Sensitivity to different antimicrobials of salmonella typhimurium isolated from eggs for human consumption

The genus Salmonella spp. is comprised of micro-organisms that are widely spread around the world (Ruiz B et al., 2006). Approximately 2000 serotypes of Salmonella have been associated with enterocolitis, and Salmonella enterica, subspecies enterica, serovar Typhimurium (ST) and Salmonella enterica, subspecies enteric, serovar Enteritidis (SE), the main etiological agents of human food-borne salmonellosis (Yang et al., 2002). Most infections caused by Salmonella spp. are the result of the ingestion of contaminated meat of chicken, beef, pork, eggs and milk (Ruiz B et al., 2006). The impact on public health due to ST infections is increasing, due to the emergence of resistance to antimicrobials by this serovar, related to the circulation of multi-resistant clones (Graziani et al., 2007). The main cause of resistant bacteria is the excessive use of antibiotics in animal rations, as growth promoters, and also the indiscriminate treatment of people and animals through medical or veterinary prescription (Casawell et al., 2003). Detection and monitoring of the ST and SE multi-resistence is important to modify the choice of antibiotics for the treatment of salmonellosis, and to assess the risk of expansion of multi-resistant strains (Yang et al., 2002). For these reasons, it is important to study profiles of ST resistance of both animal and human origin, because it will allow a better understanding of the epidemiology of the involved clones (Graziani et al., 2007).

The aim of our work was to assess the sensitivity to different antimicrobials used in human and veterinary medicine, of ST strains isolated from eggs for human consumption.

Bacterial strains. 47 ST strains isolated from pools of pools (2), egg white and yolk (12), egg white (15) and/or yolks (18) of eggs sold in supermarkets in the province of Entre Ríos, Argentina were used. Escherichia coli ATCC 25922 was used as control strain.

Analysis of antimicrobial susceptibility. We used the method of on-plate dissemination, according to the CLSI (National Committee for Clinical Laboratory Standards, 2010) recommendations. From a bacterial suspension corresponding to a 0.5 tube in the Mc Farland scale, a sterile swab was absorbed and planted on Mueller-Hinton (Difco, USA) agar plates, and incubated aerobically at 37 ° C for 24 hours. 25 antimicrobials were analyzed (OXOID, England): ceftazidime (CAZ 30µg), cefotaxime (CTX 30µg), cefoxitim (FOX 30µg), cefixime (CFM 5µg), cephalothin (KF 30µg), gentamicin (CN 10µg), streptomycin (S 300µg), amicacin (AK 30µg), neomycin (N 30µg), kanamycin (K 30µg), nalidixic acid (NA 30µg), norfloxacin (NOR 10µg), ciprofloxacin (CIP 5µg), enrofloxacin (ENR 5µg), doxycycline (OJ 30µg), tetracycline (TE 30µg), imipenem (IMP 10µg), ampicillin (AMP 10µg), sulfamethoxazole-trimethoprim (SXT 25µg), tigecycline (GCT 15µg), Chloramphenicol (C 30µg) florfenicol (FFC 30µg), phosphomycin (FOS 50µg), colistin sulfate (CT 10µg) and amoxicillin - clavulanic acid (AMC 30µg). The diameter of the growth inhibition halo was measured and sensitivity was determined according to the recommendations of the CLSI. On this basis, the strains were classified as sensitive, of intermediate sensitivity or resistant.

Table 1. Sensitivity of Salmonella Typhimurium to different antimicrobials used in human and veterinary medicine.

The ST strains showed different sensitivity to the antimicrobial drugs tested (Table 1). The resistance went from 0% (AMC, NOR, CIP, ENR, FFC, CN, IMP, TGC, FOS) to 97.9% (S). In the group of β-lactam and β-lactam + β-lactamase inhibitors, it was observed that 100% of the strains were sensitive to AMC; however, 44.7 per cent were sensitive to AMP. With regard to antimicrobials of the quinolones group (NA NOR, CIP, ENR), most of the strains were sensitive to one or more antibiotics in this group. 100% were sensitive to NOR and CIP, both 2nd generation quinolones; while for NA and ENR sensitivity was 95.7% and 87.2%, respectively. For the tetracycline group (DO and TE), a high pattern of resistance ranging from 68.1% for DO to 93.6% for TE was observed. With regard to phenicols (C and FFC), a sensitivity higher than 93% was observed.
In the group of aminoglycosides, all strains were sensitive to CN, while 80.9% of the bacteria were sensitive to AK. For N and K, we found a sensitivity of less than 30%. 97.9% were resistant to S. In the cephalosporin group, 1st generation (KF), 2nd generation (FOX), and 3rd generation (CAZ, CTX and CFM) cephalosporins were evaluated. The sensitivity of strains to CAZ, CFM, and FOX was 70.2%, 76.6 and 87.2%, respectively. For CTX a 51.1% intermediate sensitivity was observed and 25.5% of drug-resistant strains, while 48.9% of strains were sensitive strains to KF and 42.6% of strains showed intermediate behavior. 100% of strains were sensitive to the Carbapenemes (IMP) group. Antimicrobial folate pathway inhibitors (SXT), glycylcyclines (GCT), fosfomicyns (FOS) showed a sensitivity higher than 78%. In the polimixines group (CT) 51.1% and 46.8% of intermediate and sensitive strains was observed, respectively.
The presence of multi-resistance to two or more antibiotics was observed in 98% of the tested strains; multi-resistances occurred within the same group or in different groups of antibiotics; however, the average resistance was 15%; no cross-resistance was observed between the quinolones and Phenicols groups (data not shown).
In the β-lactam group, Ruiz B et al. (2006) assessed the susceptibility of different salmonella isolated in farms of commercial layer hens to AMP and AMP/Sulbactam with 100% of sensitivity to the strains. In our study, sensitivity to AMP was less than 50%, while sensitivity to AMC was 100%. For the quinolones group, Akoachere et al. (2009) and Esaki et al. (2004) reported a marked susceptibility to this group of antimicrobials of salmonella isolated from bird feed and production animal feed, which is consistent with our result. On the other hand, Ruiz B et al. (2006), observed a sensitivity of 100% to TE, in contrast to the results obtained in our study. However, these authors tested this antibiotic with serovars of Salmonella different from ST. To evaluate the phenicols group, our results are consistent with those obtained by Ruiz B et al. (2006) for C, while Khan et al. (2000) found that strains of ST showed a high resistance to the phenicols.
They also noted that strains resistant to C were also resistant to FFC. To evaluate the Group of aminoglycosides, Ruiz B et al. (2006) found a sensitivity of 100% to AK and CN, which is consistent with our findings.
With regard to the aminoglycosides group, Graziani et al. (2007), found a high resistance to S, as observed in our tests. In regards to sensitivity to the cephalosporins group, Ruiz B et al. (2006) found a sensitivity of 100% to CAZ, CTX in salmonella other than Typhimurium. In our study, we tested 5 cephalosporins, including CAZ and CTX, obtaining a sensitivity ranging from 23.4% (CTX) to 87.2% (FOX).
In the carbapenemes group, we obtained similar results to those reported by Ruiz B et al. (2006). Akoachere et al. (2009) described a high resistance to SXT in strains of ST, which does not match our results, since we observed sensitivity higher than 78% in the strains tested for the same antimicrobial.

Due to the impact of ST in public health and its multi-drug resistance, any treatment of infections caused by this organism should consider CN, NOR, CIP, IMP or AMC. Although our results indicate that the majority of the antimicrobials tested showed a good sensitivity in strains of ST, it would not be advisable to use tetracyclines and streptomycin, as there showed a high resistance. The emergence of multi-resistant strains is of great importance because it allows us to gain greater knowledge and make a better choice of the antimicrobial agent to be used, in order to avoid failures in treatment.

Akoachere J-FTK, Tanih NF, Ndip LM, Ndip RN. 2009. Phenotypic characterization of Salmonella Typhimurium isolates from food-animals and abattoir drains in Buea, Camerron. J. Health Popul. Nutr. 27:612-618.

Casawell M, Friis C, Marco E, McMullin P, Philips I. 2003. The European ban on growth-promoting antibiotics and emerging consequences for human and animal health. J. of Antimicrobial Chemoterapy 52:159-161.

Esaki H, Morioka A, Ishihara K, Kojima A, Shiroki S, Tamura Y, Takahashi T. 2004. Antimicrobial susceptibility of Salmonella isolated from cattle, swine and poultry (2001-2002): report from the Japanese Veterinary Antimicrobial Resistance Monitoring Programme. J. Antimicrob. Chemother. 53:266-270.

Graziani C, Busani L, Dionisi AM, Lucarelli C, Owczarek S, Ricci A, Mancin M, Caprioli A, Luzzi I. 2007. Antimicrobial resistance in Salmonella enterica serovar Typhimurium from human and animal sources in Italy. Vet. Microbiol. 128:414-418.

Khan AA, Nawaz MS, Khan SA, Cerniglia CE. 2000. Detection of multidrug-resistant Salmonella Typhimurium DT 104 by multiplex polymerase chain reaction. Microbiol. Letters 182:355-360.

National Committee for Clinical Laboratory Standards. 2010. Performance Standards for antimicrobial susceptibility testing; Twentieth Informational Supplement. NCCLS, 30:1-153.

Ruiz B JD, Suárez MC, Uribe C. 2006. Susceptibilidad antimicrobiana in vitro de cepas de Salmonella spp. en granjas de ponedoras comerciales del departamento de Antioquia. Rev. Col. Cienc. Pec. 19:297-305.

Yang SJ, Park KY, Kim SH, No KM, Besser TE, Yoo HS, Kim SH, Lee BK, Park YH. 2002. Antimicrobial resistance in Salmonella enterica serovars Enteritidis and Typhimurium isolated from animals in Korea: comparision of phenotypic and genotypic resistance characterization. Vet. Microbiol. 86:295-301.