Effect of Antimicrobials on Salmonella Spp. Strains Isolated from Poultry Processing Plants

The routine use of antimicrobials in animal production for the treatment of infections, disease prevention, or as growth promoters is a predisposing factor for the development and dissemination of antimicrobial resistance. In food industries, sanitizers are used for the control of microbial colonization, and their efficacy depends on contact time and on the dilution of the products used. The present study assessed the effect of 12 antimicrobials and four commercial sanitizers on 18 Salmonella spp. strains isolated from poultry processing plants. None of the evaluated antimicrobials was 100% effective against the tested Salmonella spp. strains; however, 94% of the isolates were susceptible to ciprofloxacin, 77% to amoxicillin + clavulanic acid and to ampicillin, and 72% to enrofloxacin, whereas 100% of the isolates were resistant to penicillin G, 16% to tetracycline, and 11% to sulfonamide. The tested Salmonella spp. strains were 100% inhibited by peracetic acid after five minutes of contact, 0.5% by quaternary ammonium after 15 minutes, and 85.7% by chlorhexidine after 15 minutes. The results indicate the importance of testing of efficacy of antimicrobials used in animal production and in public health to monitor their action and the development of resistance.


INTRODUCTION
The routine use of antimicrobials in animal production for the treatment of infections, disease prevention, and as growth promoters, may lead to the development and dissemination of antimicrobial resistance, which is later transmitted to human beings in the food chain (EFSA, 2014).
The sanitation standard operating procedures (SSOP) applied in processing plants aim at controlling the dissemination of microorganisms and the formation of biofilm on the surfaces with which the food has contact; however, bacteria of the genus Salmonella are known for their adhesion to surfaces and resistance to sanitizers (Rodrigues et al., 2013).The most commonly products used in SSOP in poultry processing plants are peracetic acid, quaternary ammonium, and chlorhexidine.The selection and use of sanitizers are crucial for the reduction of microbial counts.However, the degree of hygiene and type of surfaces of processing plants bacterial growth (Baltreme, 2014), and should be known when testing the efficacy of sanitizers applied in such facilities.
This study evaluated the efficacy of 12 antimicrobials and four commercial sanitizers on 18 Salmonella spp.strains isolated from poultry processing plants.Sul, Brazil, between 2012 and2014.A total of 1,071 samples were collected, out of which 18 (1.68%)were positive for Salmonella spp.The samples positive for Salmonella spp were determined in cloacal swabs (n=6), transport crate swabs after washing (n=3), carcasses after plucking (n=1), carcasses after the first wash (n=1), plucked and washed carcasses (n=1), eviscerated carcasses (n=1), at the exit of the chiller (n=1), after final wash (n=1), carcasses chilled at 4ºC (n=1), carcasses frozen at -12º for 24 hours (n=1), and carcasses frozen at -12ºC for 60 days (n=1).The samples were processed at the Laboratory of Bacteriology and Mycology of the Veterinary Hospital of the University of Passo Fundo (HV-UPF), Brazil, using Salmonella Enteritidis ATCC 13076 as positive control.

MATERIALS AND METHODS
Cloacal swabs were taken from 300 broilers in each collection, using one swab for every two birds, making up a pool of 50 swabs that were stored in flasks containing 50 mL of buffered peptone water (BPW 1.0%, HiMedia ® ).In the laboratory, pools were homogenized and 10-mL aliquots were used for Salmonella spp isolation.
Transport crates were sealed with official labels and sampled by rubbing 3M ® sponge sticks with neutralizing buffer across the inner side of the crate before and after washing and disinfection.The sponges were placed the collection bags provided by the manufacturer and 50 mL of 1.0% BPW was added; a 10-mL aliquot was used for Salmonella spp isolation.
Birds and carcasses were placed in individual plastic bags sealed with official labels and rinsed with 400 mL of 1.0% BPW, and a 10-mL aliquot was used for analyses.
For testing the efficacy of sanitizers, according to Beltrame (2009), sanitizers were tested at the dilutions routinely used in processing plants, as follows: 0.5% chlorhexidine, 0.5% and 1% quaternary ammonium, and 1% peracetic acid, were utilized.The bacteria previous stored at -20 o C in BHI broth with 20% glycerin were inoculated in BHI broth (HiMedia ® ) and incubated at 37°C for 18 hours.Subsequently, 100μL of the Salmonella spp.culture were added to sterile tubes containing 9 mL of the sanitizer at the tested concentration and 1 mL of UHT whole milk (to simulate the presence of organic matter).After contact times of 1, 5, 10 and 15 min, 10-μL aliquots were transferred to 5.0 mL of BHI broth and incubated for 96 hours at 37°C.The bacteria were considered resistant (R) when the culture medium presented turbidity, film formation on the surface, or precipitation, and susceptible (S) in the absence of turbidity.In this case, positive samples were seeded onto brilliant green agar plates to check bacterial viability.

RESULTS AND DISCUSSION
None of the tested antimicrobials was 100% effective against the isolated Salmonella spp.(Figure 1).
Chloramphenicol is used to treat human salmonellosis, due to its low cost and adequate therapeutic response (Alecrim et al., 2002).Colla et al. (2012)   Tetracyclines are one of the most widely classes of antimicrobials therapeutically in livestock (Wilson, 2004).In the present study, 61.1% of the isolates were resistant to this drug, indicating that its continuous use has increased microbial resistance, consequently reducing the available therapeutic options.Tetracyclines and aminoglycosides (gentamicin and streptomycin), amoxicillin with clavulanic acid, ampicillin, and ciprofloxacin are considered by the World Health Organization to be critically important for human medicine (WHO, 2011).The resistance of bacteria to such drugs has increased (EFSA, 2014), and represent a substantial cost to public health as resistant bacteria are more harmful to patients than susceptible strains of the same species (Balsalobre et al., 2014).Therefore, increasing rates of multi-resistant bacteria represent a potential public health hazard (Chiappini et al. 2002, Zimermann 2008).
In a survey carried out in 2010 in the European Union, Salmonella spp.isolated from poultry meat and showed 27%, 24%, 24%, 21%, 20%, 4%, 3% and 2% resistance to sulfonamide, ciprofloxacin, nalidixic acid, ampicillin, tetracycline, cefotaxime, chloramphenicol, and gentamicin respectively, which are similar to the rates detected in Salmonella strains isolated from humans.In the European Community, fluoroquinolones are used as first-line treatment against salmonellosis in adults, while third-generation cephalosporins are used in children, yielding resistant strains due to misuse and resulting in inefficient therapy, also causing multiresistance (EFSA 2014).
Drug multiresistance is described by the National Antimicrobial Resistance Monitoring System (USDA, 2012) as resistance to three or more classes of antimicrobials, and the ACSSuT R-type stands for the resistance to ampicillin (A), chloramphenicol (C), streptomycin (S), sulfonamide (Su), and tetracycline (T) (Reis et al., 2011).In the present study, a Salmonella spp.isolate from a cloacal swab presented intermediate resistance to ampicillin and resistance to ceftiofur, chloramphenicol, streptomycin, gentamicin, neomycin, penicillin g, sulfonamide, and tetracycline, suggesting that broiler flocks on the farm may host ACSSuT multiresistant Salmonella strains (Figure 2).
Considering the possible transfer of such resistance to the human population, the European Union banned the use of antibiotics as growth promoters in animal production as of January 2006.Because Brazil is one of the main poultry exporters to the EU, Brazilian companies needed to comply with EU legislation (Lorenço et al., 2007).In July 2009, the Brazilian Ministry of Agriculture (Brasil, 2012) banned the use of amphenicols, tetracycline, beta-lactams (benzylpenicillins and cephalosporins), quinolones, and sulfonamides as performance enhancers or as food preservers, limiting their use to veterinary treatments.Finally, in May 2012, the use of spiramycin and erythromycin as performance enhancers in animal production was also banned (Brasil, 2012).
The in-vitro assessment of the effect of commercial sanitizers against Salmonella spp.strains showed that only peracetic acid was 100% effective after 5 min of contact (Figure 3).Peracetic acid quickly acts against bacteria and it is more effective against biofilm formation because of its higher inhibitory potential of bacteria and in a shorter time of exposure compared with other sanitizers (Rodrigues et al., 2013).The stronger inhibitory effect of 1% quaternary ammonium (42.9% to 100%) compared with 0.5% (57.2% to 85.7%), as shown in Figure 3, emphasizes the need to test the efficacy of disinfectants sold by different manufacturers, as commercial formulations may present different dilutions of the active ingredients.The weak effect of chlorhexidine may be due to its continuous use in dental, medical, and veterinary treatments since the 1950s, producing resistance because of its prolonged use and its inappropriate contact time and dilutions (Colla et al., 2014).These results highlight that the effect of sanitizers against Salmonella strains is associated with practical situations, particularly with the presence of organic matter, contact time, and with the resistance developed by bacteria to the most common disinfectants.

CONCLUSION
None of the tested antimicrobials was 100% effective against the Salmonella spp.strains.Peracetic acid presented the best sanitizing performance.These results show that strategies to reduce contamination risks in the food chain based in careful use of antimicrobial drugs and monitoring of resistance to antimicrobials and sanitizers in animal production need to be applied.
isolated from swine carcasses, whileMion et al. (2014) observed 100% efficacy of chloramphenicol, enrofloxacin, ciprofloxacin, and streptomycin against Salmonella Heidelberg isolated from poultry processing plants between 2005 and 2009.However, in the present study, only ciprofloxacin was more than 90% effective against Salmonella spp isolated between 2012 and 2014, suggesting a possible development of resistance of this bacterium during this period.

Figure 1 -
Figure 1 -Antimicrobial sensitivity of 18 Salmonella spp.samples isolated from poultry processing plants.

Figure 2 -Figure 3 -
Figure 2 -Multiresistance of 18 Salmonella spp.samples isolated from poultry processing plants according to the number of antibiotics (ATBs)

Effect of Antimicrobials on Salmonella Spp. Strains Isolated from Poultry Processing Plants
Salmonella spp.strains were isolated from seven broiler processing plants under federal inspection services in the state of Rio Grande do reported 25.6% resistance and 64.1% of intermediate resistance to this drug in Salmonella Typhimurium and Panama serovars, respectively,