ANTIMICROBIAL RESISTANCE OF ACTINOBACILLUS PLEUROPNEUMONIAE FIELD ISOLATES IN AUTONOMOUS PROVINCE OF VOJVODINA, REPUBLIC OF SERBIA

Actinobacillus pluropneumoniae (APP) is one of the most important bacterial respiratory pathogens in swine. It is the only etiological agent of porcine pleuropneumoniae (PPP) or appears as a secondary bacterial infection of the swine’s respiratory disease complex (PRDC). Antibiotics are still the most eff ective measure for reducing mortality and severity of clinical symptoms in most parts of the world. Nevertheless, in recent years, resistance to certain antibiotics such as tetracycline, penicillin, aminoglycosides and others has been increasing. Th e aim of this study was to examine the resistance of A. pleuropneumoniae isolates in the area of AP Vojvodina. Th e samples were collected from dead pigs’ lung tissue from 14 farms. Bacterial strains were isolated on agar with 5% sheep blood and Staphylococcus aureus culture as a source of factor V and for subculture chocolate agar enriched with 10 mg/L NAD was included as well. Aft er the examination of morphological and cultural characteristics, biochemical identifi cation was performed using standard bacteriological tests. APP was confi rmed by PCR. A total of 35 bacterial isolates were tested on antimicrobial resistance using the disc diff usion method with 11 antibiotics. Antibiotics


INTRODUCTION
Swine pleuropneumonia is one of the most important swine bacterial respiratory pathogens occurring worldwide. Th e occurrence of acute epidemics characterized by high mortality results in economic damage, loss in production, reduced growth and high medical costs. Many herds get infected with several strains. High virulence strains can be present in a herd for a long period of time without clinical symptoms or lesions in the slaughterhouse.
Th e causative agent of the disease, Actinobacillus pleuropneumoniae (A. pleuropneumoniae) is a small, Gram-negative, encapsulated rod with typical coccobacillary morphology. A. pleuropneumoniae isolates can be classifi ed into two biovars depending on the need for nicotinamide dinucleotide phosphate (NADP) for growth, with biovar 1 dependent on NADP and signifi cantly less common biovar 2 being independent of NADP (Gottschalk, 2012). Th ere are currently 19 recognized serovars of A. pleuropneumoniae, based on their capsule synthesis genes (cps), with certain diff erences in geographical distribution and pathogenicity Sassu et al., 2018). In most European countries, serovar 2 is a dominant strain causing most disease outbreaks . Th is was confi rmed recently in a German study based on more than 200 APP isolates originating from the same geographical area from the years 2010-2019, where 64% were found to belong to serovar 2 as the predominant serotype (Schuwerk et al., 2021). Th e most important virulence factors of A. pleuropneumoniae are capsular polysaccharides, LPS (endotoxin), and outer membrane proteins and exotoxins (Apx -toxins). A. pleuropneumoniae produces protein cytotoxins ApxI, ApxII, ApxIII and ApxIV belonging to the so-called RTX toxin family, which secrete diff erent serotypes in various combinations, through type 1 secretory mechanism (Schaller et al., 2000).
Antimicrobial resistance is becoming a major problem both in Serbia and worldwide. Th e aim of this study is to examine the susceptibility of pathogens to antibiotics that are most commonly used in the treatment of this disease. Infections in humans caused by antimicrobial resistance bacteria of animal's origin is becoming an urgent threat to the control of bacterial infections. Identifi cation of antibiotic-resistant or susceptible strains is essential in the fi ght against antibiotic-resistant pathogens. Determination of susceptibility to antimicrobial drugs is performed by disk diff usion method or by determining the minimum inhibitory concentration (MIC).

MATERIAL AND METHODS
Th e isolates used in this study were from lung tissue of pigs' carcasses from 14 diff erent farms in the territory of AP Vojvodina ( Figure 1). Lung tissue samples were collected from carcasses with typical lesions on the lungs (elevated cherry-coloured red areas/red areas of haemorrhagic-necrotic pneumonia) with pronounced interlobular edema and covered with fi brin deposits. A. pleuropneumoniae isolates were plated on Columbia Blood Agar with 5% defi brinated sheep blood. Culture of Staphylococcus aureus strain was used as a source of factor V and for subculture chocolate agar with PolyVitex (Bi-oMerieux, Marcy l'Etoile, France) enriched with 10 mg/L NAD was included as well. Incubation lasted for 24 h at 37 °C in the presence of 5% CO 2 . Aft er examination of morphological and cultural characteristics, biochemical identification was performed using standard bacteriological tests (Žutić et al., 2009).
APP was also confi rmed by PCR. Five pairs of oligonucleotide primers described by Rayamajhi et al., 2005, are used for the amplifi cation of 4 "Apx" genes encoding exotoxin synthesis ("ApxI", "ApxII", "ApxIII" and "ApxIV") characteristic of all serotypes and biovars of A. pleuropneumoniae. Amplification of the appropriate parts of the genome are performed using the polymerase chain reaction (PCR) in multiplex format (mPCR) using the set for performing the mPCR reaction (EURx). A "primer-mix" is prepared for a total reaction volume of 50 μl, with an oligonucleotide-primer concentration of 0.5 μM and a maximum number of oligonucleotide-primer pairs of 5 per one reaction according to the following formula: 25 μl master mix + 25 μl (primer mix + sample).
Urease activity and positive Christie-Atkins-Munch-Peterson (CAMP) test further refer to A. pleuropneumoniae. From fresh clinical samples, 70% of the strains were 'sticky' and diffi cult to remove from the plate, which correlates with their ability to form a biofi lm in vitro.
A total of 85 isolates were isolated from 14 farms across AP Vojvodina ( Figure 1). Th irty-fi ve isolates were tested for their susceptibility to a panel of antimicrobials by disk diff usion method on Mueller Hinton Agar Plate (MH agar) with the addition of 5% defi brinated horse blood for fastidious bacteria. Th e following antibiotics were used: penicillin G (6 mg), amoxicillin (25 mg), ampicillin (10 mg

RESULTS AND DISCUSSION
Altogether, 51.4% of isolates were susceptible to tetracyclines. Less than half of the isolates, more precisely, 48.6% were sensitive to penicillin. Th e ef-fi cacy results within the aminoglycoside group diff er markedly. Only 28.6% of isolates were sensitive to streptomycin and 34.3% to neomycin. Gentamicin, on the other hand, shows eff ectiveness in 65.7% of isolates (Table 1.). Our results show an identical effi ciency of 74.3% and a low rate of resistance to fl uoroquinolone (enrofl oxacin) and sulpha drug. With 80% of sensitive isolates, 3 rd generation cephalosporin was in the second place in terms of effi cacy. Florfenicol shows best results and the lowest rates of antimicrobial resistance. Only 17.1% of isolates were resistant (including intermediates). Antimicrobials are intensively used in swine production for therapeutic, metaphylactic and prophylactic purposes, which has led to an increased risk of bacterial resistance over time (Jensen et al., 2006;Aarestrup et al., 2008). Moreover, the movement of pigs between herds or between countries is another key factor that can contribute to the spread of antimicrobial-resistant isolates in swine populations.
Antimicrobials that are widely used in the treatment of swine pleuropneumonia were tested. Penicillin, tetracycline, amoxicillin, cephalosporin and fl orfenicol are recommended by the latest guidelines for pleuropneumonia therapy.
As already reported, β-lactams have shown high levels in vitro activity against A. pleuropneumoniae (Yoshimura et al. 2002;Matter et al., 2007). However, in recent years, a relatively large number of resistant isolates have been reported, mainly penicillin, while amoxicillin and ampicillin are slightly eff ective. Our results confi rm this rising trend mostly for penicillin. Minor antimicrobial activity of aminoglycosides against A. pleuropneumoniae isolates in our study are confi rmed by the results in other studies such as Gutierrez-Martin (2006) and Matter et al. (2007). Tetracyclines and sulpha drugs are still among the recommended antimicrobial agents for the treatment of swine pleuropneumonia . However, these antimicrobials have been used extensively to treat several swine diseases over decades, so increased resistance rates have been reported in several European countries

CONCLUSION
Isolates collected from the territory of AP Vojvodina from 2015 to 2022 retained high sensitivity to certain antimicrobial drugs used in therapy of A. pleuropneumoniae such as fl orfenicol, cephalosporin and fl uoroquinolones. With other antimicrobials such as penicillin, tetracycline and aminoglycosides, increasing antibiotic resistance is observed. Th is confi rms the importance of continuous monitoring of clinical isolates. In order for therapy to be successful and the use of antibiotics rational, it is necessary to have reliable data on the antimicrobial resistance for any pathogenic microorganisms. All farms should perform an antibiogram test before treating pleuropneumonia. It is an additional cost, but it pays off in the eventually. Th e therapy will be more successful, and the occurrence of antibiotics resistance will be reduced.
For years, the cause of the disease and the disease itself have been studied both together and separately. Th e acquired knowledge has enabled the improvement of diagnostic tests, vaccines and relatively eff ective eradication strategies. However, the A. pleuropneumoniae remains a signifi cant cause of large economic losses for the swine industry and there is more space for the improvement of control and eradication of this pathogen.