Antimicrobial Resistance of Actinobacillus pleuropneumoniae, Streptococcus suis, and Pasteurella multocida Isolated from Romanian Swine Farms

Antimicrobial resistance is an important health issue in human and veterinary medicine. The aim of this study was to monitor the antimicrobial resistance of three of the most important bacteria involved in porcine respiratory disease. A total of 465 isolates were tested during the 2017–2022 period for antimicrobial susceptibility for Actinobacillus pleuropneumoniae (n = 137), Streptococcus suis (n = 207), and Pasteurella multocida (n = 121) by disk diffusion method. The results were interpreted by CLSI breakpoints, where available. High rates of susceptibility (from 90 to >99%) were observed for cefquinome, ceftiofur, amoxicillin + clavulanic acid, amoxicillin, penicillin, ampicillin, florfenicol, enrofloxacin, marbofloxacin, and trimethoprim–sulfamethoxazole. A. pleuropneumoniae isolates showed high resistance to streptomycin (77%), gentamycin (45%), tilmicosin (39%) erythromycin (33%), oxytetracycline (19%), and tetracycline (18%). For S. suis, the highest rates of resistance were observed for streptomycin (98%), tetracycline (75%), oxytetracycline (72%), doxycycline (52%), and erythromycin (51%). P. multocida presented a high rate of resistance for streptomycin (63%), tilmicosin (29%), oxytetracycline (13%), and tetracycline (14%). Bacteria isolates maintained high susceptibility against antimicrobial agents usually used against the mainly respiratory tract pathogens of swine. Resistance for streptomycin, tetracycline, oxytetracycline, and tilmicosin was high for all the tested pathogens


Introduction
The porcine respiratory disease complex (PRDC) is the result of the interaction of many factors, being a typical polymicrobial production disease and one of the most significant health and production problems, considered to be a leading cause of economic losses in the swine industry [1][2][3][4].Several factors contribute to the development of respiratory disease along with viral and bacterial infections, such as genetics, environmental conditions, production system, or management [1,2].A variety of bacterial pathogens is associated with PRDC, primary pathogens, such as Mycoplasma hyopneumoniae, Actinobacillus pleuropneumoniae, and Bordetella bronchiseptica, and secondary pathogens, including Pasteurella multocida, Streptococcus suis, Glaeserella parasuis, Actinobacillus suis, and Salmonella spp.[1,3,5,6].
Respiratory disease management is a major source of antibiotic use in the swine industry.It has been estimated that 60% of swine nurseries and 70% of the swine growerfinisher sites use injectable antibiotics to treat respiratory disease in the USA, while in Canada, approximately 44% to 67% of the grower-finisher pigs received antimicrobials in feed for preventive purposes [7][8][9].
Actinobacillus pleuropneumoniae (A.pleuropneumoniae) is a Gram-negative, facultative anaerobic bacteria and the causative agent of porcine pleuropneumonia, one of the most important life-threatening respiratory diseases in pigs, the most virulent strains determining fatal fibrinohemorrhagic and necrotizing pleuropneumonia in swine of all ages and chronic lung lesions in survivors [6, [10][11][12].Antimicrobials are the most effective control measure of A. pleuropneumoniae, leading to increasing levels of acquired resistance [10].
Streptococcus suis (S. suis) causes pneumonia and a diverse range of septic diseases, such as meningitis, arthritis, valvular endocarditis, polyserositis, abortion, and sudden death, mostly in the 5-10-week-old pigs [6,11,13].When antimicrobials are being used as a prophylactic or metaphylactic measure, the incidence of disease is generally lower than 5%; otherwise, the mortality may rise to 20%, but the antimicrobials that are efficient against S. suis are those that the industry is trying to reduce the usage of, because of their importance in both human and veterinary medicine [13].
Pasteurella multocida (P.multocida) is among the most commonly found and costly bacteria involved in PRDC, causing progressive atrophic rhinitis, with the destruction of the turbinate bones, and pneumonia [6,11,13].It is a commensal bacteria of the upper respiratory tract that can also cause pneumonia in growing and finishing pigs.P. multocida is normally considered a secondary agent, but it has also been described as a primary agent of hemorrhagic septicemia in pigs [14].Antimicrobials are the most used veterinary products for the management of P. multocida in animals including first-generation antibiotics [15].
Increasing antimicrobial resistance for many microorganisms has been reported in the past decades, so the interest in monitoring the trends of antimicrobial susceptibility of veterinary pathogens has extended [16].The aim of this study was to evaluate the antimicrobial susceptibility evolution of A. pleuropneumoniae, S. suis, and P. multocida isolated from swine in Romania from 2017 to 2022.

Sample Collection
This study was conducted on 465 bacterial isolates, 137 for A. pleuropneumoniae, 207 for S. suis, and 121 for P. multocida, as seen in Table 1, recovered from samples collected from lung tissue of swine with respiratory disease.Usually, the samples are collected with swabs by the veterinarian from the farm, and the administration of antibiotics and types of antibiotics given are not constantly reported.The bacterial isolates included in this study were obtained in a six-year period, between 2017 and 2022, in the Synevovet laboratory from Romania, and the farms are located all over the country, mainly in the south and west areas (Figure 1).

Isolation and Identification
The samples were cultured on chocolate agar and blood agar mediums and then incubated at 35-37 °C for 20-24 h in anaerobic conditions (CO2 5% thermostat, MMM Group, Munich, Germany).The colonies were selected based on their morphological characteristics, and then the pathogens were identified using MALDI-TOF technology (Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, Bruker Daltonics, Bremen, Germany).

Antimicrobial Susceptibility Testing
Antimicrobial susceptibility was tested using the disk diffusion method on Mueller-Hinton agar supplemented with 5% sheep blood (for S. suis and P. multocida) and chocolate agar (for A. pleuropneumoniae) [17,18].The inhibition zone sizes around the antibiotic disks were read with an ADAGIO automated system (Bio-Rad, Marnes-la-Coquette, France, 3.1).The interpretation of the resistance profiles was performed according to the CLSI (Clinical and Laboratory Standards Institute) standards, if available, and the antibiotic manufacturer.The following antimicrobials and concentrations were used and then classified as susceptible, intermediate, and resistant, as seen in Table 2.

Isolation and Identification
The samples were cultured on chocolate agar and blood agar mediums and then incubated at 35-37 • C for 20-24 h in anaerobic conditions (CO 2 5% thermostat, MMM Group, Munich, Germany).The colonies were selected based on their morphological characteristics, and then the pathogens were identified using MALDI-TOF technology (Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, Bruker Daltonics, Bremen, Germany).

Antimicrobial Susceptibility Testing
Antimicrobial susceptibility was tested using the disk diffusion method on Mueller-Hinton agar supplemented with 5% sheep blood (for S. suis and P. multocida) and chocolate agar (for A. pleuropneumoniae) [17,18].The inhibition zone sizes around the antibiotic disks were read with an ADAGIO automated system (Bio-Rad, Marnes-la-Coquette, France, 3.1).The interpretation of the resistance profiles was performed according to the CLSI (Clinical and Laboratory Standards Institute) standards, if available, and the antibiotic manufacturer.The following antimicrobials and concentrations were used and then classified as susceptible, intermediate, and resistant, as seen in Table 2.
The resistance rates of A. pleuropneumoniae are reported in Table 4, and the trends of resistance over the study period can be observed in Figure 2. A. pleuropneumoniae resistance for penicillin, tetracycline, trimethoprim-sulfamethoxazole, enrofloxacin, marbofloxacin, tilmicosin, reached the highest level in 2020, followed by a decrease in the following years.Streptomycin and gentamycin resistance rates were at the highest level in 2021, with lower prevalence next year, while oxytetracycline and doxycycline showed increasing trends over the tested period.

Discussion
Following the extent of use in human and veterinary medicine, antibiotic-resistan microorganisms are contaminating the soil and water, and their pattern of resistance correlated with the degree of antibiotic use [19].Because of the urgent need for antimicr bial treatment in acute infections, the treatment is based very often only on the suscept bility pattern of bacterial pathogens to antibiotics [11].
Numerous retrospective studies have been published for antibiotic susceptibili testing for A. pleuropneumoniae, S. suis, and P. multocida across the world.

Discussion
Following the extent of use in human and veterinary medicine, antibiotic-resistant microorganisms are contaminating the soil and water, and their pattern of resistance is correlated with the degree of antibiotic use [19].Because of the urgent need for antimicrobial Microorganisms 2023, 11, 2410 9 of 14 treatment in acute infections, the treatment is based very often only on the susceptibility pattern of bacterial pathogens to antibiotics [11].
Numerous retrospective studies have been published for antibiotic susceptibility testing for A. pleuropneumoniae, S. suis, and P. multocida across the world.
In a study from the Czech Republic, 242 A. pleuropneumoniae isolates showed low levels of antimicrobial drug resistance, except for tetracycline (23.9%).Resistance was low to florfenicol (0.8%), tiamulin (1.7%), tilmicosin (1.2%), ampicillin (3.3%), and amoxi-cillin/clavulanic (0.8%).Resistance to ceftiofur was not detected in any isolate [24].The results are similar to ours, except also for tiamulin and tilmicosin, which show a higher level of resistance in Romania.In a previous study from Romania for antibiotic resistance to A. pleuropneumoniae, the results showed an increasing trend of resistance to cefaclor, amoxicillin/clavulanic acid, oxytetracycline, ampicillin, and enrofloxacin and high susceptibility for florfenicol and erythromycin [25].
A. pleuropneumoniae trends of resistance in our study showed that for six antibiotics (penicillin, tetracycline, trimethoprim-sulfamethoxazole, enrofloxacin, marbofloxacin, and tilmicosin), the highest levels were reached in 2020, followed by a decrease in the following years.Streptomycin and gentamycin resistance rates were at the highest level in 2021, with lower prevalence next year, while oxytetracycline and doxycycline showed increasing trends over the tested period.In a report from Japan, no significant increase in resistance to the antimicrobials tested in this study was observed during the last decade when compared with published data on the prevalence of resistant strains of A. pleuropneumoniae collected from 1989 to 2005 [26].
Antibiotic resistance of our tested isolates of S. suis decreased over time for tetracycline from 95% to 63%, for oxytetracycline from 86% to 63%, and for doxycycline from 76% to 44%.For erythromycin, the level of resistance remained high over the years, but still with lower rates in 2022 compared with 2017.Low susceptibility rates were observed for tetracycline, 0 to 1.3% for S. suis in a study from the United States and Canada [5], with a significant difference from our study.Another recent study conducted in 10 European countries showed that the resistance of S. suis to ceftiofur, enrofloxacin, and florfenicol was absent or lower than 5%, while resistance to tetracycline was 82.4% in S. suis [27].
In the VetPath study, a tendency to increase the resistance was observed for ceftiofur in isolates of S. suis [16].In the Netherlands, it was reported that the resistance of S. suis to ampicillin, ceftiofur, clindamycin, enrofloxacin, florfenicol, penicillin, trimethoprim/sulfamethoxazole, and tetracycline was 0.3%, 0.5%, 48.1%, 0.6%, 0.1%, 0.5%, 3.0%, and 78.4%, respectively.In the same report, an increase in susceptibility to several antibiotics, similar to our study, was observed when comparing antimicrobial susceptibility for successive quarters.It was demonstrated that a significant decrease in susceptibility for clindamycin was observed in the fourth quarter of 2013 (compared with the third quarter of the same year), followed by a significant increase in the second quarter of 2014 (compared with the fourth quarter of 2013).The susceptibility of trimethoprim/sulfamethoxazole decreased significantly in the fourth quarter of 2013, compared with the third quarter of the same year, followed by a significant increase in the first quarter of 2014.For tetracycline, a significant decrease (p < 0.05) in susceptibility of S. suis was observed in the fourth quarter of 2013, compared with the third quarter of the same year and the second quarter of 2014, compared with the second quarter of 2013.Subsequently, a significant increase in tetracycline susceptibility of S. suis was seen in the fourth quarter of 2014.The reason for the decrease, followed by the increase in susceptibility for clindamycin, trimethoprim/sulfamethoxazole, as well as tetracycline, remains unknown [28].
In the United Kingdom, 405 S. suis isolates tested for antimicrobial susceptibility, and high rates of resistance were reported to tetracycline (91%), followed by erythromycin Since 2017, Romania has been the most African swine fever-affected country in the European Union, with 90% of the recorded outbreaks in domestic pigs [31].The ongoing evolution of African swine fever led to many changes in the Romanian swine industry.There were more than 6000 outbreaks, and many farms were affected and went through stamping out, leading to a renewal of pig populations.Many farms took the opportunity to improve the management conditions and even repopulated with specific pathogen-free animals.This could explain the variation in results from recent years.The effect of stamping out measures on the bacteria prevalence in the farms and the evolution of antimicrobial resistance could be further studied.
Examining the results of the current study, it is important to understand that the data were collected from isolates submitted for bacteria identification, and a much larger study would be necessary for obtaining an insight into the national picture, with more information regarding farms' antibiotic usage, clinical signs and lesions, vaccination programs, and strict sampling criteria.
Many European countries have developed surveillance systems for AMR in diseased animals, but antimicrobial resistance in animal bacterial pathogens is still considered a gap in the European One Health strategy on AMR surveillance [32].In a review of the national monitoring systems for ABR in animal bacterial pathogens in Europe, 12 countries reported having a national monitoring system: the Czech Republic; Denmark; Estonia; Finland; France; Ireland; Germany; the Netherlands; Norway; Spain; Sweden; and the United Kingdom [33].In Romania, there is no national surveillance program for antimicrobial susceptibility testing, even though many articles report an increase in bacteria resistance [25,[34][35][36].AMR is a priority public health issue.The country has one of the highest levels of antibiotic consumption in the human healthcare sector in Europe and faces some of the highest levels of antimicrobial resistance worldwide, as reported by the World Health Organization [37].Even though some legislative measures were taken, such as establishing the National Committee for Prevention and Limitation of Healthcare-Associated Infections and elaborating the National Guidelines Regarding Prudent Use of Antimicrobials in Veterinary Medicine, monitoring of AMR in Romania should be coordinated at a national level and follow the European trend and recommendation.Also, it is necessary to harmonize the antibiotic susceptibility testing interpretation criteria within the European Union, and we should use EUCAST ECOFFs (European Committee on Antimicrobial Susceptibility Testing epidemiological cut-off values) in the future.

Conclusions
Bacteria isolates coming from Romanian swine farms, collected from 2017 to 2022, maintained high susceptibility against antimicrobial agents usually used against the mainly respiratory tract pathogens of swine, such as cefquinome, ceftiofur, florfenicol, amoxicillin + clavulanic acid, amoxicillin, penicillin, ampicillin, and fluoroquinolones.The resistance to streptomycin, tetracycline, oxytetracycline, and tilmicosin was high for all the tested pathogens.The responsible use of antimicrobials, according to laboratory diagnosis results, after antimicrobial susceptibility testing is advisable.

Figure 1 .
Figure 1.Map of Romania showing the geographic location of the farms from which the pathogens included in this study were identified.

Figure 1 .
Figure 1.Map of Romania showing the geographic location of the farms from which the pathogens included in this study were identified.

Table 1 .
Number of isolates included in the study per year.

Table 2 .
Antimicrobials used for susceptibility testing, their concentration, and the inhibition diameters.

Table 3 .
The mean results of antimicrobial susceptibility testing for A. pleuropneumoniae, S. suis, and P. multocida, over 6-year period, classified as susceptible (S), intermediate (I), and resistant (R), expressed as percent (%) from the number of tested isolates.

Table 4 .
Antimicrobial resistance of 137 A. pleuropneumoniae isolated over 2017-2022, expressed as percent (%) from the number of tested isolates.

Table 4 .
Antimicrobial resistance of 137 A. pleuropneumoniae isolated over 2017-2022, expressed as percent (%) from the number of tested isolates.

Table 5 .
Antibiotic resistance of S. suis over 2017-2022, expressed as percent (%) from the number of tested isolates.

Table 6 .
Antibiotic resistance of Pasteurella multocida over 2017-2022, expressed as percent (%) from the number of tested isolates.

Table 6 .
Antibiotic resistance of Pasteurella multocida over 2017-2022, expressed as percent (%) from the number of tested isolates.