Renata Garcia Costa

Vibrio spp. and Salmonella spp., presence and susceptibility in crabs Ucides cordatus

The presence of Vibrio spp. and Salmonella spp. in crabs marketed at the Bezerra de Menezes Ave., Fortaleza, State of Ceará, Brazil, was assessed between February and May, 2003. The number of individuals sampled in each one of the fifteen weekly samplings ranged between four and eight. Seven strains of Salmonella, from four different samplings, were identified, being five of them identified as serotype S. Senftenberg and two as S. Poona. All strains of Salmonella were sensitive to the tested anti-microbial drugs, with the exception of tetracycline and nalidixic acid, for which an intermediary sensibility was found. The MPNs for Vibrio ranged between 110/g and 110,000/g. Of the forty five Vibrio strains isolated from the crab samples, only 10 were identified up to the species level: two V. alginolyticus and eight V. parahaemolyticus. Bacteria belonging to the Enterobacteriaceae and Pseudomonaceae families were also identified, namely Escherichia coli, Klebsiella pneumoniae, Enterobacter cloacae, Pantoea agglomerans and Pseudomonas aeruginosa. The proper cooking of the animals is recommended in order to avoid problems for the consumers of this crustacean.

Phage typing and Multidrug resistance profile in S. Typhimurium isolated from different sources in Brazil from 1999 to 2004

Salmonella Typhimurium has become a widespread cause of salmonellosis among humans and animals worldwide. In Brazil, Salmonella Typhimurium (STM) is one of the most prevalent serovars isolated from food for human consumption. The uncontrolled sale and use of antimicrobials in agriculture and for treating human patients contributes to increase multidrug resistance of this serovar. In the present study, a total of 278 STM isolates from different sources and regions of Brazil over the period 1999 to 2004 were phage typed and analyzed for their antimicrobial resistance profile at Laboratory of Enterobacteria, Oswaldo Cruz Institute, FIOCRUZ. The main STM phage types isolated were DT 193 (64.3%), DT 19 (17.4%) and DT 18 (4%). Others phage types as DT 10 (2%), DT 27 (3.24%), DT 13 (0.36%), DT 22 (0.36%), DT 28 (0.36%), DT 29 (0.36%) and DT 149 (0.36%) were obtained in low percentages. A total of 54% STM strains were resistant to three or more antimicrobial classes, while no resistance to third generation cephalosporin or ciprofloxacin was identified in these strains. Those results show the STM phage types circulating among animals, food for human consumption and humans in Brazil as well as the increasing of multidrug resistance. The surveillance of STM strains based on phage typing and antimicrobial resistance profile are useful for detecting outbreaks, identifying sources of infection and implementing prevention and control measures.

Antimicrobial susceptibility and serovars of salmonella circulating in commercial poultry carcasses and poultry products in Brazil.

Antimicrobial resistance was evaluated for 12,582 strains of Salmonella isolated by public and private laboratories from commercial poultry carcasses and poultry products from different regions in Brazil between 2007 and 2011. These isolates were submitted unsolicited to the National Reference Laboratory for Bacterial Enteroinfections, Oswaldo Cruz Institute, Oswaldo Cruz Foundation, Rio de Janeiro, Brazil, for conclusive antigenic characterization. The prevalent Salmonella enterica serogroups were O:4 (B) (28.9%), O:9 (D1) (22.3%), O:21 (L) (15.9%), and O:7 (C1) (14.3%); the most prevalent serovars were Enteritidis, Minnesota, Typhimurium, Schwarzengrund, and Mbandaka. The antimicrobial susceptibility profiles of 1,234 strains indicated that 54.5% were resistant to up to eight antimicrobial drugs. Multidrug resistance (three or more classes) was observed in 16.4% of the strains, with 190 distinct patterns. The results showed increased resistance to ampicillin (12.4 to 18.9%), tetracycline (≈ 15.2 to ≈ 18.9%), and gentamicin (f7.0 to ≈ 9.6%) during the study period. Decreased resistance to nitrofurans (61.9 to 9.2%), quinolones (44.4 to 15.5%), and folate inhibitors (11.7 to 7.2%) was observed. Resistance to third-generation cephalosporins was detected in 5.5% of the strains throughout the study period; resistance to fluoroquinolones has been observed in 0.3% of the strains since 2009. These findings highlight the importance of surveillance along the food chain for detection of altered patterns among foodborne zoonotic bacteria that are important to public health.

Characteristics of Quinolone Resistance in Salmonella spp. Isolates from the Food Chain in Brazil

Salmonella spp. is an important zoonotic pathogen related to foodborne diseases. Despite that quinolones/fluoroquinolones are considered a relevant therapeutic strategy against resistant isolates, the increase in antimicrobial resistance is an additional difficulty in controlling bacterial infections caused by Salmonella spp. Thus, the acquisition of resistance to quinolones in Salmonella spp. is worrisome to the scientific community along with the possibility of transmission of resistance through plasmids. This study investigated the prevalence of plasmid-mediated quinolone resistance (PMQR) in Salmonella spp. and its association with fluoroquinolone susceptibility in Brazil. We evaluated 129 isolates, 39 originated from food of animal sources, and 14 from environmental samples and including 9 from animals and 67 from humans, which were referred to the National Reference Laboratory of Enteric Diseases (NRLEB/IOC/RJ) between 2009 and 2013. These samples showed a profile of resistance for the tested quinolones/fluoroquinolones. A total of 33 serotypes were identified; S. Typhimurium (63) was the most prevalent followed by S. Enteritidis (25). The disk diffusion test showed 48.8% resistance to enrofloxacin, 42.6% to ciprofloxacin, 39.53% to ofloxacin, and 30.2% to levofloxacin. According to the broth microdilution test, the resistance percentages were: 96.1% to nalidixic acid, 64.3% to enrofloxacin, 56.6% to ciprofloxacin, 34.1% to ofloxacin, and 30.2% to levofloxacin. Qnr genes were found in 15 isolates (8 qnrS, 6 qnrB, and 1 qnrD), and the aac(6′)-Ib gene in 23. The integron gene was detected in 67 isolates with the variable region between ±600 and 1000 bp. The increased detection of PMQR in Salmonella spp. is a serious problem in Public Health and must constantly be monitored. Pulsed-field gel electrophoresis was performed to evaluated clonal profile among the most prevalent serovars resistant to different classes of quinolones. A total of 33 pulsotypes of S. Typhimurium were identified with a low percentage of genetic similarity (≤65%). This result demonstrates the presence of high diversity in the resistant clones evaluated in this study.

Salmonella Hadar phage types isolated from different sources of foodchain in Brazil

The gastroenteritis incidence caused by Salmonella Hadar has increased over the last decades worldwide. The uncontrolled use of antimicrobials for treating human patients and veterinary field contributes to increase the multidrug resistance of this serovar. In the present investigation, a total of 179 S. Hadar isolates from different sources of foodchain in Brazil were phage typed and analyzed for their antimicrobial resistance profile. The main S. Hadar phage types isolated were PT 38, PT 39, PT 40, PT 11, PT 34, PT 1 and PT 22. Others phage types as PT 13, PT 19, PT 21, PT 23, PT 31, PT 33 and PT 37 were obtained in low percentages. A total of 35,7% S. Hadar strains were resistant to two or more antimicrobials drugs. Furthermore, no resistance to third generation cephalosporin or ciprofloxacin was identified in these strains. Those results appoint to S. Hadar phage types circulating among animals, food and humans, as well as the increasing of multidrug resistance. The surveillance and monitoring of S. Hadar strains based on phage typing and antimicrobial resistance profile are useful for detecting outbreaks, identifying sources of infection and implementing prevention and control measures of salmonellosis.

Genetic diversity, anti-microbial resistance, plasmid profile and frequency of the Vi antigen in Salmonella Dublin strains isolated in Brazil

Salmonella Dublin is strongly adapted to cattle causing enteritis and/or systemic disease with high rates of mortality. However, it can be sporadically isolated from humans, usually causing serious disease, especially in patients with underlying chronic diseases. The aim of this study was to molecularly type S. Dublin strains isolated from humans and animals in Brazil to verify the diversity of these strains as well as to ascertain possible differences between strains isolated from humans and animals. Moreover, the presence of the capsular antigen Vi and the plasmid profile was characterized in addition to the anti‐microbial resistance against 15 drugs. For this reason, 113 S. Dublin strains isolated between 1983 and 2016 from humans (83) and animals (30) in Brazil were typed by PFGE and MLVA. The presence of the capsular antigen Vi was verified by PCR, and the phenotypic expression of the capsular antigen was determined serologically. Also, a plasmid analysis for each strain was carried out. The strains studied were divided into 35 different PFGE types and 89 MLVA‐types with a similarity of ≥80% and ≥17.5%, respectively. The plasmid sizes found ranged from 2 to >150 kb and none of the strains studied presented the capsular antigen Vi. Resistance or intermediate resistance was found in 23 strains (20.3%) that were resistant to ampicillin, ciprofloxacin, chloramphenicol, imipenem, nalidixic acid, piperacillin, streptomycin and/or tetracycline. The majority of the S. Dublin strains studied and isolated over a 33‐year period may descend from a common subtype that has been contaminating humans and animals in Brazil and able to cause invasive disease even in the absence of the capsular antigen. The higher diversity of resistance phenotypes in human isolates, as compared with animal strains, may be a reflection of the different anti‐microbial treatments used to control S. Dublin infections in humans in Brazil.

Genotypic Resistance to Quinolone and Tetracycline in Salmonella Dublin Strains Isolated from Humans and Animals in Brazil

Resistance of Salmonella Dublin strains to quinolones and tetracycline has been increasing worldwide. Studies regarding the genotypic resistance traits of strains of this serovar isolated in Brazil are scarce. This study aims to examine the genetic characteristics of Salmonella Dublin strains isolated in Brazil, which are associated with resistance to quinolone and tetracycline. The minimum inhibitory concentrations (MICs) of nalidixic acid, ciprofloxacin, and tetracycline of the 10 strains sensitive and 21 strains resistant to quinolone and tetracycline were determined using Etest.® The mutation profiles of the gyrA, gyrB, parC, and parE genes were accessed by sequencing, while the presence of plasmid-mediated quinolone resistance and tet genes was analyzed by PCR. Quinolone-resistant strains presented the amino acid substitutions Ser96→Tyr, Ser96→Phe, Asp107→Asn, or Asp108→Gly on the gyrA gene, and the Ser224→Phe and Glu231→Asp mutations on the gyrB gene. The qnrA, tet(A), and tet(B) genes were detected in 5, 13, and 6 strains, respectively. Analysis of the MIC values revealed that 1 and 3 strains presented intermediate and resistant MIC profiles to nalidixic acid, respectively; 6 strains presented intermediate MIC profile to ciprofloxacin; and 13 strains presented resistant MIC profile to tetracycline. In the Salmonella Dublin strains studied, quinolone resistance was mainly related to mutation points that led to target alteration in the gyrA and gyrB genes, while tetracycline resistance was associated with the presence of tet(A) and/or tet(B) genes, with the highest resistance levels detected in strains bearing the tet(B) gene. The presence of the aforementioned genotypic resistance traits in Salmonella Dublin strains isolated over 33 years in Brazil indicates that ciprofloxacin or tetracycline therapy against such strains may fail.

Genetically distinct lineages of Salmonella Typhimurium ST313 and ST19 are present in Brazil

In sub-Saharan Africa, two genetically distinct lineages of multi-drug resistant non-typhoidal Salmonella (NTS) serovar Typhimurium sequence type 313 (ST313) are known to cause invasive disease among people. S. Typhimurium ST313 has evolved to become more human-adapted and is commonly isolated from systemic sites (eg., blood) from febrile patients in sub-Saharan Africa. Epidemiological studies indicate that S. Typhimurium is frequently isolated from systemic sites from human patients in Brazil, however, it is currently unknown if this pathogen has also evolved to become more invasive and human-adapted in this country. Here we determined genotypic and phenotypic divergence among clinical S. Typhimurium strains isolated from systemic and non-systemic sites from human patients in Brazil. We report that a subset (8/38, 20%) of epidemiologically diverse human clinical strains of S. Typhimurium recovered from systemic sites in Brazil show significantly higher intra-macrophage survival, indicating that this subset is likely more invasive. Using the whole genome sequencing and phylogenetic approaches, we identified S. Typhimurium ST313-lineage in Brazil that is genetically and phenotypically distinct from the known African ST313-lineages. We also report the identification of S. Typhimurium ST19-lineage in Brazil that is evolving similar to ST313 lineages from Africa but is genetically and phenotypically distinct from ST19-lineage commonly associated with the gastrointestinal disease worldwide. The identification of new S. Typhimurium ST313 and ST19 lineages responsible for human illnesses in Brazil warrants further epidemiological investigations to determine the incidence and spread of a genetically divergent population of this important human pathogen.

Draft Genome Sequences of 11 Salmonella enterica Serovar Typhimurium Strains Isolated from Human Systemic and Nonsystemic Sites in Brazil

ABSTRACT Salmonella enterica serovar Typhimurium strains isolated from systemic sites outside sub-Saharan Africa have been rarely sequenced. Here, we report the draft genome sequences of S. Typhimurium sequence type 19 (ST19) (n = 9), ST1649 (n = 1), and ST313 (n = 1) strains isolated from human systemic (e.g., blood) and nonsystemic (e.g., stool and wounds) sites in Brazil.