Salmonella resistant to different classes of antibiotics

The Brazilian Ministry of Health received reports of 11,524 epidemics of food-borne illnesses between 2000 and 2015, with 219,909 sick people and 167 deaths due to the diseases in question. Bacteria have caused most outbreaks of such diseases, including diarrhea and gastroenteritis. The most common were Salmonella spp., With 31,700 cases diagnosed during this period (14.4% of total), Staphylococcus aureus (7.4%) and Escherichia coli (6.1%).

According to a survey by the Ministry of Social Development, bacteria of the Salmonella genus were etiological agents in 42.5% of confirmed laboratory disease outbreaks reported in Brazil between 1999 and 2009.

The genome sequencing of the main bacteria causing acute diarrhea is the research center of a group at the University of Sao Paulo, led by Dr. Juliana Pfrimer Falcão, a professor at the Ribeirão Preto University of Pharmaceutical Sciences School (FCFRP-USP).

In an article published in PLUS UNU, biomedical scientists Amanda Aparecida Seribelli and Fernanda Almeida, belonging to the Falcão lab, describe how they sequenced and investigated the genomes of 90 strains of a particular serovar Salmonella enterica known as S. Typhimurium.

The 90 strains were isolated at the Adolfo Lutz Institute in Ribeirão Preto (São Paulo, Brazil) and Oswaldo Cruz (Fiocruz) Foundation in Rio de Janeiro. They provide a portrait of salmonella epidemiology in Brazil over the last 30 years, coming from all regions of the country and collected from patients with food infections or contaminated food such as poultry, pork or salad and other vegetables .

"From humans, we have received blood samples, cerebral abscess and diarrheal feces," Seribelli said.

When the antibiotic action was tested in each of the 90 strains, it was found that the vast majority were resistant to different classes of antibiotics that are part of the drug arsenal. The study also identified 39 genes responsible for antibiotic resistance.

The study was attended by affiliated researchers of Fiocruz, the School of Agricultural and Veterinary Sciences at the State University of São Paulo (FCAV-UNESP) and the Adolfo Lutz Institute. The 90 strains of S. Typhimurium were sequenced at the United States Food and Drug Administration (FDA) during Almeida's doctoral stay.

Comparative analysis of genomes, transcriptoms and phenotypes of S. Typhimuriumhuman isolates and food in Brazil were supported by the São Paulo Research Foundation – FAPESP, the FDA and the Department of Development of the Faculty of Education of the Ministry of Education (CAPES).

salmonellosis

Salmonella comprises two species, S. bongori and S. enterica. The latter is the type of species with a large number of subspecies and serovars that cause more food infections than any other species in Brazil and around the world. The human and animal intestinal tract is the main natural reservoir for this pathogen, poultry, pork, and related food products that serve as major transmission vectors.

The six subspecies of S. enterica are subdivided into 2,600 serovars. A serovar (short for the serological variant) is a distinct variation in a species of bacteria or virus characterized by having the same number of specific surface antigens.

The most important subspecies of S. enterica from an epidemiological point of view S. entericathe enteric subtype, which causes the food infection known as salmonellosis. Symptoms include diarrhea, fever, stomach cramps and vomiting.

S. enterica ssp. enterica was the main cause of the 31,700 salmonellosis cases reported in Brazil between 2000 and 2015. The most frequently isolated serovars of this subspecies are S. Typhimurium and S. Enteritidis.

S. Enteritidis is one of the most important serovars that cause salmonellosis. It first spread to a pandemic that began in Europe in the 1990s. S. Typhimurium was the most prevalent serovar before the pandemic and continued to cause infections.

According to Almeida, all 90 strains analyzed in the study belonged S. Typhimurium. Another researcher at FCFRP-USP (who is also working at the University's Clinical, Toxicology and Bromatology Laboratory) is currently sequencing and analyzing samples containing serovar S. Enteritidis.

Almeida took the 90 strains of S. Typhimurium for the US in 2015. Their genomes were sequenced at the FDA Center for Food Safety and Nutrition Applied in Maryland under the supervision of researcher Marc W. Allard, he said.

S. TyphimuriumThe genome contains 4.7 million pairs of bases. Short-term reflection tells us that the study has generated a wave of data, namely 423 million bases corresponding to 90 genomes.

After returning to Ribeirão Preto, Almeida worked with Seribelli on a comparative analysis of the different strains of the Salmonella genome to understand their diversity and the evolutionary relationships between them.

According to Almeida, the technique used was SNP genotyping (pronounced "snips" and short for single nucleotide polymorphisms), which allowed them to identify the genetic composition (genotype) of each strain by DNA sequencing. SNPs are the most common markers of genetic variation. The phylogenetic results separated the 90 strains S. Typhimurium in two groups, A and B.

"The group of samples collected from food was different from the group collected from humans," Seribelli explained. "Food isolates were distributed between groups A and B in relatively similar numbers, suggesting that several subtypes circulate in Brazilian foods. Human isolates were more prevalent in group B, suggesting that a specific subtype was probably adapted to humans."

In another important part of their research funded by FAPESP, scientists measure antibiotic resistance in each of the 90 strains of Salmonella. According to the study, 65 (72.2%) of the strains were resistant to sulfonamides, 44 (48.9%) to streptomycin, 27 (30%) to tetracycline, 21 (23.3%) to gentamycin, and seven cent) to ceftriaxone, a cephalosporin antibiotic.

The origin of resistance

SNP analysis has identified 39 genes for resistance to various classes of antimicrobials or antibiotics, such as aminoglycoside, tetracycline, sulfonamide, trimethoprim, beta-lactam, fluoroquinolone, phenicol and macrolide. Point-specific mutations have also been found in some of the genes, such as gyrA, gyrB, parC and parE.

"It's striking S. Typhimurium is resistant to antibiotics that can be used to treat the disease, "said Seribelli.This medication is available to doctors for use in fighting resistance-induced infections.This is a second line of defense when micro-organisms are not killed by the immune system of the patient because salmonellosis is self-limiting and does not require the use of antibiotics. The main problem is when this fails and bacteria become invasive. "

Another point that drew the attention of scientists was the difference between strain resistance in the 30-year sampling period. "Samples of S. Typhimurium collected in the mid-1990s showed greater resistance to antibiotics than samples in the years to come. This could be explained by the appearance of the sergeant in the early 1990s S. Enteritidis, which has become one of the main causes of salmonella infection, "Seribelli said.

S. Enteritidis has been known since the 1950s, but for a while caused fewer cases of disease. This has been radically changed by S. Enteritidis pandemic, which began in the late 1980s and early 1990s in Europe and then spread throughout the world.

"Since then, S. Enteritidis was one of the most widespread serfs in Brazil and around the world. As a result, it is a serovar that can also be fought with antibiotics if necessary, "Seribelli said.

According to Almeida, S. Typhimurium remains one of the main human, animal and food serfs in Brazil and around the world.

Starting with S. Enteritidis pandemic in the mid-1990s, the number of resistant strains appears to have decreased compared to the number predated by the 1990s, but if the virulence of these strains increased to allow them to adapt to this new niche is not known.

The main finding of this research is the discovery of a large number of resistance genes in samples, considering that they were isolated from humans and food. This indicates the very significant risk of contamination in today's Brazil from foods containing Salmonella strains that are resistant to antibiotics, "Almeida said.