In order to better explain the occurrence of a foodborne disease outbreak in a city in the northwestern region of the State of São Paulo in November 2012, an epidemiological investigation was carried out by the local health surveillance team, with the collection of samples of the ingested food – mayonnaise salad and galinhada (a traditional Brazilian dish of chicken and rice) – and stool samples from five patients. All the tests were performed at the Regional Laboratory Center of Instituto Adolfo Lutz in São José do Rio Preto (RLCIAL).

The food was analyzed according to the methods described in the Compendium of Methods for the Microbiological Examination of Foods – APHA15 for contamination by coliform group bacteria, Staphylococcus aureus, Bacillus cereus, Clostridium perfringens and Salmonella.

The procedures for isolation and identification of Salmonella, were carried out through pre-enrichment of 25g samples by homogenization with 225mL lactose broth (10−1 dilution), and incubation overnight at 36±1°C. Selective enrichment was performed in tetrathionate (TT) broth and modified Rappaport-Vassiliadis (RV) broth, followed by incubation at 36±1°C for 24h and 42°C for 24–48h, respectively. Each enrichment broth was streaked onto selective plates: Salmonella-Shigella agar (SS), brilliant green agar (BG) and xylose lysine deoxycholate agar (XLD), and incubated for 24h at 36±1°C.15

Even though no other biochemical tests were performed, characteristic colonies of each plate were biochemically tested using only IAL medium16 for the presumptive identification of Enterobacteriaceae and incubated for 24h at 36±1°C. Strains with presumptive identification of Salmonella were submitted to serological tests using polyvalent somatic (O) and flagellar (H) antisera produced by the Laboratory of Enteric Pathogens of the Instituto Adolfo Lutz.

The standard methodology for the study of Salmonella15 recommends the use of the presence/absence method in 25g of a food sample. The highest dilution in which Salmonella is demonstrably present in the food sample was used as a complement to the testing, with the purpose of determining which food had the highest microbial load. Albeit important, this is not a quantitative method. For this, we started with 10mL of a 10−1 dilution, serial dilutions of the food samples were performed in tubes containing lactose broth up to a dilution of 10−9. After incubating the tubes for 18–24h at 36±1°C, the presence of turbidity was verified in the different dilutions. The inoculum from all tubes that presented turbidity was submitted to selective enrichment, isolation and identification procedures, pursuant to the APHA methodology.15

Stool samples were collected from a total of five patients by swab and transported in Cary-Blair medium to investigate Escherichia coli, Aeromonas spp., Shigella spp. and Salmonella spp. At RLCIAL, the swabs were seeded in plates with MacConkey Agar (MC), Salmonella-Shigella Agar (SS) and Sorbitol MacConkey Agar (MCS), and incubated for 24h at 36±1°C. Subsequently, the swab was placed in a tube containing 10mL of Tetrathionate (TT) broth and incubated for 24h at 36±1°C for selective enrichment. After this period, the TT broth was inoculated on plates with MC and Brilliant Green Agar. After incubation for 18–24h at 36±1°C, all the plates with the selective medium were examined for colony morphology and for utilization of lactose/sorbitol, which are used to inoculate the IAL medium16 for the presumptive identification of the researched microorganisms.

Strains with presumptive identification of Salmonella were submitted to serological tests using polyvalent somatic (O) and flagellar (H) antisera produced by the Laboratory of Enteric Pathogens, Instituto Adolfo Lutz.

All the isolates of Salmonella from the food and stool samples were sent to the Central Laboratory of the Instituto Adolfo Lutz (CLIAL) for complete serotyping on the basis of somatic O and phase 1 and phase 2 of the H flagellar antigens by agglutination tests with antisera prepared in the Laboratory of Enteric Pathogens, Institute Adolfo Lutz, São Paulo as specified in the Kauffmann–White protocol for Salmonella serotyping.17

Antimicrobial susceptibility testing was performed for all isolates using the disk diffusion method according to the guidelines of the Clinical and Laboratory Standards Institute – CLSI.18 The following antimicrobial disks (Oxoid) were used: nalidixic acid (30μg), amoxicillin–clavulanic acid (20/10μg), amikacin (30μg), ampicillin (10μg), aztreonam (30μg), ceftazidime (30μg), cefotaxime (30μg), ceftriaxone (30μg), cefepime (30μg), ciprofloxacin (5μg), chloramphenicol (30μg), streptomycin (10μg), gentamicin (10μg), imipenem (10μg), trimethoprim–sulfamethoxazole (1.25/23.75μg), sulfonamide (250μg), and tetracycline (30μg). Categorization of the diameter of halos in susceptible, intermediate or resistant followed CLSI recommendations.18

Minimum inhibitory concentrations (MIC) were determined for nalidixic acid and ciprofloxacin by Etest (AB Biodisk, Solna, Sweden) according to the manufacturer's recommendations. The range of MIC of ciprofloxacin for Salmonella was recently changed to susceptible: ≤0.06μg/mL; intermediate susceptible: 0.12–0.5μg/mL; resistant: ≥1μg/mL.18

E. coli ATCC 25922 and E. coli ATCC 35218 were used as reference strains for antimicrobial susceptibility testing.

Pulsed field gel electrophoresis (PFGE) analysis was performed for all the isolates at CLIAL according to the Centers for Disease Control and Prevention (CDC) PulseNet protocol (www.cdc.gov/pulsenet/pathogens/index.html). Briefly, cell lysis was followed by proteinase K treatment and DNA restriction with XbaI (New England Biolabs, Ipswich, MA). Electrophoresis was performed with a CHEF DRIII system (BioRad Laboratories Inc., Hercules, CA) using the following run parameters: a switch time of 2.2–63.8s and a run time of 20h. Salmonella Braenderup H9812 was used as a molecular size marker.19 TIFF images were analyzed using the BioNumerics 5.0 software (Applied Maths). Dice's coefficient with tolerance of 1.5 was used to calculate similarity using the Unweighted Pair Group Method with arithmetic averages (UPGMA).

Of the 94 people interviewed after the foodborne outbreak, the epidemiological investigation found that the consumption of mayonnaise salad and galinhada was common to the entire group; 66, both children and adults, had become ill. The median incubation period was 72h, and the main symptoms observed were: diarrhea 63/66 (95.4%), abdominal pain 50/66 (75.7%), nausea 40/66 (60.6%), fever 27/66 (40.9%), vomiting 23/66 (34.8), and headache 22/66 (33.3%). Attack rates by age group are shown in Table 1.

According to the investigation, a 60-year old female patient who was hospitalized in a serious condition, developed septic shock and died two days after consuming the food.

The presence of Salmonella was confirmed in all the analyzed stool samples and in both types of food, consequently it was isolated in dilutions of 10−7 and 10−2 of the salad mayonnaise and galinhada, respectively. No other pathogens were isolated from the food or stool samples.

The Most Probable Numbers (MPN) of thermotolerant coliforms found in the mayonnaise salad and galinhada samples were >2400/g and 240/g, respectively.

All the strains isolated from human and food sources were identified as Salmonella enterica serovar Alachua by agglutination tests.

All the Salmonella Alachua strains demonstrated resistance to nalidixic acid and reduced susceptibility to ciprofloxacin (intermediate resistant). However, all of them were susceptible to the other antimicrobials tested.

The seven strains showed higher MIC values for nalidixic acid (≥256μg/mL) and reduced ciprofloxacin susceptibility (MIC=0.5μg/mL).

A dendrogram, generated by PFGE patterns of Salmonella Alachua strains using XbaI as the restriction enzyme, is shown in Fig. 1. One PFGE pattern was identified among the Salmonella Alachua clinical and food isolates analyzed. The genetic relatedness among the strains was 100%.

Fig. 1.

Dendrogram pulsed field gel electrophoresis patterns of Salmonella Alachua strains. LabID (identification number) and source type (human or food) of the Salmonella Alachua strains analyzed. Marker: Salmonella Braenderup H9812 digested with XbaI enzyme was used as a molecular size.

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