Comparative genomics reveals high genetic similarity among strains of Salmonella enterica serovar Infantis isolated from multiple sources in Brazil.

Background: Salmonella enterica serovar Infantis (Salmonella Infantis) is a zoonotic, ubiquitous and foodborne pathogen of worldwide distribution. Despite Brazil's relevance as a major meat exporter, few studies were conducted to characterize strains of this serovar by genomic analyses in this country. Therefore, this study aimed to assess the diversity of 80 Salmonella Infantis strains isolated from veterinary, food and human sources in Brazil between 2013 and 2018 by comparative genomic analyses. Additional genomes of non-Brazilian countries (n = 18) were included for comparison purposes in some analyses. Methods: Analyses of whole-genome multi-locus sequence typing (wgMLST), using PGAdb-builder, and of fragmented genomes, using Gegenees, were conducted to compare the 80 Brazilian strains to the 18 non-Brazilian genomes. Pangenome analyses and calculations were performed for all Salmonella Infantis genomes analyzed. The presence of prophages was determined using PHASTER for the 80 Brazilian strains. The genome plasticity using BLAST Ring Image Generator (BRIG) and gene synteny using Mauve were evaluated for 20 selected Salmonella Infantis genomes from Brazil and ten from non-Brazilian countries. Unique orthologous protein clusters were searched in ten selected Salmonella Infantis genomes from Brazil and ten from non-Brazilian countries. Results: wgMLST and Gegenees showed a high genomic similarity among some Brazilian Salmonella Infantis genomes, and also the correlation of some clusters with non-Brazilian genomes. Gegenees also showed an overall similarity >91% among all Salmonella Infantis genomes. Pangenome calculations revealed an open pangenome for all Salmonella Infantis subsets analyzed and a high gene content in the core genomes. Fifteen types of prophages were detected among 97.5% of the Brazilian strains. BRIG and Mauve demonstrated a high structural similarity among the Brazilian and non-Brazilian isolates. Unique orthologous protein clusters related to biological processes, molecular functions, and cellular components were detected among Brazilian and non-Brazilian genomes. Conclusion: The results presented using different genomic approaches emphasized the significant genomic similarity among Brazilian Salmonella Infantis genomes analyzed, suggesting wide distribution of closely related genotypes among diverse sources in Brazil. The data generated contributed to novel information regarding the genomic diversity of Brazilian and non-Brazilian Salmonella Infantis in comparison. The different genetically related subtypes of Salmonella Infantis from Brazil can either occur exclusively within the country, or also in other countries, suggesting that some exportation of the Brazilian genotypes may have already occurred.

The impact of COVID-19 on antimicrobial prescription and drug resistance in fungi and bacteria.

Secondary infections are one of the complications in COVID-19 patients. We aimed to analyze the antimicrobial prescriptions and their influence on drug resistance in fungi and bacteria isolated from severely ill COVID-19 patients. Seventy-nine severely ill COVID-19 hospitalized patients with secondary bacterial or fungal infections were included. We analyzed the prescribed antimicrobial regimen for these patients and the resistance profiles of bacterial and fungal isolates. In addition, the association between drug resistance and patients' outcome was analyzed using correlation tests. The most prescribed antibacterial were ceftriaxone (90.7% of patients), vancomycin (86.0%), polymyxin B (74.4%), azithromycin (69.8%), and meropenem (67.4%). Micafungin and fluconazole were used by 22.2 and 11.1% of patients, respectively. Multidrug-resistant (MDR) infections were a common complication in severely ill COVID-19 patients in our cohort since resistant bacteria strains were isolated from 76.7% of the patients. Oxacillin resistance was observed in most Gram-positive bacteria, whereas carbapenem and cephalosporin resistance was detected in most Gram-negative strains. Azole resistance was identified among C. glabrata and C. tropicalis isolates. Patients who used more antimicrobials stayed hospitalized longer than the others. The patient's age and the number of antibacterial agents used were associated with the resistance phenotype. The susceptibility profile of isolates obtained from severely ill COVID-19 patients highlighted the importance of taking microbial resistance into account when managing these patients. The continuous surveillance of resistant/MDR infection and the rational use of antimicrobials are of utmost importance, especially for long-term hospitalized patients with COVID-19.

Campylobacter coli strains from Brazil can invade phagocytic and epithelial cells and induce IL-8 secretion.

Campylobacter spp. have been a predominant cause of bacterial foodborne gastroenteritis worldwide, causing substantial costs to public healthcare systems. This study aimed to assess the invasion and pro-inflammatory cytokine production capacity of Campylobacter coli strains isolated in Brazil. A total of 50 C. coli isolated from different sources in Brazil were analyzed for their capacity of invasion in Caco-2 and U-937 cell lines. The production of pro-inflammatory cytokines was quantitatively measured in response to C. coli. All the strains studied showed invasion percentage ≥ 40% in polarized Caco-2 cells. In U-937 cells assay, 35 of 50 C. coli strains studied showed invasion percentage ≥ 50%. A significant increase in IL-8 production by infected U-937 cells was observed for 17.5% of the C. coli isolates. The high percentages of invasion in Caco-2 and U-937 cells observed for all studied strains, plus the increased production of IL-8 by U-937 cells against some strains, highlighted the pathogenic potential of the C. coli studied and bring extremely relevant data since it has never been reported for strains isolated in Brazil and there are a few data for C. coli in the literature.

Partial Correlation Between Phenotypic and Genotypic Antimicrobial Resistance of Salmonella enterica Serovar Enteritidis Strains from Brazil.

Whole-genome sequencing analyses have provided important data and information on the repertoire of resistance genes in several bacterial species. This study examined to what extent the antimicrobial resistance genes found in a set of whole-genome-sequenced Salmonella Enteritidis strains from Brazil correlated with the phenotypic antimicrobial resistance possibly related to these genes. The genotypic resistance data from the strains studied were compared with publicly available data from strains isolated in other countries. The genotypic resistance profiles were accessed on the NCBI Pathogen Detection website, and the phenotypic resistance profiles were determined by the disk diffusion technique according to the Clinical and Laboratory Standards Institute guidelines. Fourteen of the 256 sequenced strains presented antimicrobial resistance genes, with the highest prevalence of resistance genes to aminoglycosides-with 16 genes detected in seven strains-not only in Brazilian strains but also in the strains from other parts of the world. The strongest correlation between phenotypic and genotypic resistance was found for tetracycline (75%). The genotypic and phenotypic profiles of the S. Enteritidis strains studied only partially matched, and they diverged in some antimicrobial classes more strongly than in other classes. The advances on whole-genome sequencing analyses associated with a better understanding of the correlation between phenotypic and genotypic resistance data may improve this powerful tool for antimicrobial resistance prediction.

Molecular Epidemiology and Resistance Profile of Campylobacter jejuni and Campylobacter coli Strains Isolated from Different Sources in Brazil.

Aims: The objectives of this study were to genotype a total of 48 Campylobacter jejuni and 39 Campylobacter coli strains isolated in Brazil from 1995 to 2016 by multilocus sequence typing (MLST) and to determine their resistance profile. The presence or points of mutation in the related resistance genes was verified. Results: By MLST, C. jejuni strains were typed into 36 STs and C. coli strains were typed into 27 STs. A total of 70.8% of C. jejuni and 35.9% of C. coli were resistant to at least one antimicrobial tested. The tet(O) gene was detected in 43.7% C. jejuni and in 12.8% C. coli. The ermB gene was not detected and one C. jejuni presented the mutation in the 23S rRNA gene. Besides, 58.3% C. jejuni presented the substitution T86I in the quinolone resistance-determining region of gyrA and 15.4% C. coli presented the substitution T38I. The cmeB gene was detected in 97.9% C. jejuni and in 97.4% C. coli. Conclusion: The presence of C. jejuni and C. coli resistant to some antimicrobial agents of clinical use is of public health concern. The presence of STs shared between Brazilian strains and isolates of different countries is of concern since it might suggest a possible spread of these shared types.

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.

Heart failure developed after myocardial infarction does not affect gut microbiota composition in the rat.

There is a body of evidence that supports the notion that gut dysbiosis plays a role in the pathogenesis of cardiovascular diseases. Decreased cardiac function can reduce intestinal perfusion, resulting in morphological alterations, which may contribute to changes in the gut microbiota composition in patients with heart failure (HF). In this regard, a germane question is whether changes in gut microbiota composition are a cause or consequence of the cardiovascular disturbance. We tested the hypothesis that the development of HF, after myocardial infarction, would cause gut dysbiosis. Fecal samples were collected before and 6 wk after myocardial infarction or sham surgery. Gut microbiota were characterized by sequencing the bacterial 16S ribosomal DNA. The composition of bacterial communities in the fecal samples was evaluated by calculating three major ecological parameters: 1) the Chao 1 richness, 2) the Pielou evenness, and 3) the Shannon index. None of these indices was changed in either sham or HF rats. The Firmicutes/Bacteroidetes ratio was not altered in HF rats. The number of species in each phylum was also not different between sham and HF rats. ß-Diversity analysis showed that the composition of gut microbiota was not changed with the development of HF. Bacterial genera were grouped according to their major metabolic end-products (acetate, butyrate, and lactate), but no differences were observed in HF rats. Therefore, we conclude that HF induced by myocardial infarction does not affect gut microbiota composition, at least in rats, indicating that the dysbiosis observed in patients with HF may precede cardiovascular disturbance.NEW & NOTEWORTHY Our study demonstrated that, following myocardial infarction in rats, heart failure (HF) development did not affect the intestinal microbiota despite distinct differences reported in the gut microbiota of humans with HF. Our finding is consistent with the notion that dysbiosis observed in patients with HF may precede cardiovascular dysfunction and therefore offers potential for early diagnosis and treatment.

High survival rates of Campylobacter coli under different stress conditions suggest that more rigorous food control measures might be needed in Brazil.

Campylobacter spp. have been the most commonly reported gastrointestinal bacterial pathogen in many countries. Consumption of improperly prepared poultry meat has been the main transmission route of Campylobacter spp. Although Brazil is the largest exporter of poultry meat in the world, campylobacteriosis has been a neglected disease in the country. The aim of this study was to characterize 50 Campylobacter coli strains isolated from different sources in Brazil regarding the frequency of 16 virulence genes and their survival capability under five different stress conditions. All strains studied presented the cadF, flaA, and sodB genes that are considered essential for colonization. All strains grew at 4 °C and 37 °C after 24 h. High survival rates were observed when the strains were incubated in BHI with 7.5% NaCl and exposed to acid and oxidative stress. In conclusion, the pathogenic potential of the strains studied was reinforced by the presence of several important virulence genes and by the high growth and survival rates of the majority of those strains under different stress conditions. The results enabled a better understanding of strains circulating in Brazil and suggest that more rigorous control measures may be needed, given the importance of contaminated food as vehicles for Campylobacter coli.

Molecular typing and occurrence of beta-lactam resistance genes of Shigella sonnei strains isolated from 1983 to 2014 in the São Paulo state of Brazil.

Shigella sonnei, which has generally been associated with dysentery in developed countries, has recently been emerging in developing countries. Specifically, in Brazil few published studies have that molecularly characterized this species. The aims of this study were to analyze the efficacy of typing using multiple-locus variable-number tandem-repeat analysis (MLVA), study the phylogeny by multi-locus sequence typing (MLST) and assess the presence of some beta-lactam resistance genes in S. sonnei strains isolated from human diarrhoeic faeces in the São Paulo State in Brazil between 1983 and 2014. Seventy-two such S. sonnei strains were typed by MLVA and grouped into two clusters. The discrimination index of MLVA was found to be 0.996. Twenty strains were typed by MLST as ST152. In addition, the blaTEM gene was detected in eight (72.7%) of the 11 S. sonnei strains that had previously been shown to be resistant to ß-lactams. However, blaCTX-M-1group , blaCTX-M-9group and blaSHV genes were not found. MLVA results suggested the existence of two prevalent subtypes in the S. sonnei strains studied, confirming previous results. Moreover, MLVA efficiently discriminated monomorphic S. sonnei species. Because the S. sonnei strains studied belonged to clonal complex 152 and all isolates were typed as ST152, MLST is not a suitable method for studying the population structure of S. sonnei. Although, the rates of ß-lactam resistance were not high in the present study, the frequency of blaTEM may represent a risk for patients receiving antimicrobial treatment. Taken together, the results provide better molecular characterization of this globally clinically important pathogen.

Antimicrobial resistance and plasmid replicons in Yersinia enterocolitica strains isolated in Brazil in 30 years

Abstract Some studies evaluated the resistance profile of the Y. enterocolitica strains isolated in diverse countries. However, in Brazil the isolation and the study of Y. enterocolitica are not common and therefore information about the antimicrobial resistance profile of this species in this country is scarce. Therefore, the aim of this study was to evaluate the antimicrobial resistance of Y. enterocolitica of biotypes 1A, 2 and 4 isolated from clinical and non-clinical sources between 1979 and 2012, in Brazil. This study showed that some Yersinia enterocolitica of different biotypes remain susceptible to antimicrobials used for gastroenteritis treatment. Moreover, neither acquired resistance genes nor diversity of plasmids replicons were found; however, variation in the in vitro intrinsic resistant pattern was detected, except the non-resistance to cefoxitin in all strains. Notwithstanding, due to epidemiological link between Y. enterocolitica and the pork production chain, monitoring plasmid acquired resistance in Y. enterocolitica could also be considered for antimicrobial resistance control purposes and food safety measures.

Antimicrobial resistance and plasmid replicons in Yersinia enterocolitica strains isolated in Brazil in 30 years.

Some studies evaluated the resistance profile of the Y. enterocolitica strains isolated in diverse countries. However, in Brazil the isolation and the study of Y. enterocolitica are not common and therefore information about the antimicrobial resistance profile of this species in this country is scarce. Therefore, the aim of this study was to evaluate the antimicrobial resistance of Y. enterocolitica of biotypes 1A, 2 and 4 isolated from clinical and non-clinical sources between 1979 and 2012, in Brazil. This study showed that some Yersinia enterocolitica of different biotypes remain susceptible to antimicrobials used for gastroenteritis treatment. Moreover, neither acquired resistance genes nor diversity of plasmids replicons were found; however, variation in the in vitro intrinsic resistant pattern was detected, except the non-resistance to cefoxitin in all strains. Notwithstanding, due to epidemiological link between Y. enterocolitica and the pork production chain, monitoring plasmid acquired resistance in Y. enterocolitica could also be considered for antimicrobial resistance control purposes and food safety measures.

Virulence-related genes, adhesion and invasion of some Yersinia enterocolitica-like strains suggests its pathogenic potential.

Yersina enterocolitica-like species have not been extensively studied regarding its pathogenic potential. This work aimed to assess the pathogenic potential of some Y. enterocolitica-like strains by evaluating the presence of virulence-related genes by PCR and their ability to adhere to and invade Caco-2 and HEp-2 cells. A total of 50 Y. frederiksenii, 55 Y. intermedia and 13 Y. kristensenii strains were studied. The strains contained the following genes: Y. frederiksenii, fepA(44%), fes(44%) and ystB(18%); Y. intermedia, ail(53%), fepA (35%), fepD(2%), fes(97%), hreP(2%), ystB(2%) and tccC(35%); Y. kristensenii, ail(62%), ystB(23%), fepA(77%), fepD(54%), fes(54%) and hreP(77%). Generally, the Y. enterocolitica-like strains had a reduced ability to adhere to and invade mammalian cells compared to the highly pathogenic Y. enterocolitica 8081. However, Y. kristensenii FCF410 and Y. frederiksenii FCF461 presented high invasion potentials in Caco-2 cells after five days of pre-incubation increased by 45- and 7.2-fold compared to Y. enterocolitica 8081, respectively; but, the ail gene was not detected in these strains. The presence of virulence-related genes in some of the Y. enterocolitica-like strains indicated their possible pathogenic potential. Moreover, the results suggest the existence of alternative virulence mechanisms and that the pathogenicity of Y. kristensenii and Y. frederiksenii may be strain-dependent.

Molecular and phenotypic characterization of strains of Shigella sonnei isolated over 31 years suggests the circulation of two prevalent subtypes in São Paulo State, Brazil.

Shigella sonnei is an important causative agent of bacillary dysentery worldwide that has recently emerged in developing countries. However, there are few studies that have characterized strains ofS. sonnei isolated in Brazil. The aims of this study were to assess the presence of 12 virulence genes, the antimicrobial resistance profile against 16 drugs and the genotypic diversity of strains of S. sonnei isolated in this country. Seventy-two strains of S. sonnei isolated from human diarrhoeic faeces in São Paulo State, Brazil from 1983-2014 were studied. All of the strains contained the ipaH, iuc and sigA genes. The ipaBCD gene was detected in 19 % of the strains, the ial and virF genes in 18 % and the sen gene in 10 % of the strains. The set1A, set1B, pic,sepA and sat genes were not detected. A total of 42 (58.3 %) strains were resistant to trimethoprim-sulfamethoxazole. Thirty (41.6 %) strains were resistant to tetracycline. The S. sonnei strains were grouped in two clusters called A and B by PFGE and ERIC-PCR, and the majority of the strains comprised in each cluster presented ≥80 % similarity. In conclusion, the pathogenic potential of the strains studied was highlighted by the presence of important virulence genes. The high rates of resistance to trimethoprim-sulfamethoxazole and tetracycline are alarming once those drugs can be used in the treatment of shigellosis. The PFGE and ERIC-PCR results suggest that there are two prevalent subtypes in the studied strains of S. sonnei that differed little over 31 years and have been contaminating humans and causing diseases in São Paulo State, Brazil.

Genotyping of Campylobacter coli strains isolated in Brazil suggests possible contamination amongst environmental, human, animal and food sources.

Campylobacter coli and Campylobacter jejuni are two of the most common causative agents of food-borne gastroenteritis in numerous countries worldwide. In Brazil, campylobacteriosis is under diagnosed and under-reported, and few studies have molecularly characterized Campylobacter spp. in this country. The current study genotyped 63 C. coli strains isolated from humans (n512), animals (n521), food (n510) and the environment (n520) between 1995 and 2011 in Brazil. The strains were genotyped using pulsed-field gel electrophoresis (PFGE), sequencing the short variable region (SVR) of the flaA gene ( flaA-SVR) and high-resolution melting analysis (HRMA) of the clustered regularly interspaced short palindromic repeat (CRISPR) locus to better understand C. coli genotypic diversity and compare the suitability of these three methods for genotyping this species. Additionally, the discrimination index (DI) of each of these methods was assessed. Some C. coli strains isolated from clinical and non-clinical origins presented ≥80 % genotypic similarity by PFGE and flaA-SVR sequencing. HRMA of the CRISPR locus revealed only four different melting profiles. In total, 22 different flaA-SVR alleles were detected. Of these, seven alleles, comprising gt1647­gt1653, were classified as novel. The most frequent genotypes were gt30 and gt1647. This distribution reveals the diversity of selected Brazilian isolates in comparison with the alleles described in the PubMLST database. The DIs for PFGE, flaA­SVR sequencing and CRISPR-HRMA were 0.986, 0.916 and 0.550, respectively. PFGE and flaA-SVR sequencing were suitable for subtyping C. coli strains, in contrast to CRISPR-HRMA. The high genomic similarity amongst some C. coli strains confirms the hypothesis that environmental and food sources potentially lead to human and animal contamination in Brazil.

Rapid and efficient differentiation of Yersinia species using high-resolution melting analysis.

The primary goal of clinical microbiology is the accurate identification of the causative agent of the disease. Here, we describe a method for differentiation between Yersinia species using PCR-HRMA. The results revealed species-specific melting profiles. The herein developed assay can be used as an effective method to differentiate Yersinia species.

Comparison of four molecular methods to type Salmonella Enteritidis strains.

This study compared the pulsed-field gel electrophoresis (PFGE), enterobacterial repetitive intergenic consensus-PCR (ERIC-PCR), multilocus variable-number of tanden-repeat analysis (MLVA), and multilocus sequence typing (MLST) methods for typing 188 Salmonella Enteritidis strains from different sources isolated over a 24-year period in Brazil. PFGE and ERIC-PCR were more efficient than MLVA for subtyping the strains. However, MLVA provided additional epidemiological information for those strains. In addition, MLST showed the Brazilian strains as belonging to the main clonal complex of S. Enteritidis, CC11, and provided the first report of two new STs in the S. enterica database but could not properly subtype the strains. Our results showed that the use of PFGE or ERIC-PCR together with MLVA is suitable to efficiently subtype S. Enteritidis strains and provide important epidemiological information.

Phenotypic and genetic features of enteropathogenic Escherichia coli isolates from diarrheal children in the Ribeirão Preto metropolitan area, São Paulo State, Brazil.

This study was designed to characterize a collection of 60 enteropathogenic Escherichia coli (EPEC) isolates from diarrheic feces of patients in the Ribeirão Preto metropolitan area regarding different phenotypic and molecular features. We examined antibiotic resistance profiles, occurrence of virulence factors-encoding genes, intimin subtypes and the correlation of serotypes among typical (tEPEC) and atypical (aEPEC) EPEC isolates. The results demonstrated that atypical EPEC was more heterogeneous than typical EPEC concerning the characteristics investigated and 45.2% do not belong to classical EPEC serogroups. Intimin subtype ß was the most frequent among the EPEC isolates (46.7%), being detected in both tEPEC and aEPEC. The majority of aEPEC isolates presented localized adherence-like (LAL) pattern to HEp-2 cells, although aEPEC isolates displaying diffuse adherence (DA) or non-adherent were also detected. High prevalence of antimicrobial resistance was found for ampicillin, cephalothin, sulfonamide and tetracycline. In general, tEPEC isolates were more resistant to the antimicrobials tested than aEPEC isolates.

A novel high-resolution melting analysis-based method for Yersinia enterocolitica genotyping.

Pathogenic Yersinia enterocolitica strains are associated with biotypes 1B, 2-5, while environmental strains with biotype 1A. In this work a method for Y. enterocolitica genotyping based on HRMA to determine SNPs was developed and the genetic diversity of 50 strains was determined. The strains were clustered into three groups consistent with the pathogenic profile of each biotype. The results provided a better understanding of the Y. enterocolitica genetic variability.

Genotyping of Yersinia enterocolitica biotype 1A strains from clinical and nonclinical origins by pulsed-field gel electrophoresis.

Yersinia enterocolitica biotype 1A (B1A) strains are considered mainly nonpathogenic. However, some studies considered strains of this biotype to be the causal agents of infections in humans and animals. In South America, there are no studies that have compared clinical and nonclinical strains of B1A typed by pulsed-field gel electrophoresis (PFGE) and none that have compared the capability of different enzymes on typing these strains. This study typed 51 Y. enterocolitica B1A strains isolated in Brazil and Chile by PFGE, testing the enzymes XbaI, NotI, and XhoI. The resulting dendrograms discriminated the strains in 47, 40, and 49 pulsotypes generated by the cleavage with the enzymes XbaI, NotI, and XhoI, respectively. The majority of the strains were grouped independently of their clinical or nonclinical origins. The high discriminatory power of PFGE confirmed the heterogeneity of B1A strains but could not divide the strains studied into clusters that differed in the frequency of some virulence genes as observed in studies using other methodologies.

Pulsed-Field Gel Electrophoresis characterization of Listeria monocytogenes isolates from cheese manufacturing plants in São Paulo, Brazil.

This study aimed to evaluate the occurrence of Listeria monocytogenes in cheese and in the environment of three small-scale dairy plants (A, B, C) located in the Northern region state of São Paulo, Brazil, and to characterize the isolates using conventional serotyping and PFGE. A total of 393 samples were collected and analyzed from October 2008 to September 2009. From these, 136 came from dairy plant A, where only L. seeligeri was isolated. In dairy plant B, 136 samples were analyzed, and L. innocua, L. seeligeri and L. welshimeri were isolated together with L. monocytogenes. In dairy plant C, 121 samples were analyzed, and L. monocytogenes and L. innocua were isolated. Cheese from dairy plants B and C were contaminated with Listeria spp, with L. innocua being found in Minas frescal cheese from both dairy plants, and L. innocua and L. monocytogenes in Prato cheese from dairy plant C. A total of 85 L. monocytogenes isolates were classified in 3 serotypes: 1/2b, 1/2c, and 4b, with predominance of serotype 4b in both dairy plants. The 85 isolates found in the dairy plants were characterized by genomic macrorestriction using ApaI and AscI with Pulsed Field Gel Electrophoresis (PFGE). Macrorestriction yielded 30 different pulsotypes. The presence of indistinguishable profiles repeatedly isolated during a 12-month period indicated the persistence of L. monocytogenes in dairy plants B and C, which were more than 100 km away from each other. Brine used in dairy plant C contained more than one L. monocytogenes lineage. The routes of contamination were identified in plants B and C, and highlighted the importance of using molecular techniques and serotyping to track L. monocytogenes sources of contamination, distribution, and routes of contamination in dairy plants, and to develop improved control strategies for L. monocytogenes in dairy plants and dairy products.