Brazil's Ministry of Health received
reports of 11,524 outbreaks of foodborne
diseases between 2000 and 2015, with 219,909 individuals falling sick and 167
dying from the diseases in question. Bacteria caused most outbreaks of such
illnesses, including diarrhea and gastroenteritis. The most frequent were
Salmonella spp., with 31,700 cases diagnosed in the period (14.4% of the
total), Staphylococcus aureus (7.4%), and Escherichia coli (6.1%).
According to a survey by the
Ministry of Social Development, bacteria of the genus Salmonella were the etiological
agents in 42.5% of the laboratory-confirmed foodborne disease outbreaks
reported in Brazil between 1999 and 2009.
Whole-genome sequencing of the
main bacteria that cause acute diarrhea is the research focus for a group at
the University of São Paulo led by Juliana Pfrimer Falcão, a professor at the
university's Ribeirão Preto School of Pharmaceutical Sciences (FCFRP-USP).
In an article published in PLOS
ONE, biomedical scientists Amanda Aparecida Seribelli and Fernanda
Almeida, who belong to Falcão's lab, describe how they sequenced and
investigated the genomes of 90 strains of a specific serovar of Salmonella
entericaknown as S. Typhimurium (an abbreviation
of Salmonella entericasubspecies serovar Typhimurium).
The 90 strains were isolated
between 1983 and 2013 at Adolfo Lutz Institute in Ribeirão Preto (São Paulo
State, Brazil) and Oswaldo Cruz Foundation (Fiocruz) in Rio de Janeiro. They
provide a portrait of the epidemiology of salmonellosis in Brazil in the last
30 years, coming from all regions of the country and having been collected from
patients with foodborne infections or from contaminated food such as poultry,
pork, or lettuce and other vegetables.
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"From humans, we received
samples of blood, brain abscesses, and diarrheic feces," Seribelli told.
When the action of antibiotics in
each of the 90 strains was tested, it was discovered that the vast majority
were resistant to different classes of antibiotics that are part of the arsenal
of medicine. The study also identified 39 genes responsible for resistance to
antibiotics.
Researchers affiliated with
Fiocruz, São Paulo State University's School of Agrarian and Veterinary
Sciences (FCAV-UNESP) and Adolfo Lutz Institute participated in the study. The
90 strains of S. Typhimurium were sequenced at the United
States Food and Drug Administration (FDA) during Almeida's doctoral stay.
The comparative analysis of the
genomes, transcriptomes, and phenotypes of S. Typhimurium strains
isolated from humans and food in Brazil was supported by São Paulo Research
Foundation -- FAPESP, the FDA, and the Ministry of Education's Office for
Faculty Development (CAPES).
Salmonellosis
Salmonella comprises two
species, S. bongori and S. enterica. The
latter is the type species, with a large number of subspecies and serovars that
cause more foodborne infections than any other species in Brazil and worldwide.
The human and animal intestinal tract is the main natural reservoir for this
pathogen, with poultry, pork and related food products serving as major
transmission vectors.
The six subspecies of S.
enterica are subdivided into 2,600 serovars. A serovar (short for
serological variant) is a distinct variation within a species of bacteria or
virus characterized by having the same number of specific surface antigens.
The most important subspecies
of S. enterica from the epidemiological standpoint is S.
enterica subspecies enterica, which causes the foodborne infection
known as salmonellosis. The symptoms are diarrhea, fever, stomach cramps, and
vomiting.
S. enterica subsp. enterica was the main cause of the 31,700
cases of salmonellosis 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 leading salmonellosis-causing
serovars. It first spread in a pandemic that started in Europe in the
1990s. S. Typhimurium was the most prevalent serovar before
the pandemic and has continued to cause infections.
According to Almeida, all 90
strains analyzed in the study belonged to S. Typhimurium. Another
researcher at FCFRP-USP (also working at the university's Clinical,
Toxicological and Bromatological Analysis Laboratory) is currently sequencing
and analyzing samples containing the serovar S. Enteritidis.
Almeida took the 90 strains
of S. Typhimurium to the US in 2015. "Their genomes
were sequenced at the FDA's Center for Food Safety and Applied Nutrition in
Maryland under the supervision of researcher Marc W. Allard," he said.
S. Typhimurium's genome contains 4.7 million base pairs. Brief
reflection tells us that the study generated a mountain of data, more
specifically 423 million bases corresponding to the sum of 90 genomes.
After his return to Ribeirão
Preto, Almeida worked with Seribelli on a comparative analysis of the various
strains' genomes to understand their diversity and the evolutionary
relationships between them.
According to Almeida, the
technique used was high-throughput genotyping with SNPs (pronounced
"snips" and short for single-nucleotide polymorphisms), which enabled
them to identify the genetic composition (genotype) of each strain by means of
DNA sequencing. SNPs are the most common markers of genetic variation. The
phylogenetic results separated the 90 strains of S. Typhimurium into
two groups, A and B.
"The group of samples
collected from food differed from the group collected from humans,"
Seribelli explained. "Food isolates were distributed between groups A and
B in relatively similar numbers, suggesting that more than one subtype is
circulating in foods in Brazil. Human isolates were more prevalent in group B,
suggesting that a specific subtype has probably adapted to humans."
In another important part of
their research funded by FAPESP, the scientists measured antibiotic resistance
in each of the 90 strains. According to the study, 65 (72.2%) of the strains
proved resistant to sulfonamides, 44 (48.9%) to streptomycin, 27 (30%) to
tetracycline, 21 (23.3%) to gentamicin and seven (7.8%) to ceftriaxone, a
cephalosporin antibiotic.
Origin of resistance
The analysis of SNPs identified
39 genes for resistance to different classes of antimicrobial or antibiotic,
such as aminoglycoside, tetracycline, sulfonamide, trimethoprim, beta-lactam,
fluoroquinolone, phenicol and macrolide. Point mutations were also found in
some of the genes, such as gyrA, gyrB, parC and parE.
"It's striking that S.
Typhimurium is resistant to antibiotics that can be used to treat the
disease," Seribelli said. "These drugs are available to physicians
for use in combating infections that display resistance. They're a second line
of defense when microorganisms aren't killed by the patient's immune system
since salmonellosis is normally self-limiting and doesn't require the use of
antibiotics. The main problem is when this fails and the bacteria become
invasive."
Another point that drew the
scientists' attention was the difference between the strains' resistance over
the 30-year sample collection period. "The samples of S.
Typhimurium collected in the mid-1990s showed more resistance to
antibiotics than samples from later years. This could be explained by the
emergence in the early 1990s of the serovar S. Enteritidis, which
has since become one of the main causes of salmonella infection,"
Seribelli said.
S. Enteritidis has been known since the 1950s, but for a time, it
caused fewer cases of the disease. This was radically changed by the S.
Enteritidis pandemic, which began in the late 1980s and early 1990s
in Europe and then spread around the world.
"Since then, S.
Enteritidis has been one of the most prevalent serovars in Brazil and
worldwide. As a result, it's a serovar that can also be combated with
antibiotics if necessary," Seribelli said.
According to Almeida, S. Typhimurium remains
one of the main serovars isolated from humans, animals and food in Brazil and
worldwide.
As of the S. Enteritidis pandemic in
the mid-1990s, the number of resistant strains apparently decreased compared
with the number prevalent before the 1990s, but whether the virulence of these
strains increased to allow them to adapt to this new niche is unknown.
"The key finding of this research is the discovery
of a large number of resistance genes in the samples, considering that they
were isolated from humans and food. This points to the very significant risk of
contamination in Brazil today from food containing strains of Salmonella that
are resistant to antimicrobials," Almeida said.
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Posted by Dr. Tim Sandle, Pharmaceutical Microbiology
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Pharmaceutical Microbiology