≥1 mg/L) and belonging to serotypes Typhimurium, I. 4,5,12:i:-, Enteritidis, and Heidelberg. Amplification and DNA sequencing of topoisomerases genes identified multiple amino acid substitutions in GyrA and ParC. No mutations were identified in GyrB, and 1 amino acid substitution was identified in ParE. Among the 16 Salmonella strains resistant to ciprofloxacin, 8 S. I. 4,5,12:i:- presenting mutations in gyrA and parE genes were grouped into the same pulsotype. Plasmid-mediated quinolone resistance (PMQR) determinants: qnrB, aac(6')-lb-cr, and oqxA/B were detected among 13 strains. To the best of our knowledge, this is the first work to report Salmonella isolates resistant to ciprofloxacin in Brazil. Indeed, this is the first detection of PMQR determinants in Salmonella strains from Sao Paulo State. These findings alert for the potential spread of quinolone resistance of Salmonella strains, particularly in S. I. 4,5,12:i:-, a prevalent serotype implicated in human disease and foodborne outbreaks.

In vivo influence of in vitro up-regulated genes in the virulence of an APEC strain associated with swollen head syndrome.

Avian Pathogenic Escherichia coli is responsible for significant economic losses in the poultry industry by causing a range of systemic or localized diseases collectively termed colibacillosis. The virulence mechanisms of these strains that are pathogenic in poultry and possibly pathogenic in humans have not yet been fully elucidated. This work was developed to study if over-expressed genes in a microarray assay could be potentially involved in the pathogenicity of an Avian Pathogenic Escherichia coli strain isolated from a swollen head syndrome case. For this study, five over-expressed genes were selected for the construction of null mutants [flgE (flagellar hook), tyrR (transcriptional regulator), potF (putrescine transporter), yehD (putative adhesin) and bfr (bacterioferritin)]. The constructed mutants were evaluated for their capacity for the adhesion and invasion of in vitro cultured cells, their motility capacity, and their pathogenic potential in one-day-old chickens compared with the wild-type strain (WT). The Δbfr strain showed a decreased adhesion capacity on avian fibroblasts compared with WT, in the presence and absence of alpha-D-mannopyranoside, and the ΔpotF strain showed decreased adhesion only in the absence of alpha-D-mannopyranoside. The ΔtyrR mutant had a reduced ability to invade Hep-2 cells. No mutant showed changes in invading CEC-32 cells. The mutants ΔflgE and ΔtyrR showed a decreased ability to survive in HD-11 cells. The motility of the mutant strains Δbfr, ΔyehD and ΔpotF was increased, while the ΔtyrR mutant showed reduction, and the ΔflgE became non-motile. No mutant strain caused the same mortality of the WT in one-day-old chickens, showing attenuation to different degrees.

Overlapped sequence types (STs) and serogroups of avian pathogenic (APEC) and human extra-intestinal pathogenic (ExPEC) Escherichia coli isolated in Brazil.

Avian pathogenic Escherichia coli (APEC) strains belong to a category that is associated with colibacillosis, a serious illness in the poultry industry worldwide. Additionally, some APEC groups have recently been described as potential zoonotic agents. In this work, we compared APEC strains with extraintestinal pathogenic E. coli (ExPEC) strains isolated from clinical cases of humans with extra-intestinal diseases such as urinary tract infections (UTI) and bacteremia. PCR results showed that genes usually found in the ColV plasmid (tsh, iucA, iss, and hlyF) were associated with APEC strains while fyuA, irp-2, fepC sitDchrom, fimH, crl, csgA, afa, iha, sat, hlyA, hra, cnf1, kpsMTII, clpVSakai and malX were associated with human ExPEC. Both categories shared nine serogroups (O2, O6, O7, O8, O11, O19, O25, O73 and O153) and seven sequence types (ST10, ST88, ST93, ST117, ST131, ST155, ST359, ST648 and ST1011). Interestingly, ST95, which is associated with the zoonotic potential of APEC and is spread in avian E. coli of North America and Europe, was not detected among 76 APEC strains. When the strains were clustered based on the presence of virulence genes, most ExPEC strains (71.7%) were contained in one cluster while most APEC strains (63.2%) segregated to another. In general, the strains showed distinct genetic and fingerprint patterns, but avian and human strains of ST359, or ST23 clonal complex (CC), presented more than 70% of similarity by PFGE. The results demonstrate that some "zoonotic-related" STs (ST117, ST131, ST10CC, ST23CC) are present in Brazil. Also, the presence of moderate fingerprint similarities between ST359 E. coli of avian and human origin indicates that strains of this ST are candidates for having zoonotic potential.