Prevalence, antimicrobial resistance and staphylococcal toxin gene of blaTEM-1a-producing Staphylococcus aureus isolated from animals in Chongqing, China CURRENT STATUS: POSTED

Background Livestock-associated Staphylococcus aureus is one of the most important etiological agents in both human and animals. It has been reported with high antimicrobial resistance and multiple staphylococcal superantigen genes in many countries and several provinces of China. However, large-scale investigation of this organism has not been documented in Chongqing, China. The aim of this study is to demonstrate the prevalence, antimicrobial susceptibility and some molecular characteristics of S. aureus acquired from animals in Chongqing. Results A total of 89 S. aureus isolates were cultured from 1371 samples picked up from March 2014 to December 2017. The isolates were originated from pigs (25), cattle (6), goats (10), rabbits (16) and chicken (32). Four MRSA strains were identified from 3 pig samples and 1 chicken sample. The isolates showed high resistance to penicillin (93.3%) and ampicillin (92.1%), but were more susceptible to amikacin and ofloxacin, since the resistance rates of these two drugs were less than 10%. Meanwhile, 74.2% isolates exhibited varying degree of MDR. Almost all strains, except for 3 chicken-originated isolates, were positive for blaTEM-1a, but did not harbor other ESBL genes. Nineteen staphylococcal SE/SEl/TSST-1 genes, except seq, were detected in isolates. The predominant genes were sei (58.4%), tst-1 (56.2%) and seg (51.7%). The

Although there have been a great deal of reports on the epidemiological studies of S. aureus originated from food producing animals in China in recent years [1,29,30], little is known about the prevalence and occurrence of this organism in Chongqing, China. In this study, we investigated the prevalence of S. aureus isolated from food animals in Chongqing. Furthermore, antimicrobial susceptibility, MRSA strains, ESBL genes and staphylococcal toxin genes were also characterized.

Results
Prevalence of S. aureus from animals A total of 89 S. aureus isolates were recovered from 1371 samples during March 2014 and December 2017 in Chongqing, and the overall prevalence was 6.5% ( Table 1). The isolation frequency varied in different animals. Of these, 25 (7.3%) were isolated from 343 pig samples, 1 (0.6%) were isolated from 165 beef cattle samples, 5 (2.6%) were isolated from 190 dairy cattle samples, 10 (11.4%) were isolated from 88 goat samples, 16 (15.2%) were obtained from 105 rabbit samples, and 32 (6.7%) were obtained from 480 chicken samples. The prevalence rate of S. aureus among rabbits was the highest. However, the prevalence rate in cattle was low, and the rate in beef cattle was the lowest and no more than 1%.
Four strains carrying mecA gene were classified as MRSA and the prevalence was 0.3% from samples.
The positive rate of MRSA in S. aureus isolates was 4.5%. Three MRSA were pig-originated and all these strains were obtained from the same farm. The prevalence of MRSA in pigs was 0.9% and the rate in pig-associated isolates was 12.0%. Additionally, 1 was isolated from a chicken farm. Therefore, the prevalence of MRSA in chicken was 0.2% and the rate in chicken-associated isolates was 3.1%.
The MRSA isolation rate was higher in pigs than that in chicken. Conversely, MRSA was not detected in other animal samples.

Susceptibility of S. aureus to antimicrobials
The result of the antimicrobial susceptibility of all 89 S. aureus isolates were summarized in Table 2.
Four MRSA strains had high MDR. The chicken-associated MRSA was resistant to 8 classes of drugs.
However, 2 pig-associated isolates were resistant to 9, and 1 was resistant to 10 classes of antimicrobials.

Prevalence of ESBL genes
In all, 86 (96.6%) isolates harbored bla TEM genes with the exception of 3 chicken-associated strains, and all bla TEM belonged to TEM-1a. No other ESBL gene was detected with listed primers in Table S1.
Interestingly, 1 MRSA strain isolated from chicken sample did not contain bla TEM gene.
The toxin patterns were variable, from 1 to 10 except for one rabbit-associated strain that no toxin gene was amplified ( Table 5, Table S3). Four isolates only had 1 gene. More than 1 gene were detect in 8, 13,18,12,9,15 and 5 isolates, and they were positive for 2, 3, 4, 5, 6, 7 and 8 toxin genes. Two pig-associated isolates and 1 rabbit-associated isolate harbored 9 toxin genes. Ten toxin genes were observed from 1 strain isolated from a pig.

Discussion
In this study, we investigated the prevalence of S. aureus in different food producing animals, indicated that the prevalence (46%) of goat-associated S. aureus was higher than isolation rate (11.4%) of S. aureus obtained from goats in our study. The differences of isolation rates can be interpreted because of the culture media and methods, samples types, different regions, animal species, farm hygienic conditions, and so on. In our study, the isolation rate varied significantly between different animals, from 0.6% of beef cattle samples to 15.2% of rabbit samples.
In the present study, 4 MRSA were isolated from 3 pig samples and 1 chicken sample, but not other animals. The prevalence of MRSA in our studywas lower than most researches, both in China [1,29,33] and foreign countries [14,34]. However, the study developed in Chongqing did not detect mecA gene in all 32 identified goat-originated S. aureus [31]. This study was identical to our result about the goat-associated isolates. Although the prevalence of MRSA was at a low level, the resistance of S. aureus isolates to penicillins was serious. Therefore, LA-MRSA might become very popular and the prevalence rate might be much higher in animals in the future under the selective pressure of βlactam antibacterial drugs. As the LA-MRSA is a reservoir of health-threatening organism for people [19][20][21]33], longitudinal surveillance of MRSA in animals, especially in pigs and chicken, should be performed periodically.
Most of isolates, particularly obtained from pigs and chicken, in this study, were high resistance and multi-drug resistant. The high prevalence of resistance to antimicrobials in our study was similar to that reported in previous studies [1,29,33,35]. Antimicrobial agents are often used for the prevention and treatment of infectious diseases and growing promotion of food producing animals in intensive production. Based on records of antimicrobial usage at each farm, overuse of antimicrobials is the possible explanation of extensive drug resistance. The isolates were most resistant to penicillins, but seemed susceptible to amikacin, ofloxacin, cephalothin, gentamicin and imipenem. On the other hand, most antimicrobials showed less resistance in herbivores than in pigs and chicken.
The trend was also observed in MDR strains in different animals. For example, the ratio of MDR isolates obtained from pigs and chicken was higher than herbivore-originated strains. This might be related to the more frequencies of bacterial infectious diseases, thereby leading to more usage of antimicrobials, in pigs and chicken than in herbivores. More importantly, some isolates were resistant to animal-prohibited antimicrobials, such as imipenem, clarithromycin, chloramphenicol, clindamycin and enoxacin, for the selective pressure from the corresponding same class of antimicrobials, which were widely used in animal production. For example, florfenicol was applied in veterinary infectious disease treatment universally. Although chloramphenicol was inhibited in application on animal diseases, the resistance rate was 31.5% in our study. The reason might be that chloramphenicol and florfenicol were both resistant to the productions of chloramphenicol/florfenicol exporter genes [36].
We found a great proportion of isolates were resistant to cephalosporins. This suggested that these strains have one or more ESBL genes, although no similar assay has been reported. In this study, we discovered that almost all strains, except for 3 chicken-originated isolates, were positive for bla TEM-1a , but did not harbor other ESBL genes, even though more than one pair primers published in articles were attempted (Data not shown). In the further study, we will isolate more S. aureus clinical strains and try to find more ESBL genes. Meanwhile, we will explore the conjugation machanism of TEM-1a gene from Enterobacteriaceae to S. aureus and from S. aureus to S. aureus.
The prevalence of staphylococcal toxin genes in this study was different from other reports. The prevailing toxin genes was sei (56.2%), tst-1 (56.2%) and seg (51.7%), which were more than 50% in

Conclusions
In conclusion, this was the first investigation of prevalence, antimicrobial susceptibility and molecular characterization of S.aureus isolates from food producing animals in Chongqing. The high antimicrobial resistance and prevalence of bla TEM-1a seriously reminded that it was urgent to standardize the use of antimicrobials. Meanwhile, optimization of management practices and housing conditions should be applied to animals, such that reduction of veterinary infectious diseases in herds will be effective and the usage of antimicrobials can be cut down. The universal existence of staphylococcal SE/SEl/TSST-1 genes in isolated strains implied a risk of staphylococcal food poisoning that livestock-associated S. aureus were transmitted from animals to human through the food chain.

Samples collection
From March 2014 to December 2017, a total of 1371 samples were collected from healthy animals in Chongqing, China. Samples were collected in a random manner from pigs (n = 343) and rabbits (n = 105) by nasal swabs, beef cattle (n = 165) and goats (n = 88) by feces, chicken (n = 480) by anal swabs, and dairy cattle (n = 190) by milk. The farmers of each farm gave permission for sample collection. The animal did not suffer from any diseases and administer any drugs. All samples were stored in low temperature with ice and were taken back to laboratory within 6 h.

Isolation and identification of S. aureus
Isolation and identification of S. aureus were performed after the samples arrived at laboratory immediately. Briefly, each fecal sample was mixed with 2 mL PBS for 2 h in order to release bacteria.
About 0.2 mL milk or PBS mixture was added to 10 mL Mueller-Hinton broth containing 10% NaCl and cultured at 37℃ for 10 h. The medium was streaked onto mannitol salt plate and incubated at 37℃ for 20 h. Presumptive colonies were transferred into Luria-Bertani medium for enrichment at 37℃ for 8 h on a rotary incubator. DNA was extracted from culture, and S. aureus isolate was confirmed by amplifying its specific gene nuc. The primers and annealing temperature were listed in Table S1. S. aureus ATCC 25923 was used as positive control. All confirmed S. aureus clinical strains were cultured in Luria-Bertani medium until reached to exponential grown phase and stored with 40% glycerol at -80℃.

Bacterial DNA extraction
Culture was centrifuged at 5 000 r/min for 5 min, and the pellet was resuspended in 1 mL sterile water. Suspension was centrifuged and the supernatant was discarded. The pellet resuspended with 0.1 mL sterile water was treated in a thermostat at 105℃ for 10 min and immediately frozen at -20℃ for 30 min three times, followed by centrifugation at 10 000 r/min for 10 min. The supernatant was removed without disturbing the pellet and the extracted genomic DNA was stored at -20℃ as the template for PCR.

Determination of SE/SEl/TSST-1 genes
The presence of SE, SEl and TSST-1 genes was confirmed by PCR using specific primers described in

Availability of data and materials
All data generated or analysed during this study are included in this published article and its supplementary information files.

Competing interests
The authors declare that they have no competing interests. Outbreak of staphylococcal food poisoning among children and staff at a Swiss boarding school due to soft cheese made from raw milk.

Supplementary Files
This is a list of supplementary files associated with this preprint. Click to download. Table S3.docx Table S1.docx Table S2.docx