Antibiotic sensitivity of common respiratory bacteria of pig from Hubei province, China

16 Nong Lam University, Ho Chi Minh City Antibiotic sensitivity of common respiratory bacteria of pig from Hubei province, China Anh L. L. Nguyen1∗, An T. T. Vo1, & Qigai He2 1Faculty of Animal Science and Veterinary Medicine, Nong Lam University, Ho Chi Minh City, Vietnam 2Huazhong Agricultural University, Wuhan, China ARTICLE INFO Research Paper Received: February 27, 2020 Revised: April 17, 2020 Accepted: May 19, 2020 Keywords Antibiotic resistance Pigs Respiratory bacteria ∗Corresponding author Nguyen Luong Lam Anh Email: lamanhft@gmail.com ABSTRACT The use of antimicrobials for feeding and treatment is crucial to animal health. However, continuous use of antibiotics is contribut- ing to emergence and widespread of antibiotic resistance. This study aimed to investigate the antimicrobial resistance of five major respi- ratory pathogens in pigs of Hubei province, China, from October to December, 2019. Antibiotic susceptibility testing for Streptococcus suis, Haemophilus parasuis, Pasteurella multocida, Bordetella bron- chiseptica and Actinobacillus pleuropneumoniae was determined to representatives of relevant antibiotic classes. Streptococcus suis isolates were mostly sensitive to beta-lactams, whereas high levels of resistance were observed to quinolones, gen- tamycin, doxycycline, trimethoprime and lincomycin. For H. para- suis, P. multocida and A. pleuropneumoniae of Pasteurellaceae family, the susceptibility to beta-lactams and quinolones was dis- played. Most B. bronchiseptica isolates were sensitive to doxycy- cline, azithomycin, polymycin whereas high resistance levels to beta- lactams, aminoglycosides and quinolones were recorded. This study obtained practical data for later studies and usage to combat infections due to respiratory bacteria. Cited as: Nguyen, A. L. L., Vo, A. T. T., & He, Q. (2020). Antibiotic sensitivity of common respiratory bacteria of pig from Hubei province, China. The Journal of Agriculture and Development 19(3), 16-21. 1. Introduction Porcine respiratory diseases complex is caused by multifactorial aetiologies, including the viral and bacterial pathogens, the environment, man- agement and genetic factors. Within this com- plex, Streptococcus suis, Haemophillus parasuis, Pasteurella multocida, Actinobacillus pleurop- neumoniae and Bordetella bronchiseptica have been known to be ubiquitous in almost all pig farms. S. suis is as a major respiratory com- mensal and pathogen of pigs and an emerging zoonotic agent of meningitis in human (Goyette- Desjardins et al., 2014). Haemophillus parasuis produces Gla¨sser’s disease as well as pneumo- nia (Nedbalcova et al., 2006). Pasteurella mul- tocida causes atrophic rhinitis, particularly when combined with B. bronchiseptica (Jeffrey et al., 2013). Actinobacillus pleuropneumoniae gener- ates contagious hemorrhagic pleuropneumonia in pigs (Brownfield, 2013). Due to their complex- ity and indeterminacy, bacterial diseases are very challenging to control. Antimicrobial agents are important for effec- tive production of food animals as growth pro- moter or/and disease prevention. As the world’s largest pork producer and consumer, China has been reported for the massive use of antibiotic in food animal production. Zhao et al. (2011) showed antimicrobial susceptibility tests on B. bronchiseptica isolates from Chinese farms that were highly resistant to ampicillin, cefazolin, The Journal of Agriculture and Development 19(3) www.jad.hcmuaf.edu.vn Nong Lam University, Ho Chi Minh City 17 streptomycin, amoxicillin and tetracycline. Zhang et al. (2015) found the most antibiotics consumed in China’s swine farming were fluoroquinolones and β-lactams. Therefore, antimicrobial surveil- lance is necessary to provide a better understand- ing of antibiotic resistance in the animal popula- tion. This study aimed to contribute the comprehen- sion of the antibiotic susceptibility pattern of S. suis, H. parasuis, P. multocida, A. pleuroplneu- moniae and B. bronchiseptica, the five important pathogens found in the respiratory tract of pigs in Hubei province, China, using disk diffusion test. 2. Materials and Methods 2.1. Sample collection From October to December 2019, a total of 155 samples from 14 different pig farms in Hubei province were sent to the Animal Diagnostic Cen- ter of Huazhong University. The collected sam- ples included lungs, spleen, synovial fluid, brain, tracheal effusion etc. Lived pigs were observed for evaluating clinical signs and endured necropsy to collect samples. For every individual pig, lung and spleen samples were sealed in a clean zipper bag; brain and synovial fluid were kept in an eppendorf tubes (EP tube). Nasal samples were collected by using sterile cotton swabs and placed in sterilized EP tubes. The samples were clearly marked. After the period of three months, 133 strains of the five concerned bacteria species from 155 samples were isolated and identified by using multiplex PCR assays. For the identification of the five bacteria, the primers of following tar- get genes were used: 16S rRNA to detect S. suis (Cheung, 2008), 16S rRNA for H. parasuis, apxIV for A. pleuropneumoniae, fla for B. bron- chiseptica (Xue, 2009) and ktm1 for P. multo- cida (Nagai et al., 1994). The greatest number of isolated strains were obtained from S. suis (40%, 62/155), followed by H. parasuis (18.71%, 29/155), P. multocida (14.83%, 23/155), B. bron- chiseptica (8.39%, 13/155), and A. pleuropneu- moniae (3.87%, 6/155). 2.2. Kirby-Bauer antibiotic testing Twenty antibiotic agents (Hangzhou Binhe Mi- croorganism Reagent Co., Ltd) were used, in- cluding cefotazime (30 µg), cephradine (30 µg), ceftriaxone (30 µg), ceftazidime (30 µg), amox- icillin (20 µg) and ampicillin (10 µg), ofloxacin (5 µg), ciprofloxacin (5 µg), enrofloxacin (10 µg), norfloxacin (10 µg), spectinomycin (100 µg), gentamicin (10 µg), streptomycin (10 µg), amikacin (30 µg), kanamycin (30 µg), doxycy- cline (30 µg), lincomycin (30 µg), azithromycin (15 µg), polymyxin B (300 µg) and trimethoprim (23.75/1.25 µg). Each purified isolates of tested bacteria were evenly spread onto a tryptic soy agar plate (TSA, BDTM, USA) that had been coated with nicotinamide adenine dinucleotide liquid (NAD, Guangzhou Saiguo Biotech, China) and bovine serum (Zhejiang Tianhang Biotechnology, China). The antimicrobial discs were placed onto the surface of the agar. The plates were then incu- bated at 37oC for about 24 h. The inhibition zone diameter was measured and compared with stan- dardized CLSI interpretive criteria to designate the isolate as sensitive, intermediate or resistant to the drug (CLSI, 2018). In this study, the iso- lates that showed intermediate were classified as resistant. 2.3. Results and Discussion The resistant and sensitive rates of the five bac- teria species to 20 antibiotic agents are presented in Table 1. Results showed the resistance rates of S. suis strains to quinolones, aminoglycosides, macrolides, lincomycins, tetracyclines, polymyx- ins and sulfonamides were all over 60%. H. pasasuis strains were sensitive to majority of the drugs but highly resistant to amoxicillin, strepto- mycin, amikacin, kanamycin and lincomycin. The resistance of P. multocida strains to aminoglyco- sides and lincosamides were apparently high com- pared to other antibiotic groups (Table 1). With the small number of isolates being tested, the two purified isolates of A. pleuropneumo- niae were sensitive to beta-lactams, quinolones and aminoglycosides. In contrast, all of the three isolates of B. bronchiseptica resisted to those drugs and only sensed to doxycycline, gentam- icin, azithromycin and polymyxin B. The drug-resistance pattern of bacterial iso- lates obtained in this study indicates that S. suis, H. parasuis, P. multocida, B. bronchisep- tica and A. pleuropneumoniae displayed high antibiotic resistance rates to 8 tested antibi- otics/antimicrobial classes. The resistance pro- portion of S. suis to these antibiotics were all www.jad.hcmuaf.edu.vn The Journal of Agriculture and Development 19(3) 18 Nong Lam University, Ho Chi Minh City Table 1. Antibiotic susceptibility rates (%) and number of S. suis, H. parasuis and P. multocida isolates (in brackets) from infected pigs of Hubei province Antibiotics S. suis H. pasasuis P. multocidaSensitive Resistant Sensitive Resistant Sensitive Resistant Amoxicillin 62.5 37.5 20.0 80.0 25.0 75.0(5) (3) (1) (4) (1) (3) Ampicillin 75.0 25.0 50.0 50.0 40.0 60.0(6) (2) (2) (2) (2) (3) Ceftiaxone 75.0 25.0 80.0 20.0 60.0 40.0(6) (2) (4) (1) (3) (2) Cefotaxime 100.0 0.0 75.0 25.0 50.0 50.0(6) (0) (3) (1) (2) (2) Ceftazidime 25.0 75.0 66.7 33.3 0.0 100.0(1) (3) (2) (1) (0) (1) Cefradine 80.0 20.0 60.0 40.0 100.0 0.0(4) (1) (3) (2) (4) (0) Ofloxacin 25.0 75.0 80.0 20.0 80.0 20.0(2) (6) (4) (1) (4) (1) Ciprofloxacin 0.0 100.0 50.0 50.0 80.0 20.0(0) (8) (2) (2) (4) (1) Enrofloxacin 50.0 50.0 75.0 25.0 100.0 0.0(4) (4) (3) (1) (4) (0) Norfloxacin 0.0 100.0 60.0 40.0 60.0 40.0(0) (8) (3) (2) (3) (2) Spectinomycin 37.5 62.5 80.0 20.0 40.0 60.0(3) (5) (4) (1) (2) (3) Gentamicin 0.0 100.0 40.0 60.0 20.0 80.0(0) (8) (2) (3) (1) (4) Streptomycin 0.0 100.0 0.0 100.0 0.0 100.0(0) (6) (0) (1) (0) (2) Amikacin 0.0 100.0 0.0 100.0 20.0 80.0(0) (8) (0) (5) (1) (4) Kanamycin 0.0 100.0 0.0 100.0 20.0 80.0(0) (7) (0) (5) (1) (4) Doxycycline 25.0 75.0 100.0 0.0 60.0 40.0(2) (6) (5) (0) (3) (2) Lincomycin 0.0 100.0 0.0 100.0 0.0 100.0(0) (8) (0) (5) (0) (5) Azithromycin 12.5 87.5 60.0 40.0 60.0 40.0(1) (7) (3) (2) (3) (2) Polymyxin B 0.0 100.0 60.0 40.0 60.0 40.0(0) (8) (3) (2) (3) (2) Trimethoprim 12.5 87.5 60.0 40.0 60.0 40.0(1) (7) (3) (2) (3) (2) over 60% except for β-lactam group. Some an- tibiotics that used to effectively deal with Gram- negative bacteria (H. parasuis, P. multocida, B. bronchiseptica and A. pleuropneumoniae) such as macrolides and beta-lactams were indicated to be less sensitive, especially lincomycin could not be used for any bacterial isolates. Polymycin B, which is known to use in human treatment, pre- sented 100% resistance by S. suis and A. pleu- ropneumoniae, and 40% by H. parasuis and P. multocida. As a result, only a narrow spectrum of effective antibiotic drugs can be used for the treatment of infection in Hubei pigs. This study also revealed the number of bacte- The Journal of Agriculture and Development 19(3) www.jad.hcmuaf.edu.vn Nong Lam University, Ho Chi Minh City 19 *Note: Beta-lactams (AMOX: amoxicillin, AMP ampicillin, CEFTI: ceftriaxone, CEFRA: cefradine, CEFO: cefotaxime, CEFTA: ceftazidime). Quinolones (OFL: ofloxacin, CIP: ciprofloxacin, ENRO: enrofloxacin, NOR: norfloxacin). Aminogly- cosides (SPEC: spectinmycin, GEN: gentamycin, STREP: streptomycin, AMI: amikacin, KANA: kanamycin). Tetracyclines (DOX: doxycycline). Lincosamide (LIN lincomycin). Macrolides (AZI: azithromycin). Polymycin (POLY: polymyxin B). Sulfonamide (TRIME: trimethoprime). SS1 – SS6: S. suis isolates number 1 to 6. HPS1 – HPS6: H. parasuis isolates number 1 to 5. PM1 – PM5: P. multocida isolates number 1 to 5. Figure 1. The number of bacterial isolates resistant to antimicrobial agents (A) S. suis isolates. rial isolates that exhibited multi-drug resistance (MDR) (Figure 1). According to these data, each isolate of S. suis were resistant to at least one antimicrobial drug in more than six antimicro- bial categories. Each isolate of H. parasuis and P. multocida were resistant to at least one an- timicrobial drug in two or more antimicrobial cat- egories. The three B. bronchiseptica isolates were www.jad.hcmuaf.edu.vn The Journal of Agriculture and Development 19(3) 20 Nong Lam University, Ho Chi Minh City also against to at least one antimicrobial agent of beta-lactams, quinolones, aminoglycosides and lincosamides. Similarly, A. pleuropneumoniae iso- lates were resistant to at least one antimicro- bial agent of seven tested drug classes, except for macrolides. The results suggested that five species of bac- teria were highly multi-resistant to the eight common drug classes. Multi-drug resistance is a problem that continues to challenge the health- care sector. Different countries have reported the widespread of clinical resistance due to the mas- sive of antimicrobial drugs (Jong et al. 2018). The transmission of MDR bacteria into the commu- nity is seriously associated with increased mor- bidity, mortality, healthcare costs and antibiotic use. Together with many European countries and the USA, China is preparing a national action to deal with antibiotic resistance. Current tech- nology makes possible the identification of new drugs or inhibitors of resistance mechanisms to extend the life of existing antibiotics, or alterna- tives like plant extracts (Laxminarayan, 2013). However, these tend to take time and require fur- ther efforts. Initial steps to prevent the spreading of MDR is use antibiotics only when needed and correctly, control the usage by reducing antibi- otics in livestock management. Due to different antibiotic usage of differ- ent farms, more difficulty and complication have raised in the aspect of antibiotic control of the area. The temporary solution is giving drug reg- imen based on susceptibility result of individual farms. Long-term plan with a detailed guideline of antibiotic implication should be developed for the control of bacterial disease and protect public health from antimicrobial resistance. 3. Conclusions The results demonstrated high multi-resistance among the five bacterial species to the eight tested antimicrobial classes. The results empha- size the need for continuous surveillance of resis- tance patterns. Antibiotic prescription guidelines and infection control through the early detection of clinical should be carried out to prevent trans- mission of pathogens, as well as in the possible incorporation of the prevalent serotypes in the development of new vaccines. Acknowledgments I would like to express my gratitude to Prof. He Qigai and Assoc. Prof. Vo Thi Tra An for being my research supervisors, for their valuable sup- port and advice. I would like to send my special thanks to Dr. Sun Qi for his enthusiastic guid- ance. This study was supported by the Diagnos- tic Center for Animal Disease, College of Vet- erinary Medicine, Huazhong Agricultural Univer- sity, China. This research was supported by the China Agriculture Research System (No. CARS- 35). 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