Extended Spectrum Beta-Lactamase Production and Biofilm Formation in Salmonella Serovars Resistant to Antimicrobial Agents

I Programa de pós-graduação em Bioexperimentação, Universidade de Passo Fundo, Passo Fundo, MG, Brazil. II Programa de pós-graduação em Farmacologia e Terapêutica, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil. III Centro de Diagnóstico e Pesquisa em Patologia Aviária, Faculdade de Veterinária, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil. IV Programa de pós-graduação em Ciências Veterinárias, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil.


INTRODUCTION
Salmonella strains adapt to adverse environmental conditions and can survive in the environment for long periods, colonizing domestic animals and humans.The most common sources of infection among humans are products of poultry origin, dairy products, and surfaces in contact with contaminated products reused without proper disinfection, which could stimulate biofilm formation (Murray et al., 2015).Biofilm promotes bacteria survival in stressful environments, like slaughterhouses and food-processing plants (Steenackers et al., 2012).Biofilms favor greater resistance to antimicrobials and disinfectants (Steenackers et al., 2012).Antimicrobial resistance is one of the major public health problems worldwide and microorganisms isolated from foods, especially Salmonella spp., show high resistance to these agents (Markle et al., 2015).A global increase in extended spectrum betalactamase (ESBL) producing is likely to be occurring (Ziech, 2015;ECDC, 2016).
Therefore, the present study assessed antimicrobial resistance, ESBL production, and biofilm formation by Salmonella serovars isolated from poultry slaughterhouses.

Salmonella strains
The tested Salmonella serovars were previously isolated (Santos et al., 2015;Mion et al., 2016) from ten poultry slaughterhouses under federal inspection system between 2012 and 2014 from the northern region of the state of Rio Grande do Sul (Table 1).The samples were stored in brain heart infusion broth (Oxoid®, United Kingdom) and frozen at -18 o C.
The disk diffusion assay was used for testing ESBL production (CLSI, 2015), using amoxicillin+clavulanate placed at the center of the Mueller Hinton and three other disks were placed within a 20 mm radius of the first one: aztreonam, cefotaxime and ceftazidime.ESBL production was considered positive when the formation of "inhibition zones" or an increase in inhibition halo size was observed.The quality control of antimicrobials was made with E. coli ATCC 25922 and the positive control for ESBL was a K. pneumoniae ATCC 700603 strain.

Biofilm-forming assay
Biofilm-forming ability was assessed on polystyrene plates (Rodrigues et al., 2009) at the following incubation temperatures: 3±1ºC (temperature used for refrigeration of meat cuts), 9±1ºC (cutting room temperature for export to the European Union), 25±1ºC (ambient temperature), 36±1ºC (optimal pattern for the growth of mesophilic), and 42 ºC±1ºC (temperature for selective enrichment due to thermotolerance of Salmonella).The arithmetic mean of the absorbance values for each sample (ODa) was compared with the average absorbance of the wells containing sterile TSB (OD) and the following classification was used to determine the level of adhesion: non-adherent (ODa≤OD), weakly adherent (OD<ODa≤2OD), moderately adherent (2OD<ODA≤4OD), strongly adherent (4OD<Oda).

RESULTS AND DISCUSSION
Table 2 displays the susceptibility profiles of Salmonella serovars, ESBL production, and their capacity to adhere to polystyrene at different incubation temperatures.
Eight out of the 10 serovars were resistant to sulfonamides.High resistance to these drugs is probably related to their extensive use, leading to an increase in the selective pressure on resistant strains and in the dissemination of resistance genes (Machinski Junior et al., 2005;WHO, 2011;ECDC, 2016).Also, 40% of serovars were resistant to enrofloxacin, which belongs to the class of fluoroquinolones and is exclusively used in veterinary medicine; however, despite its restricted use, it has been shown that enrofloxacin-resistant bacteria can develop resistance to ciprofloxacin.Fluoroquinolones and quinolones play a crucial role in human clinical practice, as they are used in the treatment of severe infections and are the main treatment option against salmonellosis (Souza et al., 2010;WHO, 2011).
Resistance to chloramphenicol was found to be 20%, even though it cannot be used in animal production in Brazil since 2003(Brasil, 2003).As this antimicrobial was used for many years in veterinary medicine, resistance to it might not have been reversed yet, or else, florphenicol, which is exclusively used by veterinarians, could be favoring the transmission of resistance genes that are common to these two antimicrobials (Nógrády et al., 2012;Mattiello et al., 2015).

Extended Spectrum Beta-Lactamase Production and Biofilm Formation in Salmonella Serovars Resistant to Antimicrobial Agents
eRBCA-2018-0913 All tested serovars were sensitive to tetracycline.Serovar Brandenburg was sensitive to all tested drugs.Serovars Anatum, Lexington, and Panamá; Agona and Tennessee; Schwarzengrund; and Infantis were considered moderately resistant to sulfonamides, enrofloxacin, chloramphenicol, and gentamicin, respectively.These are alarming findings as the assessed antimicrobials are widely used in veterinary and human medicine and also because the use of drugs with moderate resistance could result in the selection of a resistant bacterial population (Machinski Junior et al., 2005).
The number of Salmonella serotypes showing ESBL production in the current study was lower than in previous studies (Gelinski et al., 2014).Only serovars Infantis, Panamá, and Tennessee phenotypically demonstrated ESBL production, accounting for 30% of the assessed serovars.The prevalence of ESBL production strains varies worldwide.However, the incidence has increased greatly in recent years in several countries.In the study undertaken by Ziech (2015) in Brazilian samples, 45% (44/98) of Salmonella isolates collected from birds were ESBL producers.ESBL production has emerged in serovar Infantis in Italy and Switzerland (Franco et al., 2015;Hindermann et al., 2017).Serovars Panama and Tennessee have also been reported as ESBL producers (Weill et al., 2004;Gelinski et al., 2014).ESBL-producing strains have also been described in serovars Heidelberg, Senftenberg, Newport, Enteritidis, Weltevreden, Indiana, Typhimurium, Litchfield, Oranienburg and Typhi (Jure et al., 2010;González-López et al., 2014;Bae et al., 2015;Djeffal et al., 2017).
Salmonella serovars can adhere to polystyrene, leading to biofilm formation.It is of public health concern, since strains which remain in slaughter plants and in food products could produce biofilms and favor dissemination through breakage of these structures and consequent release of pathogenic microorganisms.
Note that serovars Panamá; Anatum, Infantis and Schwarzengrund were moderate biofilm producers at 3ºC and 9ºC, respectively, demonstrating a possible adaptation of these serovars to these temperatures.These findings are in line with those of Rodrigues et al. (2017), who observed biofilm formation at these temperatures, with strongly adherent S. Enteritidis strains.These findings are relevant because these serovars adhered moderately to polystyrene at unfavorable temperatures for the multiplication of Salmonella spp., which is recommended as a conventional method for food preservation under refrigeration (Gast, 2008).
The results obtained in the present study allow inferring that even if the recommended temperatures are complied with, there could be multiplication of Salmonella spp.and biofilm formation in slaughterhouses and consequent contamination of the final products, which may lead to foodborne diseases.In addition, serovars Infantis, Panamá, and Tennessee were found to produce ESBL.This is a public health problem, which highlights the need of caution in the use of antimicrobials.Legend: AMP = Ampicillin 10 μg, CLO = Chloramphenicol 30 μg, GEN = Gentamicin 10 μg, ENO = Enrofloxacin 5 μg, SUT = Sulfonamides 300 μg, TET = Tetracycline 30 μg.ESBL = Amoxicillin+Clavulanate 20/10 μg, Aztreonam μg, Ceftazidime 30 μg, Cefotaxime 30 μg.

Table 1 -
Salmonella serovars and source of poultry slaughterhouse isolates.

Table 2 -
Sensitivity of Salmonella serovars, ESBL production, and adhesion to polystyrene at different incubation temperatures.