Phenotypic and genotypic characterization of Salmonella spp . isolated from foods and clinical samples in Brazil

Increasing antimicrobial resistance in Salmonella species has been a serious problem for public health worldwide. This study examines Salmonella spp. recovered from foods and clinical samples on serotype, antimicrobial resistance and PFGE genotypes. It identified 91 salmonellae, belonging to 31 different serotypes, from 36 isolates from food and 55 clinical samples. Salmonella Infantis (16.5%) and Salmonella Enteritidis (13.7%) are the most common among food isolates, whereas Salmonella Enteritidis (29.0%) and Salmonella Typhimurium (16.0%) mainly causes human salmonellosis. Antimicrobial susceptibility data showed that 63.0% of the isolates were fully susceptible to 12 antibiotics tested. Nalidixic acid showed high resistance rates, 32.7% and 25.0% of the clinical isolates and food, respectively. Three main PFGE types: A (Salmonella Enteritidis), B (Salmonella Infantis) and C (Salmonella Schwarzengrund) comprised isolates recovered from foods and clinical samples. Our results indicate that the clonal groups were both causing diseases and food contamination, emphasizing the need to maintain a system of surveillance for foodborne disease.


INTRODUCTION
Salmonella is a leading cause of food-borne disease being an important health problem in industrialized and developing countries (Chu et al. 2009, Abbassi-Ghozzi et al. 2012, Yang et al. 2013).In recent years, serotypes of Salmonella isolated from foods have shown an increase in antimicrobial resistance, particularly those of animal origin.Moreover, an increased incidence of human salmonellosis due to multidrug-resistant strains have been reported (Lu et al. 2011, Wang et al. 2013).
There has been increasing concern over the past 30 years about the worldwide emergence of multidrug-resistant phenotypes among Salmonella ANDREA L. MIRANDA et al. serovars, in particular Salmonella Typhimurium and, more recently, Salmonella Newport as well as several other serovars.The levels and extent of resistance vary in different regions and are influenced by antimicrobial use in humans and animals as well as geographical differences in the epidemiology of Salmonella (Weinberger and Keller 2005, Zhao et al. 2008).
Most individuals with gastroenteritis present no serious clinical conditions and do not need hospitalization, so it is difficult to isolate the causative agents and identify the sources of contamination, thus underestimating the number of cases of salmonellosis worldwide.In Brazil, during the period of 2001 to 2006, a total of 3,749 cases of typhoid fever were reported with 30 deaths, the highest number recorded in the North and Northeast regions, especially in the states of Acre (741 cases), and Bahia (671 cases) (Brazilian Ministry of Health 2009, Souza et al. 2010).
However, sporadic studies and limited information on Salmonella food contamination and microbiological investigation in foodborne infections have not clarified uncertainties regard of the salmonellosis epidemiology in Brazil (Campioni et al. 2012).Therefore, the current study evaluates the prevalence of antimicrobial susceptibility and genotypes of Salmonella spp.isolated from food and human in the state of Bahia (Northeast region) from 1996 to 2012.

SAMPLES
This study analyzed 91 isolates of Salmonella spp.from various cities in the State of Bahia Brazil, 36 isolated from food (meat, fish, chicken and pork, cheeses, salads, rice, ready-to-eat foods and desserts).Food Isolates came from the Laboratory for Food Microbiology Research, Federal University of Bahia and the Central Public Health Laboratory of Bahia (LACEN / BA).Merchants and local health surveillance officers sent food samples to the laboratories with suspected food infection outbreaks, isolating them for identification of Salmonella.Isolated from clinical specimens (55 isolates) were isolated from human feces (n = 43), cerebrospinal fluid -CSF (n = 10), and blood (n = 2) of patients treated at four hospitals in the city of Salvador, Bahia.Invasive CSF samples were obtained from patients with meningitis and blood and stool samples were obtained from patients with gastroenteritis suspected.The information provided by hospitals or food laboratories were referring to the year of isolation, source (feces, blood, CSF or food) and city of origin.

SEROLOGICAL IDENTIFICATION
The isolates were serotyped by the slide agglutination method using antisera prepared at the National Reference Laboratory for Enterobacteria of the Oswaldo Cruz Foundation in Rio de Janeiro (Fundação Oswaldo Cruz -FIOCRUZ-RJ).

PFGE TYPING AND ANALYSIS
Chromosomal digests generated by XbaI were prepared and analysed as described previously (CDC 2010).Fragments were separated in 1.0% agarose gels using using CHEF-DRII apparatus (Bio-Rad Laboratories, Hercules, CA) with pulse times of 2.2 to 63.8s for 20h at 14°C and 6 V/cm in TBE buffer.Restriction profiles were analysed using GelCompar II (Applied Maths, St. Martens, Belgium).To develop the dendrogram, the study used an unweighted pair-group with arithmetic mean (UPGMA) method and the Dice similarity coefficient, with a position tolerance coefficient of 1.5%.Isolates with ≥ 90.0% relatedness were considered a cluster.

RESULTS
Out of the 91 samples investigated, it was possible to identify 20 serotypes (Table I) from a set of isolates from food, of which 16.5% (6 of the 36) corresponded to serotype Salmonella Infantis (Figure 1).Among isolates obtained from clinical samples, this study identified different serotypes.Salmonella Enteritidis was predominant with 29.1% (Figure 1).Serotype diversity was lower among clinical isolates than in foods samples (Table II).
The study identified 47 PFGE types, including 11 clonal groups, ranging from 2 to 20 isolates.The isolates were grouped in two major clusters designated as PFGE A (20 isolates) and PFGE B (11 isolates).In the PFGE A cluster, 19 isolates of Salmonella Enteritidis and one Salmonella Infantis exhibited similarity above 90.0%(Figure 3).

DISCUSSION
Salmonella is an environmentally persistent pathogen that that survives and multiplies in diverse environments, and is one of the most common bacteria in food-borne diseases in humans.The CDC estimated that, each year, there are approximately one million cases of salmonellosis caused by non-typhoidal Salmonella (CDC 2011).In Brazil, salmonellosis has been substantially underestimated, thus, this study brings important ANDREA L. MIRANDA et al.
contribution to public health in Brazil and all over the world.
Derived food products such as poultry meat and eggs are the main forms of infection by Salmonella spp. in humans (Gantois et al. 2009).A large number of serovars of Salmonella are pathogenic for humans, so the ability to genus Salmonella to cause a disease depends on several virulence determinant factors.Therefore, there is a less diverse serotypes among clinical isolates than food samples.Higher frequencies of Salmonella Enteritidis and Salmonella Typhimurium were also found in other studies in Brazil and elsewhere, and they pose a threat to human health (Silva and Duarte 2002, Mürmann et al. 2009).
Salmonella Enteritidis was the most common serotype in the country.In 1993, Salmonella Enteritidis accounted for approximately 10.0% of the Salmonella serotypes isolated from human samples, and from then on it became the dominant serotype in both human and non-human samples (Taunay et al. 1996).In 1994 and 1995, it accounted for 43% and 65% of Salmonella Enteritidis isolated from humans, respectively, in Sao Paulo   b c (Taunay et al. 1996).Modifications in serotype distribution reflect changes in animal production and the spreading of new serotypes due to world trade.Of a particular concern is the emergence of resistance to multiple antimicrobials.Resistance to quinolones, such as nalidixic acid, has become a significant problem for public health and has been increasing in recent years, mainly due to the liberal use of quinolone drugs in aquaculture and industrial animal production (Oliveira et al. 2005).
In contrast, antibiotics such as aminoglycosides, florfenicol and nalidixic acid, not commonly used in animal health and production showed no resistance (Piras et al. 2011, Li et al. 2014).
Antimicrobial resistance to at least one antibiotic was identified in 40.0% and 33.0% of clinical and food isolates, respectively.In the Southern Brazil, 82.0% of Salmonella samples of fresh pork sausage were resistant to at least one antibiotic tested (Mürmann et al. 2009).In the US, 47.0% of Salmonella Heidelberg isolates from human patients were resistant to antimicrobials (Han et al. 2011).Salmonella multidrug resistance is a growing concern since it limits treatment options for invasive disease, and it could lead to the transfer of resistance to other pathogenic organisms.In this study, a relative high frequency of multidrug-resistant Salmonella (25.0% of clinical isolates and 11.0% of food isolates) represents an additional public health risk, and Salmonella Schwarzengrund, in special, seemed to be resistant to six or more antibiotics.
The current gold standard method used to evaluate relatedness among Salmonella strains is PFGE has been established as important for the discrimination, characterization, and investigation of food-borne outbreaks (CDC 2010).Thus, PFGE fingerprinting shows several foods isolates that clustered with clinical isolates, mainly Salmonella Enteritidis and Salmonella Infantis among cities more than 500 miles apart, with persistence and geographical transmission in the Bahia State.However, other studies have described the limited discriminatory power of PFGE for Salmonella Enteritidis (CDC 2010, Mezal et al. 2014).A combination of different typing methods may be better to achieve best resolution for the discrimination of highly clonal organisms.
The combined use of different patterns of resistance and genotypes to analyze pathogenic isolates from food has been a useful way to identify the origins of outbreaks, establish sources of contamination, determine the etiological agents responsible for outbreaks, understand the epidemiology of food-borne disease and facilitate further studies on the spread of antibiotic-resistant isolates.This study provides data that indicate a probable transmission of Salmonella from food sources to cause infections in humans, establishing a serious risk to public health.
In this study, the most prevalent serotype in food samples was Salmonella Infantis and clinical samples of Salmonella Enteritidis.The most frequently observed resistance profiles were the NLX (nalidixic acid) and multi-resistance AMP / CFL / CRO / CTX / TET / TMS / NLX * (ampicillin, cephalothin, ceftriaxone, cefotaxime, tetracycline and trimethoprim / sulfametazol, nalidixic acid), common to isolates from food and clinical samples.In addition, clonal groups, found among strains epidemiologically unrelated, suggested that some isolated persisted in the environment over the years in different cities of Bahia.Thus, our results indicate that the clonal groups were both causing diseases and food contamination, emphasizing the need to maintain a system of surveillance for foodborne disease.

Figure 2 -
Figure 2 -Resistance of Salmonella serotypes in food and clinical samples to a variety of antimicrobials.X-axis represents the number of antimicrobials to which strains are resistant.Isolates from human and food were grouped according to the number of antimicrobial agents they were resistant.

Figure 3 -
Figure 3 -Dendrogram representing genetic relationships among Salmonella Enteritidis (a) strains based on PFGE.The data were sorted by the UPGMA method.NLX (nalidixic acid).

TABLE I (continuation) Figure
1 -Distribution of Salmonella serotypes in food and clinical samples.An Acad Bras Cienc (2017) 89 (2) Salmonella OF FOOD AND CLINICAL SAMPLES 1147