Prevalence and antimicrobial susceptibility of bacterial pathogens isolates from diseased swine in southwest , China

The purpose of this study was to investigate the prevalence of antimicrobial resistance in the clinical bacterial isolates from diseased swine in southwest, China during 2009-2010. A total of 504 bacterial isolates (19 species) were collected from the 364 clinical samples. The activity of 6-14 antibiotics to each bacterial species was examined. The sensitivity was tested by the disk diffusion method and performed according to CLSI guidelines in Mueller-Hinton agar. The most common pathogens were Escherichia coli (n=154; 30.56%), Staphylococcus spp. (n=110; 21.83%), Enterococcus faecalis (n=58; 11.51%), Klebsiella pneumoniae (n=44; 8.73%), Proteus mirabilis (n=43; 8.53%) and Streptococcus suis (n=30; 5.93%). All isolates revealed high level of resistance to ampicillin (47.6-100%), amoxicillin (52.6100%), cephalothin (29-100%), norfloxacin (52.6-83.3%), gentamicin (45.1-83.3%) and terramycin (61.9100%). Moreover, 93% of the isolates exhibited multiple drug resistance (MDR; resistance ≥ 3 antimicrobials). Only ticarcillin/clavulanate exhibited very high activity against E. coli (98.1%), Staphylococcus spp. (91.9%), K. pneumaniae (92.3%) and P. mirabilis (97.2%), respectively. These findings suggest that antimicrobial resistance of bacterial pathogens isolates is commonly present among diseased swine in Southwest, China, and they also suggest the need for more prudent use of antibiotics by farmers and veterinarians.


INTRODUCTION
As acute infections and outbreaks of infectious diseases in groups or herds become more common, use of an effective antimicrobial treatment as early as possible is critically important.The empirical treatment is generally based on knowledge of the resistance patterns of the different bacterial pathogens toward antimicrobial agents used in the particular animal species.Uncontrolled usage of antimicrobial agents is recognized as the most important factor that favors the development and spread of resistant microorganisms (Van den Bogaard and Stobberingh, 1999;White, 2002).The digestive tracts of *Corresponding author.E-mail: lixutingzy@163.com.pigs can harbor antimicrobial-resistant bacteria among the commensal flora, which contain a reservoir of antibiotic-resistance genes potentially transmissible to humans through the food chain and environment (Caprioli et al., 2000).In addition to the human health concerns, antimicrobial-resistant pathogens also pose a severe and costly health problem in that they may prolong illness and decrease productivity through higher morbidity and mortality (Yang, 2004).However, data on the prevalence of antimicrobial-resistant veterinary pathogens are sparse, particularly in Southwest, where animal husbandry was more developed than other area.
Therefore, the purpose of this study was to investigate the prevalence of bacterial infection and resistance to antimicrobial agents in the clinical isolates obtained from diseased swine in Southwest, China.

Clinical samples
364 Samples, including lungs, lymph nodes, livers, hearts, spleens, kidneys, andblood (obtained between May 2009 andJuly 2010) were collected from 72 pig farms in southwestern China.All the samples were obtained from diseased pigs which had at least one of the following symptoms: septicemia, arthritis, enteritis, meningitis, pneumonia, pleuritis, diarrhea, edema, fever, pneumonia, endocarditis, and dysentery.Before sampling, all samples surface were being asepsis by burning of ethanol.Aseptically collected samples were appropriately processed and seeded in chocolate agar and blood agar.All of the samples were transported to the laboratory of Sichuan Animal Science Academy for bacterial counts and isolation within 6 h.

Bacteria isolation
The strains were incubated at 37°C for 18-24 h.One loo p-full from each enrichment were streaked on tryptic soy agar, blood agar (5% fresh rabbit blood), MacConkey agar (Tianhe Microorganism Reagent Co., Ltd.) and Salmonella-Shigella agar respectively.All plates were incubated at 37°C in air for 24 h and pur ified by standard methods (Murray et al., 2003).Single colony was obtained from the isolates and stored in Luria-Bertani containing 20% glycerol, at -80°C until use.No replicate isolates f rom the same samples were used.All media agar were purchased from Tianhe Microorganism Reagent Co., Ltd.

Bacteria identification
Identification was based on colony type and morphology, Gram staining characteristics, and standard biochemical tests.All isolates were identified at species level by VITEK (Vitek System, bioMerieux).They were also confirmed using primers 27F (5' AGA GTT TGA TCC TGG CTC AG 3') and 1492R (5' TAC GGC TAC CTT GTT ACG ACT T 3') by the polymerase chain reaction (PCR) assay (Wilson, 1990).The template DNA was prepared by suspending an overnight cultured of bacteria in 400 µL Milli-Q water.The suspensions were heated at 95°C for 5 mi n and centrifuged at 13,000 rpm for 5 min.Each 50 µL of PCR mixture consisted of 4 µL of template, 5 µL 10×PCR Buffer, 1.5 mM MgCl2, 200 µM dNTP, 0.4 µM each of the seven primers and 2.5 U Taq DNA polymerase.PCR was performed in a DNA thermal cycler (Bio-Rad, Hercules, CA) using the following program: an initial denaturation step at 94°C for 5 min, 30 cycles of den aturation at 94°C for 1 min, annealing at 50°C for 1 min and exte nsion at 72°C for 1.5 min, followed by a final elongation at 72°C for 10 min.The amplified PCR products were analyzed on 0.8% (w/v) agarose gels.Subsequently, the PCR product was sequenced by Shanghai Sangon Bioengineering Co., Ltd.The nucleotide sequences were analyzed with software (BLAST) available from the National Center for Biotechnology Information website (http://www.ncbi.nlm.nih.gov).

Detection of blaTEM resistance genes
The template DNA was prepared as described above.The forward primer ATGAGTATTCAACATTTCCGTG and the reverse primer TTACCA ATGCTTA ATCAGTGAG were used to amplify the blaTEM gene by following procedure: an initial denaturation step of 94°C for 5 min, 30 cycles of denaturation at 94°C for 1 min, annealing at 50°C for 1 min and extension at 72°C for 1 min, foll owed by a final elongation at 72°C for 10 min.Amplified PCR products were analyzed on 0.8% (w/v) agarose gels.The PCR product was sequenced and analyzed as above.

bla TEM resistance genes detection
Based on the results of the susceptibility tests, 154 E. coli, 44 K. pneumoniae and 43 P. mirabilis were selected to amplify the bla TEM genes.The PCR analysis and sequencing showed that 140 (92.1%)E. coli, 44 (100%) K. pneumoniae and 42 (97.67%)P. mirabilis isolates harbored a bla TEM1 gene.

DISCUSSION
To investigate the prevalence of bacterial infections in swine in southwest, China, a total of 504 bacterial isolates (19 species) were collected and identified from the 364 clinical samples.The results revealed that E. coli and the Staphylococcus spp.were the most widespread bacterial isolates.In swine, E. coli is an important pathogenic bacteria including enterotoxigenic (ETEC) and extracellular pathogenic E. coli (ExPEC) strains which are common causes of a variety of clinical syndromes, including urinary tract infections, abdominal infections, pneumonia, neonatal meningitis, sepsis, neonatal and post weaning diarrhea and edema (Yang, 2004;Wada et al., 2004;Nazareth et al., 2007;Boerlin et al., 2005).
E. faecalis (n=58; 11.51%), K. pneumoniae (n=44; 8.73%), P. mirabilis (n=43; 8.53%) and S. suis (n=30; 5.93%) were the another four frequent isolates.E. faecalis is intrinsical not as virulent as other Grampositive organisms, such as S. aureus, S. pneumoniae and S. suis Type 2 (Bittencourt, 2004;Gaspar et al., 2009).It emerges as an opportunistic pathogen, nevertheless, it is known to cause serious infections such as bacteraemia, septicaemia, urinary tract infections, wound infections, meningitis and endocarditis (Giacometti et al., 2000;Hershberger et al., 2005;Hällgren et al., 2003;Hébert et al., 2007).K. pneumoniae is also an opportunistic pathogen that responsible for a wide range of infection in humans and animals, such as urinary tract infections, pneumonia, wound infections and septicemia (Podschun and Ullmann, 1998;Brisse and Duijkeren, 2005).P. mirabilis is also often found in human as opportunistic pathogens (Zych et al., 2001).S. suis, especially the serotype 2, is an important swine pathogen causing meningitis, septicemia, endocarditis, and arthritis (Marie et al., 2002;Lun et al., 2007;Domínguez-Punaro et al., 2007;Ma et al., 2009).In 2005, an Streptococcal Toxic Shock Syndrome (STSS) human outbreak caused by S. suis serotype 2 was found in Sichuan province with 38 human deaths and over 200 human infections, and more than 640 pigs were found to be severely infected (Yu et al., 2006).Although other zoonotic bacterial such as Salmonella spp., H. parasuis, P. multocida and Actinobacillus spp.were lesser, they showed higher mortality and morbidity.The traditional zoonotic E. coli, Staphylococcus spp., S. suis and opportunistic E. Li et al. 1581 faecalis, K. pneumoniae, P. mirabilis were the main pathogens in swine in Southwest, China.The enterobacteria were resistant to β-lactams, tetracyclines and aminoglycosides as previously described in China (Chang et al., 2002;Yang et al., 2004;Liu et al., 2007;Tian et al., 2009).The E. coli isolates assessed in this study displayed similar levels of resistance to tetracyclines, ampicillin, gentamicin and fluoroquinolones as were previously reported for E. coli strains isolated from diseased swine in China by Tian et al. (2009).High levels of resistance to tetracycline (~60-95%) have also been detected in E. coli isolates recovered from apparently healthy swine on-farm or at slaughter in other countries (Kozak et al., 2003;Kijima-Tanaka et al., 2003;Teshager et al., 2000;Blake et al., 2003).Most of E. coli isolates (67.3%) from swine were resistant to fluoroquinolones (e.g.norfloxacin and ciprofloxacin).Somewhat similar findings have been reported in a recent study of clinical E. coli isolates from swine by Wang et al., 2010.Resistance to amoxicillinclavulanic acid does not occur frequently in E. coli isolates from diseased swine in China before 2004 (Yang et al., 2004), but 21.1% of our swine isolates were resistant to this antibiotic-inhibitor combination.In the present study, amikacin exhibited moderate activity against all strains tested.
Interestingly, fewer reports of antimicrobial resistance in K. pneumoniae isolated from swine have been published in China.In the present study, K. pneumoniae was the sixth most frequently encountered pathogen in swine and showed high resistance to antimicrobial.K. pneumoniae isolates may be naturally resistant to ampicillin, amoxicillin, carbenicillin, and ticarcillin, but not to extended-spectrum β-lactam antibiotics due to a constitutively expressed chromosomal class A βlactamase (Haeggman et al., 2004).In the present study, the degree of resistance to cephalothin and ceftiofur was 100 and 48.2%, respectively.The antibiotic-inhibitor combination revealed actively to K. pneumoniae.This could be due to the wide use of these classes of cephalosporins in husbandry activities and the considerable increase in prevalence of ESBL-producing and multiple-antimicrobial-resistant isolates from pig farms (Yang et al., 2004;Tian et al., 2009).
Another important observation in this study is that the ceftiofur resistance has increased.Ceftiofur is the only extended-spectrum cephalosporin approved for veterinary use in many countries (Salmon et al., 1995).Because ceftiofur-resistant organisms are cross resistant to ceftriaxone, the use of this antimicrobial agent in food animals has come under increasing scrutiny as a selective agent potentially responsible for the emergence and dissemination of ceftriaxone resistance in Salmonella and other enteric pathogens (Alcaine et al., 2005).The rate of resistance to ceftiofur were (9.6%)E. coli, (48.2%)K. pneumoniae, (30.1%)P. mirabilis and (23.8%) S. suis isolates in this study, respectively.These findings are very significant difference to the previous reports which displayed high actively to ceftiofur (Marie et al., 2002;Yang et al., 2004;Morioka et al., 2005;Zhou et al., 2010: Wang et al., 2010).The high incidence of ceftiofur resistance in the K.pneumoniae, P. mirabilis and S. suis isolates tested herein was somewhat unexpected, as this drug was introduce into veterinary clinics for use in China only 2 to 3 years ago.Of all the 154 E. coli, 44 K. pneumoniae and 43 P. mirabilis, 140 (92.1%),44 (100%) and 42 (97.67%)isolates harboured a bla TEM1 gene.The results of the study indicated that TEM-1 was the most common βlactamase gene among the K. pneumoniae and E. coli isolates, in agreement with previous studies that reported a high prevalence of the bla TEM-1 gene among animal E. coli isolates (Liu et al., 2007;Rayamajhi et al., 2008;Li et al., 2007;Chander et al., 2011).
In conclusion, our results confirmed a different prevalence of bacterial infection in swine during 2009-2010, in Southwest, China.The E. coli, Staphylococcus spp., S. suis , E. faecalis, K. pneumoniae and P. mirabilis isolates are commonly present among diseased swine.
Here we have shown that bacterial pathogens from diseased swine exhibited high level resistance to a large number of antimicrobial agents.If this situation continues, there will be no effective antibiotic therapeutic reserve for some bacterial infections.In the future, the data from monitoring programs and resistance studies should be taken into account for the usage of antimicrobials in veterinary medicine.

Table 1 .
Antibiotic resistance in the clinical bacterial isolates.Breakpoints are those recommended by the Clinical and Laboratory Standards Institute (CLSI);.-, no CLSI breakpoints were available, b According to cefotaxime, c According to tetracycline, d including Shigella spp.,Salmonella spp., H. parasuis, A. lwoffii, P.