Resistance to Antibiotics in Strains of Staphylococcus spp., Enterococcus spp. and Escherichia coli Isolated from Rectal Swabs of Pigs

Kolář M., J. Bardoň, I. vágnerová, P. Sauer, d. Koukalová, J. Petrželová, l. Čekanová, r. Pospíšil: resistance to antibiotics in Strains of Staphylococcus spp., Enterococcus spp. and Escherichia coli Isolated from rectal Swabs of Pigs. acta Vet Brno 2008, 77: 103-1100. The study aimed at determining the level of resistance of selected bacterial species (Staphylococcus spp., Enterococcus spp., Escherichia coli) isolated from rectal swabs of pigs to antimicrobial agents. The tested strains were isolated from piglets aged 7 to 30 days. Bacterial species were identified by standard microbiological techniques and susceptibility to antibiotics was determined quantitatively by the standard microdilution method. resistance of the Staphylococcus aureus strain to oxacillin was confirmed by detection of the mecA gene and PBP2a. a total of 115 Staphylococcus spp. isolates were collected. In the case of Staphylococcus aureus, the methicillinresistant strain (MrSa) was identified. Moreover, higher frequency of coagulase-negative staphylococci with minimum inhibitory concentration of oxacillin ≥ 0.5 mg/l was noticed. Inducible resistance to clindamycin in the Staphylococcus hominis strain was also detected. The strains of Enterococcus spp. (61 isolates) exhibited high resistance to tetracycline (98.5%), erythromycin (86.8%) and chloramphenicol (54.4%). Vancomycin-resistant enterococci were not isolated. In the case of Escherichia coli strains (111 isolates), higher frequency of resistant strains to tetracycline (81.1%) and ampicillin (62.2%) was documented. resistance to fluoroquinolones and production of broad-spectrum ß-lactamases was not noticed. The presented study may be considered as a pilot project assessing the prevalence of resistant bacteria in piglets kept on a single farm. It demonstrated the presence of resistant strains of Staphylococcus spp., including one MrSa strain, Enterococcus spp. and Escherichia coli. These strains may be present as a result of postnatal colonization with both bacterial microflora of dams and environmental microflora. Pigs, rectal swabs, bacterial resistance, MRSA, Czech Republic Bacterial resistance to antimicrobial agents has become a serious problem both in human and veterinary medicine. It is related to an excessive use of antibiotics which represent an important part of therapy and, in selected cases, prevention of bacterial infections. on the other hand, they contribute to the development and spread of bacterial resistance to their effects. It must be pointed out that bacterial resistance is not a theoretical issue without a practical impact. In human medicine, multiresistant bacterial strains are associated with higher rates of death from the infections they cause. Similar data are available in animal medicine. a case of lethal infection in a dog caused by a methicillin-resistant Staphylococcus aureus strain – MrSa has been described (Waller 2005). Bacteria with prominent clinical importance and bacterial resistance include staphylococci (especially methicillin-resistant strains of Staphylococcus aureus and coagulase-negative staphylococci), enterococci (in particular vancomycin-resistant isolates) and enterobacteria producing broad-spectrum ß-lactamases (e.g. eSBl and ampC enzymes). at present, ACTA VET. BRNO 2008, 77: 103–110; doi:10.2754/avb200877010103 Address for correspondence: Mvdr. Jan Bardoň, Ph.d., MBa State Veterinary Institute olomouc Jakoubka ze Stříbra 1 779 00 olomouc Czech republic Phone: +420 585 225 641 e-mail: [email protected] http://www.vfu.cz/acta-vet/actavet.htm human medicine is characterized by a worldwide increase in the prevalence of MrSa. MrSa have also been confirmed in veterinary practice, e.g. in cattle, chickens, cats, dogs, horses and a seal (gortel et al. 1999; lee 2003; lilenbaum et al. 1998; o’Mahony et al. 2005). there is concern about increasing data on the interspecific transfer of MrSa, i.e. between animals and humans. Seguin et al. (1999) documented prevalence of identical MrSa in horses and their breeders and a similar relation was suggested by Weese et al. (2005). also Van Duijkeren (2004) assumed a MrSa transfer between humans and dogs. MrSa strains were isolated from animals and veterinary office staff, as described by o’Mahony et al. (2005). although the impact of methicillin-resistant coagulase-negative staphylococci in veterinary medicine has not been specified, these strains may represent a reservoir of resistance genes (Vengust et al. 2006; Busscher et al. 2006). The same is true for vancomycin-resistant enterococci (Vre) as their prevalence both in animals and humans has already been proved in the Czech republic (Kolář et al. 2000, 2005). Broad-spectrum ß-lactamases in enterobacteria of animal origin have not been noticed in the Czech republic as yet. However, this is one of the most serious issues concerning antibiotic resistance in humans, with an impact on higher mortality of patients. It is not unrealistic to presume that multiresistant bacterial strains may enter the human food chain as part of foods of animal origin; they may also colonise or infect people who are in contact with animals. It is well known that pork makes up a significant part of the average Czech consumer basket. Therefore, the study aimed at determining the susceptibility of Staphylococcus spp., Enterococcus spp. and Escherichia coli strains isolated from rectal swabs of piglets to antimicrobial agents. Thus, with respect to the monitoring of antibiotic resistance, the study is a pilot project. Materials and Methods The tested strains of Staphylococcus spp., Enterococcus spp. and Escherichia coli were isolated from 100 piglets kept on a single farm (a farrowing house) in the olomouc region. The piglets, aged 7 to 30 days, were clinically healthy and had not been administered any antibiotics during their lives. all samples were obtained within the same day. according to the breeder’s data, no antibiotics were administered to the sows in the week prior to farrowing. at the time of sampling, the total number of piglets of the given age category kept at the farm was 113. The strains were isolated from 100 rectal swabs collected with a kit containing a swab and a transport medium (Venturi Transystem amies agar gel transport medium with charcoal). The piglets were randomly selected for sampling from the aforementioned group of 113 individuals. after being delivered to the laboratory, the swabs were inoculated onto a solid medium (blood agar, endo agar, XlD agar, Becton Dickinson) subsequently incubated aerobically at 37 °C for 24 h. Following isolation of enterococci, the swabs were brokenoff into meat-peptone broth with 6.5% naCl, once again incubated at 37 °C for 24 h. then the liquid medium was transferred with a calibrated loop on a blood agar. Twenty four-hour cultivation followed at 37 oC and under aerobic conditions. Identification of the aforementioned bacterial strains was carried out by standard microbiological techniques using commercially produced sets enTerotest16, en-CoCCuStest and STaPHYtest16 (Pliva-lachema Diagnostika). In the case of Staphylococcus aureus strains, another commercial set was utilized, Staphaurex Plus® (remel) capable of detecting the clumping factor, protein a and other specific antigens characteristic for Staphylococcus aureus. For identification of enterococci, the PYratest for pyrrolinyl arylamidase (Pliva-lachema Diagnostika) was used as well. When various species of Staphylococcus spp. and Enterococcus spp. were detected in a single sample, all strains of the different species were included in the database. When both haemolytic and non-haemolytic strains of Escherichia coli were present simultaneously, both isolates were included. To assess susceptibility of the isolated bacteria, the standard microdilution method determining minimum inhibitory concentrations (MIC) of the tested antibiotics (ClSI 2007; ClSI 2006) was used. at the end of the epizootic-epidemiological chain there are humans in whom multiresistant bacteria of animal origin may, under certain circumstances, cause severe diseases. therefore, the susceptibility tests utilised breakpoints (susceptibility/ resistance criteria) defined by the national reference laboratory for antibiotics, national Institute of Public Health, Prague and commonly used in the Czech republic for evaluating resistance in human medicine. The MIC50 and MIC90 values were calculated as 50% and 90%, respectively, percentiles of the MIC values of individual antibiotics. The reference strains Escherichia coli aTCC 25922, Escherichia coli aTCC 35218, Pseudomonas aeruginosa aTCC 27853, Staphylococcus aureus aTCC 29213, Staphylococcus aureus aTCC 25923 and Enterococcus faecalis atCC 29212 served for protocol quality control. Detection of the penicillin-binding protein 2a (PBP2a) in Staphylococcus aureus was performed by latex 104