Luisa Zanolli Moreno

Molecular characterization, serotyping, and antibiotic susceptibility profile of Leptospira interrogans serovar Copenhageni isolates from Brazil

Leptospira interrogans serogroup Icterohaemorrhagiae is the major serogroup infecting humans worldwide, and rodents and dogs are the most significant transmission sources in urban environments. Knowledge of the prevalent serovars and their maintenance hosts is essential to understand the epidemiology of leptospirosis. In this study, 20 Leptospira isolates were evaluated by pulsed-field gel electrophoresis (PFGE), variable number tandem-repeat analysis (VNTR), serotyping, and determination of antimicrobial resistance profile. Isolates, originated from bovine, canine, human, and rodent sources, were characterized by microscopic agglutination test with polyclonal and monoclonal antibodies and were identified as L. interrogans serogroup Icterohaemorrhagiae serovar Copenhageni. MICs of antimicrobials often used in veterinary medicine were determined by broth microdilution test. Most of tested antibiotics were effective against isolates, including penicillin, ampicillin, and ceftiofur. Higher MIC variability was observed for fluoroquinolones and neomycin; all isolates were resistant to trimethoprim/sulfamethoxazole and sulphadimethoxine. Isolates were genotyped by PFGE and VNTR; both techniques were unable to discriminate between serovars Copenhageni and Icterohaemorrhagiae, as expected. PFGE clustered all isolates in 1 pulsotype, indicating that these serovars can be transmitted between species and that bovine, rodent, and dogs can maintain them in the environment endangering the human population.

Characterization of atypical Listeria innocua isolated from swine slaughterhouses and meat markets

Atypical Listeria innocua strains presenting phenotypic characteristics similar to those of Listeria monocytogenes were recently isolated from food and the environment. These isolates also tested positive for virulence genes specific to L. monocytogenes. Here we report the isolation of atypical hemolytic L. innocua strains from the environment of pork processing plants in Brazil. The strains were positive for L. monocytogenes virulence genes hly, inlA and inlB by PCR and presented genotypic similarities with human isolates of L. monocytogenes via the AFLP technique using HindIII single enzyme protocol. Phenotypic and genotypic similarities suggest that these atypical L. innocua may be pathogenic strains.

Characterization of Haemophilus parasuis isolated from Brazilian swine through serotyping, AFLP and PFGE.

Haemophilus parasuis infection in pigs is characterized by fibrinous polyserositis, arthritis and meningitis. Despite the fact that traditional diagnosis is based on herd history, clinical signs, bacterial isolation and serotyping, molecular-based methods are alternatives for species-specific tests and epidemiological studies. The aim of this study was to characterize H. parasuis field strains from different states of Brazil, employing serotyping and genotyping methods. Serotyping revealed that serovar 4 was the most prevalent (26.1%), followed by serovars 5 (17.4%), 14 (8.7%), 13 (4.4%) and 2 (4.4%), whereas 39% of the strains were considered as untypeable. AFLP with a single enzyme and PFGE were able to type all isolates tested, generating 34 and 20 different profiles, respectively, including untypeable strains. Besides the slightly higher discrimination index presented by AFLP, PFGE with Not I restriction enzyme showed a better correlation with epidemiological data, grouping strains of the same serovar, animal or farm origin. The results indicated AFLP and PFGE as valuable tools for typing H. parasuis isolates collected in Brazil.

Characterization of antibiotic resistance in Listeria spp. isolated from slaughterhouse environments, pork and human infections

INTRODUCTION Listeria species are susceptible to most antibiotics. However, over the last decade, increasing reports of multidrug-resistant Listeria spp. from various sources have prompted public health concerns. The objective of this study was to characterize the antibiotic susceptibility of Listeria spp. and the genetic mechanisms that confer resistance. METHODOLOGY Forty-six Listeria spp. isolates were studied, and their minimal inhibitory concentrations of antibiotics were determined by microdilution using Sensititre standard susceptibility MIC plates. The isolates were screened for the presence of gyrA, parC, lde, lsa(A), lnu(A), and mprF by PCR, and the amplified genes were sequenced. RESULTS All isolates were susceptible to penicillin, ampicillin, tetracycline, erythromycin, and carbapenems. Resistance to clindamycin, daptomycin, and oxacillin was found among L. monocytogenes and L. innocua, and all species possessed at least intermediate resistance to fluoroquinolones. GyrA, parC, and mprF were detected in all isolates; however, mutations were found only in gyrA sequences. A high daptomycin MIC, as reported previously, was observed, suggesting an intrinsic resistance of Listeria spp. to daptomycin. CONCLUSIONS These results are consistent with reports of emerging resistance in Listeria spp. and emphasize the need for further genotypic characterization of antibiotic resistance in this genus.

Characterization of Yersinia enterocolitica Biotype 1A Strains Isolated from Swine Slaughterhouses and Markets

Yersinia enterocolitica is an important foodborne pathogen that causes illness in humans and animals. Y. enterocolitica is also the most heterogeneous species of the genus and is divided into distinct serotypes and over six biotypes. Y. enterocolitica biotype 1A strains are classically considered as nonpathogenic; however, some biotype 1A isolates have been considered as causative of gastrointestinal disease, yielding symptoms indistinguishable from those produced by pathogenic biotypes. Even after decades of isolation of clinical strains, the pathogenic mechanisms of these isolates are still not fully understood. In the present study, 122 Yersinia enterocolitica biotype 1A strains isolated from swine slaughterhouses and meat markets in Sao Paulo, Brazil, were characterized according to the presence of the virulence genes ail, virF, and ystA. A total of 94 strains were positive to at least one virulence gene (77.05%), and 67 were positive to all of them (54.92%). Twenty-two strains were submitted to PFGE genotyping resulting in 22 distinct pulsotypes, varying from 50% to 84% of genetic similarity. Any clustering tendency among pulsotypes related to origin, isolation site, or even virulence profile was not observed. The present study reports an important contamination of the environment in swine slaughterhouses, meat markets, and pork, by potentially virulent Y. enterocolitica biotype 1A.

Shiga toxin-producing Escherichia coli (STEC): Zoonotic risks associated with psittacine pet birds in home environments

Psittacidae are frequently bred as pets worldwide, but little is known about the zoonotic risks of these animals. The objective of this study was to investigate the presence of Shiga toxin-producing Escherichia coli (STEC) in the feces of psittacine birds housed as pets. A total of 171 fecal samples (67 cockatiels, 59 budgerigars, and 45 agapornis) were cultured. Forty-two (E. coli) strains were identified, and the presence of the eae, stx1, and stx2 genes was determined using PCR. The antimicrobial resistance profiles of the STEC strains were determined using the disk diffusion method and phylogenetic analysis according to the new Clermont phylotyping method. Using these methods, 19.4% (8/42) of the STEC strains were determined to be positive for the eae and stx2 genes. The results revealed a STEC frequency of 4.6% in the birds (8/171), with a percentage of 8.47% in budgerigars (5/59), 4.47% in cockatiels (3/67), and 0% in agapornis (0/45). None of the STEC isolates belonged to the O157 serogroup. Most of the strains were classified as sensitive to the 18 antibiotics tested. None of the strains exhibited a multiresistance profile. In the phylogenetic analysis, two strains were classified as non-typeable, three were classified as B2, two were classified as F, and one was classified as Clade I. Seven of the eight STEC strains showed a clonal profile using AFLP. E. coli strains that are stx2(+) plus eae(+) are usually associated with severe human diseases such as hemorrhagic colitis and hemolytic-uremic syndrome. The STEC-positive results indicate the zoonotic risk of breeding psittacidae in home environments.

Draft genome of the Leptospira interrogans strains, Acegua, RCA, Prea, and Capivara, obtained from wildlife maintenance hosts and infected domestic animals

In the present paper, we announce new draft genomes of four Leptospira interrogans strains named Acegua, RCA, Prea, and Capivara. These strains were isolated in the state of Rio Grande do Sul, Brazil, from cattle, dog, Brazilian guinea pig, and capybara, respectively.

Genotypic Characterization of Yersinia enterocolitica Biotype 4/O:3 Isolates from Pigs and Slaughterhouses Using SE-AFLP, ERIC-PCR, and PFGE

Yersinia enterocolitica is a foodborne pathogen that causes illness in humans and animals. The biotype 4/O:3 has been commonly associated with yersiniosis and is characterized by the presence of chromosomal and extra-chromosomal virulence genes. Molecular typing methods have been successfully used to characterize Y. enterocolitica genetic heterogeneity and to study the epidemiology of the bacteria from different origins. In this study, 320 Y. enterocolitica biotype 4/O:3 isolates originating in pigs and slaughterhouses were characterized according to the virulence profile, and 61 isolates were typified through SE-AFLP, ERIC-PCR, and PFGE techniques. The majority of the isolates originated from pigs, and the predominant virulence profile was ail+ virF+ rfbC+ ystA+, representing 83.4% of the tested isolates. All of the Y. enterocolitica 4/O:3 isolates were positive for at least ystA gene. The SE-AFLP and ERIC-PCR patterns were highly homogeneous. The SE-AFLP was more discriminative than the ERIC-PCR and tended to cluster isolates according to the slaughterhouse. Despite the limited genetic diversity of Y. enterocolitica 4/O:3, PFGE was shown to be the most discriminative technique considering one band of difference. Fattening pigs proved to be an important reservoir of Y. enterocolitica biotype 4/O:3 carrying virulence genes.

Molecular and serological characterization of the first Leptospira santarosai strain isolated from a dog

Leptospirosis is a zoonotic disease of global importance caused by pathogenic Leptospira species. Dogs can become asymptomatically infected, acting like reservoir hosts for pathogenic Leptospira, notably Leptospira interrogans serovar Canicola. Identification of such individuals and characterization of leptospires involved in chronic infections may unravel the role of dogs in the epidemiology of particular leptospiral strains. The aim of the present work was to describe the first Leptospira santarosai strain isolated from a dog. The dog was kept in a public shelter in São Paulo city, Brazil, and presented asymptomatic urinary shedding detected by PCR. Prospective evaluation was performed to fully characterize its chronic carrier state. The dog did not present anti-Leptospira titles or clinical/laboratorial abnormalities during the evaluations; nevertheless long-term urinary shedding was confirmed by PCR and leptospires were recovered from two occasions. The isolated strain was molecularly characterized by partial 16S rRNA and secY gene sequencing and MLST analysis. Serogroup identification was performed using polyclonal antibodies. The strain was identified as Leptospira santarosai, serogroup Sejroe. This is the first evidence in the literature of the isolation of L. santarosai in dogs. Our findings show that dogs can persistently harbor leptospires other than L. interrogans.

Phenotypic and Genotypic Characterization of Atypical Listeria monocytogenes and Listeria innocua Isolated from Swine Slaughterhouses and Meat Markets

In the last decade, atypical Listeria monocytogenes and L. innocua strains have been detected in food and the environment. Because of mutations in the major virulence genes, these strains have different virulence intensities in eukaryotic cells. In this study, we performed phenotypic and genotypic characterization of atypical L. monocytogenes and L. innocua isolates obtained from swine slaughterhouses and meat markets. Forty strains were studied, including isolates of L. monocytogenes and L. innocua with low-hemolytic activity. The isolates were characterized using conventional phenotypic Listeria identification tests and by the detection and analysis of L. monocytogenes-specific genes. Analysis of 16S rRNA was used for the molecular identification of the Listeria species. The L. monocytogenes isolates were positive for all of the virulence genes studied. The atypical L. innocua strains were positive for hly, plcA, and inlC. Mutations in the InlC, InlB, InlA, PI-PLC, PC-PLC, and PrfA proteins were detected in the atypical isolates. Further in vitro and transcriptomic studies are being developed to confirm the role of these mutations in Listeria virulence.