Acute diarrhea associated with Salmonella enterica in Belo Horizonte-MG: prevalence and characterization of isolates

Introduction: Acute infectious diarrhea is still regarded as a public health problem associated with a wide range of etiologic agents, from which Salmonella enterica is particularly worth mentioning inasmuch as it is a major cause of inflammatory diarrhea in both developed and developing countries. Objective: To assess the distribution of S. enterica among children with acute diarrhea in Belo Horizonte and to characterize bacterium isolates. Material and methods: The study group comprised a total of 157 children from low socioeconomic background. Stool samples were collected for leukocyte analysis and Salmonella bacterial culture. The isolates were serotyped and evaluated as to antimicrobial susceptibility profile, extended-spectrum β -lactamases (ESBL) production, and presence of virulence markers ( invA , iroB , and spvC ). Results: A total of 5/3.2% children were infected by S. enterica , 3/60% by S. enterica Typhimurium, 1/20% by S. enterica Enteritidis and 1/20% S. enterica subsp. enterica serotype 8.20:z4,z23:-. Fecal leucocytes were detected in two out of five fecal specimens positive for S. enterica . Isolates from three children were resistant to nalidixic acid, nalidixic acid + chloramphenicol, and nalidixic acid + chloramphenicol + ampicillin. ESBL production was not detected. All samples presented invA and iroB genes. spvC marker was observed in isolates from two children infected by S. Typhimurium and S. Enteritidis. Conclusion: The results demonstrate that S. enterica infection is uncommon among children from our region. Furthermore, they indicate the need for periodic monitoring of bacterial antimicrobial susceptibility profile in order to establish suitable antimicrobial therapy when required.

Acute diarrhea associated with Salmonella enterica in Belo Horizonte-MG: prevalence and characterization of isolates introDuCtion Acute infectious diarrhea still poses a great challenge for public health authorities worldwide.It is estimated the annual occurrence of approximately 1.5 to 2 billion cases of the disease and 1.5 to 2 million deaths among children aged up to five years (9,42) .Most episodes of acute diarrhea are considered to be mild and are ultimately solved with neither medical care nor a specific diagnosis.However, the etiologic diagnosis of diarrheal disease is of utmost importance to the establishment of effective prevention and eradication programs (23) .
A wide range of diarrheagenic agents have already been identified and nontyphoidal salmonellae is among them.Salmonella enterica comprises a large division of hydrogen sulfide-producing Gram negative bacteria hugely diverse from the antigenic perspective (27) .The species is regarded as a major agent of acute inflammatory diarrhea both in industrialized as well as developing countries.Approximately 94 million cases of Salmonella enteritis occur worldwide leading to around 150,000 deaths (22,31,32) annually.
We addressed the distribution of S. enterica-associated diarrhea in a pediatric population of Belo Horizonte, the association between the disease and clinical and epidemiological parameters, and the characterization of bacterium isolates as to virulence markers and antimicrobial susceptibility profile.

MAtEriAL AnD MEthoDS
This protocol was approved by the Ethics Committee of Universidade Federal de Minas Gerais (UFMG).Written informed consent was obtained from the parents or guardians of all children included in this investigation.

originAL ArtiCLE
This study comprised a total of 157 children (73 girls and 84 boys, age range 1-48 months, mean age 11.7 months, median 8.0 months) from low socioeconomic background (monthly income below U$300.00),who searched for assistance at Hospital Infantil João Paulo II, Belo Horizonte-MG, from March 2004 to March 2005.All of them presented acute diarrhea and no patient had history of hospitalization or antimicrobial therapy in the last 15 days prior to specimen collection.Clinical and epidemiological data were obtained through questionnaires filled in by the children's guardians.
Stool samples were individually transferred to two sterile leak proof wide mouth screw cap vials, one of them containing a transport medium of equal parts of glycerol and 0.033 M phosphate buffer, maintained in an ice bath, and processed within one hour.
For leukocytes search, fecal smears were stained with May-Grünwald-Giemsa and examined under bright field microscopy at 400× and 1,000×.
The specimens transported in buffered glycerol were streaked onto MacConkey Agar (Difco, Sparks, MD, USA) and SS Agar (Difco).They were also inoculated into Tetrathionate Broth (Acumedia, Baltimore, MD, USA) and, following incubation for about 18 h at 35ºC, subcultured on SS Agar.All agar cultures were incubated for up to 24 h at 35ºC.Afterwards, around five lactosenegative colonies from MacConkey Agar and five lactose-negative and five H 2 S-positive colonies from each SS Agar plate were picked and inoculated into Triple Sugar Iron Agar (Acumedia), Escola Paulista de Medicina (EPM) (36) and Citrate medium (Biobrás, Montes Claros-MG, Brazil).Whenever possible, morphologically different colonies were selected.
Following identification, Salmonella isolates were antigenically characterized by using somatic antisera directed against A, B, C1, C2, D, and E serogroups (Probac, São Paulo-SP, Brazil), according to manufacturer's instructions.Bacterium suspensions were boiled for 10 min before performing agglutination test when necessary.Subsequently, one S. enterica isolate obtained from each child was sent to Instituto Oswaldo Cruz (IOC) (Rio de Janeiro-RJ, Brazil) and serotyped by use of polyvalent and monovalent antisera against somatic and flagelar S. enterica antigens (19) .
When strains isolated from the same patient showed divergent susceptibility profiles determined by disk diffusion technique, minimum inhibitory concentration was evaluated by agar dilution technique (2) .This was the case exclusively for chloramphenicol.Drug concentrations from 4 to 128 µg/ml were employed.Escherichia coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27853 were included as controls for antimicrobial susceptibility testing.
In order to search for virulence determinants, S. enterica isolates were cultivated overnight on Tryptic Soy Agar (Difco) plates at 35ºC, bacterial cells were suspended in 500 μl sterile distilled water and centrifuged for 15 min at 6,000 g.Total deoxyribonucleic acid (DNA) was isolated by a phenol-chloroform method (12) and employed in amplification reactions targeting invA (5) and iroB (3) .Additionally, plasmidial DNA extracted by a protocol proposed by Birnboim & Doly (4) was used for detecting spvC (5) .
About 20 ng of bacterial DNA was used as template for amplification reactions.Details of the protocols are described in Amplicons were resolved in 8% polyacrylamide gels, stained with ethidium bromide, and visualized under UV light.Standard of 100 bp (Life Technologies, Gaithersburg, MD, USA) was used as molecular size marker.Positive, negative, and negative internal controls (sterile water) were included in each batch of reaction.
The results generated were analyzed by using the χ 2 test with Yates' correction or Fisher's exact test.The level of significance was set at 0.05.

rESuLtS
S. enterica was isolated from fecal specimens obtained from five out of 157 (3.2%) children.A total of 53 strains (4 to 22 isolates/child, mean 10.6, median 8.0) of S. enterica were isolated.Data regarding serotyping of S. enterica strains are shown in Table 2.There was no statistically significant association between infection by S. enterica and any epidemiological (seasonal bacterium distribution, gender, and age of the child) or clinical (volume and consistency of stools, frequency of bowel movements, presence of blood, mucus, and pus in stools, fever, vomit, and dehydration) parameters.Four out of five S. enterica-infected children were aged up to 18 months.In regard to clinical data, all S. enterica-positive patients reported unaltered fecal volume and watery stools and four of them had no blood or pus in feces and presented fever.
S. enterica strains obtained from two children (one infected by S. Typhimurium and one by S. enterica 8,20:z4,z23:-) were susceptible to all antimicrobial drugs tested.Isolates from one child (S.Enteritidis) were resistant exclusively to nalidixic acid.Isolates obtained from the other two children (S.Typhimurium) exhibited the following patterns: NAL R CLO R and NAL R CLO R AMP R .When susceptibility to chloramphenicol was evaluated by disk diffusion method, these strains showed divergent (resistant and intermediate) profiles.Data generated by agar dilution technique confirmed that all isolates were resistant to the drug minimum inhibitory concentration (MIC) ≥ 32 µg/ml.ESBL production was not detected.
All S. enterica-positive children were infected by strains harboring invA and iroB.spvC was observed in isolates recovered from two fecal specimens, one positive for S. Typhimurium and one for S. Enteritidis.There was no diversity among strains obtained from the same child concerning the presence of the three virulence markers surveyed.

DiSCuSSion
Salmonella is considered one of the three most relevant agents of inflammatory diarrhea worldwide, affecting mainly those individuals living in areas lacking safe food and water supplies, basic medical care, and adequate nutrition and hygiene practices (23,31) .Despite its relevance, few reports on the prevalence of S. enterica-associated diarrhea are available for Brazil.
Our data demonstrate a low prevalence (3.2%) of S. enterica among children with diarrhea in our region.Similar finding was recently reported for the Northeast region of our country.Nunes and coworkers (29) detected S. enterica in 2% of the study group exclusively among children with diarrhea.
Among more than 2.500 S. enterica serovars, only a few of them seem to be commonly associated with diarrheal disease.Geographical and temporal variations have been observed, but worldwide S. Enteritidis (65%) and S. Typhimurium (12%) have been considered the most common serotypes (7,10,11,13,14,17) .In agreement with these reports, four out of five isolates were serotyped as S. Typhimurium (three) and S. Enteritidis (one).For Latin America the dominance of S. Enteritidis (31%) has been reported (13) .
Patients with salmonellosis usually develop nausea, vomiting, abdominal cramping, diarrhea, and fever.Additionally, the presence of red and white blood cells in feces is common due to the inflammatory nature of the disease (11,23) .In this study we did not detect statistically significant association between S. enterica-associated diarrhea and any of the clinical and epidemiological parameters evaluated.This could be due to the low prevalence of the organism, which may compromise statistical analysis.Fecal leukocytes were detected in 40% of S. enterica-positive fecal specimens, results similarly described by Huicho and coworkers (18) .
In most cases a supportive treatment with no antibiotics is advised for patients presenting uncomplicated salmonellosis.The disease is generally self-limiting and antimicrobial therapy apparently does not alter the clinical course and increases the carrier rate.Notwithstanding, prescription of antimicrobial drugs is necessary in some specific situations (15,25,26,28,30,33) .
As observed in several other infections, the high frequency of multidrug resistance Salmonella strains has made the selection of antibiotics a medical challenge (8,16,23,34,38) .Chloramphenicol, ampicillin, and trimethoprim-sulfametoxazole could be employed for treating patients with salmonellosis.However, due to increasing resistance to these drugs, fluroquinolones and third generation cephalosporins have now been frequently selected (20,24,40,41) .
The high resistance rate to nalidixic acid corroborates data reported by other researchers (29,39) and should be considered a predictable result since the drug is frequently used in Brazil for treating patients diagnosed with inflammatory diarrhea.Only one out of five S. enterica-infected children harbored ampicillin-resistant strains, an unexpected result considering that this antimicrobial has also been largely employed in our country.Nunes and coworkers (29) recently described a 100% resistant rate to the drug.Admittedly, overall drug resistance rates described by these authors were higher than those Acute diarrhea associated with Salmonella enterica in Belo Horizonte-MG: prevalence and characterization of isolates found in this investigation.The lack of resistance to trimethoprimsulfametoxazole is also surprising considering the drug is frequently chosen for treating patients with diarrhea.
The detection of the virulence markers invA and iroB in all S. enterica strains confirms the identification of the organism at the species level and is in accordance with previously reported data.As to spvC, only isolates recovered from two children harbored the gene.spvC is located in the plasmid and it is not found in all bacterium isolates.spvC-positive S. enterica strains tend to be associated with more severe cases of the disease.In this investigation spvC-positive strains were serotyped as S. Typhimurium and S. Enteritidis, which are known to harbor the gene (1,3,5,21,35) .

ConCLuSion
Our data demonstrate that S. enterica infection is uncommon among children from our region and stress the need for monitoring the emergence of resistant strains periodically in order to guide the establishment of empirical antimicrobial therapy when required.Despite the fact that infectious diarrhea is a frequent condition, mainly in resource-poor regions, the etiology of the disease is mostly unknown.S. enterica is one of the most prevalent diarrheagenic agents worldwide commonly associated with severe cases of the disease.Considering the existence of geographic and temporal variations, periodic comprehensive surveys are needed to allow the understanding of the prevalence of different enteropathogenic bacteria.Evaluation of antimicrobial susceptibility profile is also of major relevance to the establishment of specific antimicrobial therapy whenever required.

1 .
Doctor in Microbriology by Universidade Federal de Minas Gerais (UFMG); coordinator of Hermes Pardini Microbiology Sector.2. Doctor in Science (Microbiology) by Universidade Federal do Rio de Janeiro (UFRJ); professor at the Complementary Propaedeutic Department of UFMG Medical School.3. Doctor in Pediatrics and Applied Sciences by Universidade Federal de São Paulo (UNIFESP); professor at the Pediatrics Department of UFMG Medical School.4. Doctor in Medicine (Gastroenterology) by UFMG; associate professor at the Pediatrics Department of UFMG Medical School. 5. Doctor in Science (Microbiology) by UFRJ; associate professor at the Microbiology department of UFMG Biological Science Institute.

tABLE 1 -
PCR conditions employed for the detection of virulence markers of Salmonella enterica PCR: polymerase chain reaction.

tABLE 2 -
Serotyping of 53 Salmonella enterica isolates obtained from five out of 157 children with acute diarrhea