High levels of multidrug-resistant isolates of genetically similar Salmonella 1,4, [5],12:I:- from Brazil between 1983 and 2020.

Introduction. Salmonella 1,4, [5],12:i:- strains with different antimicrobial resistance profiles have been associated with foodborne disease outbreaks in several countries. In Brazil, S. 1,4, [5],12:i:- was identified as one of the most prevalent serovars in São Paulo State during 2004-2020.Gap Statement. However, few studies have characterized this serovar in Brazil.Aim. This study aimed to determine the antimicrobial resistance profiles of S. 1,4, [5],12:i:- strains isolated from different sources in Southeast Brazil and compare their genetic diversity.Methodology. We analysed 113 S. 1,4, [5],12:i:- strains isolated from humans (n=99), animals (n=7), food (n=5) and the environment (n=2) between 1983 and 2020. Susceptibility testing against 13 antimicrobials was performed using the disc diffusion method for all the strains. Plasmid resistance genes and mutations in the quinolone resistance-determining regions were identified in phenotypically fluoroquinolone-resistant strains. Molecular typing was performed using enterobacterial repetitive intergenic consensus PCR (ERIC-PCR) for all strains and multilocus sequence typing (MLST) for 40 selected strains.Results. Of the 113 strains, 54.87 % were resistant to at least one antimicrobial. The highest resistance rates were observed against ampicillin (51.33 %), nalidixic acid (39.82 %) and tetracycline (38.05 %). Additionally, 39 (34.51 %) strains were classified as multidrug-resistant (MDR). Nine fluoroquinolone-resistant strains exhibited the gyrA mutation (Ser96→Tyr96) and contained the qnrB gene. The 113 strains were grouped into two clusters using ERIC-PCR, and most of strains were present in one cluster, with a genetic similarity of ≥80 %. Finally, 40 strains were typed as ST19 using MLST.Conclusion. The prevalence of MDR strains is alarming because antimicrobial treatment against these strains may lead to therapeutic failure. Furthermore, the ERIC-PCR and MLST results suggested that most strains belonged to one main cluster. Thus, a prevalent subtype of Salmonella 1,4, [5],12:i:- strains has probably been circulating among different sources in São Paulo, Brazil, over decades.

Virulence potential of Salmonella 1,4, [5],12:i:- strains isolated during decades from different sources in the Southeast region of Brazil.

Salmonella 1,4, [5],12:i:- is one of the most prevalent serovars associated with gastroenteritis in several countries, including Brazil. However, few studies have analyzed the virulence potential of this variant in this country. Therefore, this study aimed to characterize S. 1,4, [5],12:i:- strains isolated in Southeast Brazil. To this end, 113 S. 1,4, [5],12:i:- strains isolated from different sources between 1983 and 2020 were analyzed. For all strains, the frequencies of 11 virulence genes were investigated using PCR and the molecular typing was performed using pulsed-field gel electrophoresis (PFGE). Furthermore, 40 strains isolated from human and non-human sources were characterized by survival under acid and oxidative stress, and virulence analysis in Galleria mellonella was performed for 20 selected strains. All virulence genes were detected in more than 91% of the strains. The studied strains were grouped into four clusters using PFGE. Most strains were present in one cluster, named PFGE-A, with a genetic similarity of ≥ 79.5%. All 40 strains survived acid stress after 10 min and 1 h of exposure. Under oxidative stress, all 40 strains survived after 10 min, and 36 survived after 1 h of exposure. In the G. mellonella assay, nine isolates from non-human sources and six isolates from human showed high-to-intermediate virulence profiles. In conclusion, the pathogenic potential of the strains studied was corroborated by the high frequency of all the virulence genes identified. The PFGE results suggested that most strains belonged to one main cluster that has been prevailing in the São Paulo State, Brazil. The S. 1,4, [5],12:i:- strains isolated from human and non-human sources successfully survived the unfavorable conditions in the human gastrointestinal tract. Finally, strains isolated from non-human sources showed a higher proportion of isolates with high to intermediate virulence profiles in G. mellonella than in human isolates, suggesting a possible difference between isolates from different origins.

Antimicrobial Resistance Profiles and Phylogenetic Analysis of Campylobacter jejuni Strains Isolated in Brazil by Whole Genome Sequencing.

Aims: The objectives of this work were to use whole genome sequencing (WGS) to determine the antimicrobial resistance genotypes of 116 Campylobacter jejuni strains isolated in Brazil and to compare it with the results obtained by antimicrobial susceptibility testing (AST). In addition, WGS was used to uncover the phylogenetic relationship among those strains. Results: By AST, the C. jejuni strains resistant to ciprofloxacin, tetracycline, doxycycline, and erythromycin were 51 (44%), 41 (35.3%), 41 (35.3%), and 6 (5.2%), respectively. By WGS, the genes aph(3')III, aadE, blaOXA-449, blaOXA-184, blaOXA-61, and tet(O) were detected in 6 (5.2%), 3 (2.6%), 1 (0.9%), 10 (8.6%), 55 (47.4%), and 44 (38%) strains, respectively. Fifty-four (46.6%) strains showed the mutation T86I in the gyrA gene, and four (3.4%) strains presented the mutation A2075G in the 23S rRNA gene. The correlation between AST and WGS was 100% for ciprofloxacin, 97.5% for tetracyclines, and 66.7% for erythromycin. The whole genome single nucleotide polymorphism (SNP) tree clustered the C. jejuni strains into two clades comprising strains that were highly related from different sources, places, and years. Conclusion: The high rates of C. jejuni strains resistant to ciprofloxacin and tetracyclines are of concern and may represent a public health problem. WGS has a potential to be a powerful tool for the prediction of resistance of antibiotics used to treat campylobacteriosis. The results obtained by whole genome SNP analysis suggested the potential for transmission between clinical and nonclinical sources and between human and animal sources over the course of 20 years in Brazil.

Correction: Phenotypic and genotypic characterization of Salmonella Typhimurium isolates from humans and foods in Brazil.

[This corrects the article DOI: 10.1371/journal.pone.0237886.].

Phenotypic and genotypic characterization of Salmonella Typhimurium isolates from humans and foods in Brazil.

Salmonella enterica subsp. enterica serovar Typhimurium (S. Typhimurium) causes gastroenteritis in many countries. However, in Brazil there are few studies that have conducted a virulence characterization of this serovar. The aim of this study was to evaluate the virulence potential of S. Typhimurium strains isolated in Brazil. Forty S. Typhimurium strains isolated from humans (n = 20) and food (n = 20) from Brazil were studied regarding their invasion and survival in human epithelial cells (Caco-2) and macrophages (U937). Their virulence potential was determined using the Galleria mellonella larvae model combined with the analysis of virulence genes by whole genome sequencing (WGS). A total of 67.5% of the S. Typhimurium studied (32.5% isolated from humans and 35% isolated from food) invaded Caco-2 epithelial cells at levels similar to or greater than the S. Typhimurium SL1344 prototype strain. In addition, 37.5% of the studied strains (25% isolated from humans and 12.5% isolated from food) survived in U937 human macrophages at levels similar to or greater than SL1344. S. Typhimurium strains isolated from humans (40%) and food (25%) showed high or intermediate virulence in G. mellonella larvae after seven days exposure. Approximately, 153 virulence genes of chromosomal and plasmidial origin were detected in the strains studied. In conclusion, the ability of the S. Typhimurium to invade Caco-2 epithelial cells was strain dependent and was not related to the source or the year of isolation. However, S. Typhimurium strains isolated from humans showed greater survival rates in U937 human macrophages, and presented higher proportion of isolates with a virulent profile in G. mellonella in comparison to strains isolated from food suggesting that this difference may be related to the higher frequency of human isolates which contained plasmid genes, such as spvABCDR operon, pefABCD operon, rck and mig-5.

Molecular typing of Campylobacter jejuni strains: comparison among four different techniques.

This study compared the ability of pulsed-field gel electrophoresis (PFGE), flaA small variable region (SVR) sequencing, analysis of the clustered regularly interspaced short palindromic repeats locus by high resolution melting analysis (CRISPR-HRMA), and multilocus sequence typing (MLST) for typing 111 Campylobacter jejuni strains isolated from diverse sources during 20 years in Brazil. For this, we used previous results obtained by PFGE and flaA-SVR sequencing from our research group and performed CRISPR-HRMA and MLST typing for the first time. Furthermore, the discrimination index (DI) of each method was accessed. The DI for PFGE, flaA-SVR sequencing, CRISPR-HRMA, and MLST was 0.980, 0.932, 0.868, and 0.931, respectively. By PFGE and flaA-SVR sequencing, some strains from clinical and non-clinical sources and from humans and animals presented ≥ 80% similarity. Similarly, some strains from different origins presented the same ST and CRISPR-HRMA types. In conclusion, despite the different DI values, all assays provided the same epidemiological information suggesting that a potential transmission may have occurred between C. jejuni from clinical and non-clinical sources and from animals and humans in Brazil. Furthermore it was demonstrated the suitability of PFGE that should be used preferably together with MLST and/or flaA-SVR sequencing for typing C. jejuni strains.

Phylogenetic and antimicrobial resistance gene analysis of Salmonella Typhimurium strains isolated in Brazil by whole genome sequencing.

Whole genome sequencing (WGS) has been used as a powerful technology for molecular epidemiology, surveillance, identification of species and serotype, identification of the sources of outbreaks, among other purposes. In Brazil, there is relatively few epidemiological data on Salmonella. In this study, 90 Salmonella Typhimurium strains had their genome sequenced to uncover the diversity of Salmonella Typhimurium isolated from humans and food, between 1983 and 2013, from different geographic regions in Brazil based on single nucleotide polymorphism (SNP) analysis. A total of 39 resistance genes were identified, such as aminoglycoside, tetracycline, sulfonamide, trimethoprim, beta-lactam, fluoroquinolone, phenicol and macrolide, as well as the occurrence of point mutations in some of the genes such as gyrA, gyrB, parC and parE. A total of 65 (72.2%) out of 90 S. Typhimurium strains studied were phenotypically resistant to sulfonamides, 44 (48.9%) strains were streptomycin resistant, 27 (30%) strains were resistant to tetracycline, 21 (23.3%) strains were gentamicin resistant, and seven (7.8%) strains were resistant to ceftriaxone. In the gyrA gene, it was observed the following amino acid substitutions: Asp(87)→Gly, Asp(87)→Asn, Ser(83)→Phe, Ser(83)→Tyr. Phylogenetic results placed the 90 S. Typhimurium strains into two major clades suggesting the existence of a prevalent subtype, likely more adapted, among strains isolated from humans, with some diversity in subtypes in foods. The variety and prevalence of resistant genes found in these Salmonella Typhimurium strains reinforces their potential hazard for humans and the risk in foods in Brazil.

Draft Genome Sequences of 116 Campylobacter jejuni Strains Isolated from Humans, Animals, Food, and the Environment in Brazil.

Campylobacter jejuni is a major zoonotic pathogen that causes foodborne gastroenteritis worldwide. However, clinical cases of campylobacteriosis have been underreported and underdiagnosed in Brazil. Herein, we describe the draft genome sequences of 116 C. jejuni strains isolated from diverse sources in Brazil.

Distribution of Streptococcus pneumoniae serotypes in the northeast macro-region of São Paulo state/Brazil after the introduction of conjugate vaccine.

BACKGROUND: Infections caused by Streptococcus pneumoniae (Spn) still challenge health systems around the world, even with advances in vaccination programs. The present study evaluated the frequency of various Spn serotypes isolated in Regional Health Care Network 13 (RRAS 13), which includes the regional health departments (RHDs) of Araraquara, Barretos, Franca and Ribeirão Preto, especially after the introduction of 10-valent pneumococcal conjugate vaccine (PCV10) in 2010. METHODS: The analyzed Spn strains were isolated from patients with invasive pneumococcal diseases (IPDs) and then sent to Adolfo Lutz Institute (ALI) for further confirmative identification tests during the period from 1998 to 2013. The samples were from the cities in RRAS13, which is located in the Northeast region of São Paulo State, and totals 90 municipalities. RESULTS: We analyzed strains isolated from 796 patients. They were predominantly: men (58.9%); 20 to 60 years old (32.2%); evaluated from 2003 to 2010 (60.2%); and diagnosed with meningitis (45.7%) and pneumonia (45.0%), the most common invasive pneumococcal diseases. In 2010, serotypes 3, 19F, 1, 23F, 6A and 6B were among the most frequent, while serotypes 3, 12F, 14, 6A, 18C, 8 and 6B were more common after the introduction of PCV10. Serotypes 14, 19F and 3 were more frequent in meningitis, while serotypes 14, 3 and 1 prevailed in pneumonia. After 2010, there was a decrease in serotypes 14, 1, 23F and 5 and an increase in serotypes 3, 12F, 11A and 8, which were not present in the vaccine. CONCLUSIONS: The present study noted the increase in serotypes 3, 12F, 11A and 8 after vaccination. None of those serotypes are included in the available conjugate vaccines, which highlights the importance of continued monitoring of IPDs in order to measure the disease burden in the population in the long term and provide new epidemiological information to determine the impact of PCV10 in Brazil.

Pathogenic potential and genotypic diversity of Campylobacter jejuni: a neglected food-borne pathogen in Brazil.

Purpose and methodology.Campylobacter jejuni is a major zoonotic pathogen that causes food-borne gastroenteritis worldwide. However, there are only a few studies available that have molecularly characterized C. jejuni strains isolated in Brazil. The aim of this study was to genotype 111 C. jejuni strains isolated from sick humans (43), monkey faeces (19), chicken faeces (14), chicken meat (33) and sewage (2) between 1996 and 2016 in Brazil using flaA-SVR (short variable region) sequencing and PFGE. Furthermore, the presence of 16 virulence genes was analysed by PCR. RESULTS: Using PFGE and flaA-SVR sequencing, the 111 C. jejuni strains studied were grouped into three and two clusters, respectively, and some strains of different origin presented a similarity of ≥80 %. In total, 35 flaA-SVR alleles were detected. Alleles gt45, gt49 and gt57 were the most prevalent, in contrast with those frequently described in the PubMLST database. All 111 C. jejuni strains contained the genes flaA, flhA, cadF, docA, cdtA, cdtB, cdtC, iamA, ciaB, sodB, dnaJ, pldA, racR and csrA. The wlaN gene was detected in 11 strains (9.9 %), and the virB11 in just one strain (0.9 %). CONCLUSIONS: In conclusion, the pathogenic potential of the C. jejuni strains studied was highlighted by the high frequency of the majority of the virulence genes searched. The flaA-SVR sequencing and PFGE results showed that some of the strains studied presented a high genotypic similarity, suggesting potential for transmission between animal sources and humans in this country. Altogether, the results characterize further C. jejuni isolates from Brazil, an important producer and exporter of chicken meat.

Molecular characterization of Salmonella Typhimurium isolated in Brazil by CRISPR-MVLST.

CRISPR-multi-locus virulence sequence typing (CRISPR-MVLST) was performed to type 92 S. Typhimurium strains isolated from humans and food sources between 1983 and 2013 in Brazil and assess the suitability of this methodology comparing it with PFGE already used for subtyping the same strains. Among the 92 S. Typhimurium strains studied, we identified 25 CRISPR1 alleles, 27 CRISPR2 alleles, 2 fimH alleles and 3 sseL alleles showing that the genetic variability is much higher in the CRISPRs loci than in the virulence genes. The CRISPR-MVLST analysis provided similar results to the PFGE previously published used to type the same set of strains, demonstrating that CRISPR-MVLST is a very efficient approach for subtyping S. Typhimurium serovar and can be used to complement and validate results obtained by the PFGE methodology.

Draft Genome Sequences of 40 Salmonella enterica Serovar Typhimurium Strains Isolated from Humans and Food in Brazil.

Salmonellosis is an important health problem worldwide and Salmonella enterica serovar Typhimurium is one of the most common isolated serovars. Here, we reported the draft genomes of 40 S Typhimurium strains isolated from humans and food in Brazil. These draft genomes will improve phylogenetic analysis and will help enhance our understanding of strains of this serovar isolated in Brazil.

Clinical and microbiological implications of invasive pneumococcal disease in hospitalized patients (1998-2013)

Abstract Introduction Infections caused by Streptococcus pneumoniae (pneumococcus) still represent a challenge for health systems around the world. Objective The objective of this study was to assess microbiological and clinical aspects in hospitalized patients with invasive pneumococcus disease between 1998 and 2013. Materials and methods This was a retrospective study that analyzed the results of pneumococcus identification, serotyping, and susceptibility testing found in the Adolfo Lutz Institute databank. Personal variables, medical history and clinical outcome of patients admitted with invasive pneumococcal disease were analyzed. These were obtained from records of a public teaching hospital – Hospital das Clínicas Faculdade de Medicina Ribeirão Preto. Results The sample comprised 332 patients. Patient age ranged from less than one month to 89 years old (mean 20.3 years) and the sample was predominately male. Pneumonia (67.8%) was the most common disease, accounting for 18.2% of deaths. Serotypes 14, 1, 3, 9V, 6B, 6A, 23F, 19A, 18C, 19F, 12F, and 4 were the most common (75.3%). Most patients, or 67.5%, were cured without any complication (success), 6.9% had some type of sequela (failure), and 25.6% died (failure). In the case of deaths due to meningitis, strains of fully penicillin resistant pneumococcus were isolated. Furthermore, 68.2% of patients who died presented some type of comorbidity. The 60 and older age group presented the most significant association (Odds Ratio = 4.2), with outcome failure regardless of the presence of comorbidity. Serotype 18C was the most significant risk factor both in raw analysis (Odds Ratio = 3.8) and when adjusted for comorbidity (Odds Ratio = 5.0) or age (Odds Ratio = 5.4). The same occurred with serotype 12F (respectively, Odds Ratio = 5.1, Odds Ratio = 5.0, and Odds Ratio = 4.7) Conclusion The present findings highlight the importance of IPD among young adults and older adults. In the era of conjugate vaccines, monitoring serotypes in different age groups is essential to assess the impact and adequacy of immunization.

Clinical and microbiological implications of invasive pneumococcal disease in hospitalized patients (1998-2013).

INTRODUCTION: Infections caused by Streptococcus pneumoniae (pneumococcus) still represent a challenge for health systems around the world. OBJECTIVE: The objective of this study was to assess microbiological and clinical aspects in hospitalized patients with invasive pneumococcus disease between 1998 and 2013. MATERIALS AND METHODS: This was a retrospective study that analyzed the results of pneumococcus identification, serotyping, and susceptibility testing found in the Adolfo Lutz Institute databank. Personal variables, medical history and clinical outcome of patients admitted with invasive pneumococcal disease were analyzed. These were obtained from records of a public teaching hospital - Hospital das Clínicas Faculdade de Medicina Ribeirão Preto. RESULTS: The sample comprised 332 patients. Patient age ranged from less than one month to 89 years old (mean 20.3 years) and the sample was predominately male. Pneumonia (67.8%) was the most common disease, accounting for 18.2% of deaths. Serotypes 14, 1, 3, 9V, 6B, 6A, 23F, 19A, 18C, 19F, 12F, and 4 were the most common (75.3%). Most patients, or 67.5%, were cured without any complication (success), 6.9% had some type of sequela (failure), and 25.6% died (failure). In the case of deaths due to meningitis, strains of fully penicillin resistant pneumococcus were isolated. Furthermore, 68.2% of patients who died presented some type of comorbidity. The 60 and older age group presented the most significant association (Odds Ratio=4.2), with outcome failure regardless of the presence of comorbidity. Serotype 18C was the most significant risk factor both in raw analysis (Odds Ratio=3.8) and when adjusted for comorbidity (Odds Ratio=5.0) or age (Odds Ratio=5.4). The same occurred with serotype 12F (respectively, Odds Ratio=5.1, Odds Ratio=5.0, and Odds Ratio=4.7) CONCLUSION: The present findings highlight the importance of IPD among young adults and older adults. In the era of conjugate vaccines, monitoring serotypes in different age groups is essential to assess the impact and adequacy of immunization.

Isolamento de Campylobacter sp na região nordeste do Estado de São Paulo – Brasil / Isolation of Campylobacter sp in northeast region of São Paulo State – Brazil

As espécies de Campylobacter podem ser patogênicas ou comensais do trato gastrointestinal de animais domésticos e silvestres, sendo dispersas no ambiente contaminando água, pastos e plantações. São isoladas a partir do meio ambiente e animais, especialmente mamíferos, pássaros e aves domésticas. As espécies termófilas, principalmente C. jejuni e C. coli são reconhecidas mundialmente como agentes etiológicos de diarreia aguda em seres humanos, bem como infecção extraintestinal. Este estudo teve como objetivo avaliar a ocorrência de campilobacteriose nas análises realizadas pelo Centro de Laboratório Regional de Ribeirão Preto VI, Instituto Adolfo Lutz (CLR-RP-VI/IAL), provenientes da Rede Regional de Assistência à Saúde (RRAS-13), São Paulo - Brasil, durante o período de 1987 a 2013. Realizou-se o isolamento e a identificação de Campylobacter sp de acordo com metodologia tradicional. Avaliaram-se 2.534 amostras de fezes das quais 279 (11,0%) culturas foram positivas para Campylobacter sp: 63,1% C. jejuni; 3,9% C. coli; 0,4% C. laridis; 32,6% Campylobacter spp. Em 44 (15,8%) amostras ocorreu associação de Campylobacter sp com outras bactérias patogênicas. Isolou-se três casos em infeccções extraintestinais. É necessária vigilância constante para reduzir a ocorrência de campilobacterioses na região estudada, especialmente C. jejuni...

Genotypic diversity, pathogenic potential and the resistance profile of Salmonella Typhimurium strains isolated from humans and food from 1983 to 2013 in Brazil.

Salmonella enterica subsp. enterica serovar Typhimurium is one of the leading serovars that causes salmonellosis worldwide. However, few studies have molecularly characterized S. Typhimurium strains in Brazil. In this study, we genotyped 92 S. Typhimurium strains isolated from humans (43) and food (49) between 1983 and 2013 in Brazil using PFGE, multiple-locus variable number of tandem repeats analysis (MLVA) and enterobacterial repetitive intergenic consensus PCR (ERIC-PCR). Moreover, we assessed the frequency of 12 virulence markers by PCR and the resistance profile against 12 antimicrobials. More than 85.8% of the strains studied carried 11 of the virulence markers or more. Thirty-three strains (25%) were multidrug resistant (MDR). The 92 S. Typhimurium studied were grouped by PFGE as PFGE-A, PFGE-B1 and PFGE-B2; by MLVA as MLVA-A, MLVA-B1 and MLVA-B2; and, finally, by ERIC-PCR as ERIC-A and ERIC-B. The strains isolated from humans before the mid-1990s were allocated to all clusters. The strains isolated from humans after the mid-1990s were distributed in the PFGE-B1, MLVA-B1, MLVA-B2 and ERIC-A clusters. The strains isolated from food were distributed in all clusters, except in PFGE-B2. All typing results suggested that the S. Typhimurium strains of human clinical origin isolated before the mid-1990s were genetically more diverse, which might indicate the selection of a more adapted S. Typhimurium subtype after Salmonella Enteritidis became the most prevalent serovar in Brazil. Regarding strains isolated from food, the results suggest the current circulation of more than one subtype. Furthermore, the high frequency of virulence genes and the presence of MDR strains reinforces their potential hazard for humans and the risk of their presence in foods in Brazil.

Phenotypic and genotypic characterization of Rhodococcus equi isolated from sputum

INTRODUCTION: Rhodococcus equi is an opportunistic pathogen, causing rhodococcosis, a condition that can be confused with tuberculosis. Often, without identifying M. tuberculosis, physicians initiate empiric treatment for tuberculosis. R. equi and M. tuberculosis have different susceptibility to drugs. Identification of R. equi is based on a variety of phenotypic, chromatographic, and genotypic characteristics. OBJECTIVE: This study aimed to characterize bacterial isolates from sputum samples suggestive of R. equi. METHODS: The phenotypic identification included biochemical assays; thin-layer chromatography (TLC) and polymerase chain reaction (PCR) were used for genotypic identification. RESULTS: Among 78 Gram-positive and partially acid-fast bacilli isolated from the sputum of tuberculosis-suspected patients, 51 were phenotypically and genotypically characterized as R. equi based on literature data. Mycolic acid analysis showed that all suspected R. equi had compounds with a retention factor (Rf) between 0.4-0.5. Genotypic characterization indicated the presence of the choE gene 959 bp fragments in 51 isolates CAMP test positive. Twenty-two CAMP test negative isolates were negative for the choE gene. Five isolates presumptively identified as R. equi, CAMP test positive, were choE gene negative, and probably belonged to other bacterial species. CONCLUSIONS: The phenotypic and molecular techniques used constitute a good methodological tool to identify R. equi.