Multiple Drug Resistant Streptococcus Strains—An Actual Problem in Pig Farms in Western Romania

Streptococci are a type of bacteria that can cause severe illnesses in humans and animals. Some typical species like S. suis, or atypical species like S. porcinus and, S. dysgalactiae subsp. dysgalactiae, can cause infections like septicemia, meningitis, endocarditis, arthritis, and septic shock. S. suis is considered a newly emerging zoonotic pathogen. Although human streptococcal infection outbreaks are rare, it is appropriate to review the main streptococcal species isolated in pig farms in western Romania, due to the high degree of antibiotic resistance among most isolates commonly used in human treatment. This study examines the resistance patterns of these isolates over 5 years (2018–2023). The research investigated the antimicrobial susceptibility of 267 strains of Streptococcus spp. isolated from pigs, primarily from lung and brain tissues. This report is the first to describe the distribution of atypical Streptococcus species (SDSE, S. porcinus, S. hyovaginalis, S. pluranimalium, S. canis) in Romania, as well as the antibiotic resistance profile of these potentially zoonotic species. It is important to re-evaluate and consider the high rates of resistance of S. suis to tetracyclines, lincosamides, macrolides, and aminoglycosides, as well as the high recovery rates of S. suis from the lungs and brain when treating swine diseases.


Introduction
Streptococci can cause a range of diseases in both humans and animals, from mild to severe.Normally, these bacteria belong to the commensal microflora but can sometimes cause infections as opportunistic pathogens.In 2020, several zoonotic streptococci were identified, including S. suis, S. canis, S. dysgalactiae subsp.dysgalactiae, S. equi subsp.zooepidemicus, S. halichoeri, S. iniae, S. porcinus, and others.Under certain circumstances, these and other streptococcal species can also cause human diseases [1].
Although S. suis is the most important species for the global pig industry, other Streptococcus species may also pose a greater and often underestimated risk to pig health by causing diseases.Streptococcus suis possesses antigens somehow related to Lancefield group D streptococcus.It is considered a facultatively anaerobic, alpha-hemolytic, Grampositive, nonmotile coccus, displayed in chains of varying lengths.The number of reported human cases due to S. suis has increased significantly in recent years, mainly in China and Southeast Asian countries, especially in Vietnam and Thailand [2].
S. dysgalactiae subsp.equisimilis (SDSE) is a type of bacteria that belongs to the group of beta-hemolytic streptococci.It is typically found in the vaginal secretions and colostrum of sows and is considered to be a part of their commensal microflora.It is a common source of infection in piglets.SDSE can cause septicemia by entering the bloodstream through skin lesions or the tonsils of piglets and can lead to arthritis, meningitis, or endocarditis [3][4][5].
Streptococcus porcinus, a beta-hemolytic streptococci in Lancefield group NG1 (A1, C1), NG2, NG3, E, P, U, or V antigen, was first identified in pigs in 1984 and later detected in Out of the 267 strains of streptococci that were isolated, S. suis was the most found, accounting for 67.79% of the samples, followed by S. dysgalactiae subsp.equisimillis (SDSE) at 14.98%, S. porcinus at 8.98%, S. hyovaginalis at 4.86%, S. pluranimalium at 1.87%, and S. canis at 1.49%.The identification of different species of streptococci was confirmed by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF Biotyper System, Bruker Daltonics, Bremen, Germany).
All streptococcus species, except S. canis and S. pluranimalium, were found in samples obtained from the lower respiratory tracts and particularly from the lungs.S. suis was isolated mostly from the lungs and brain, and, to a lesser extent, from the surface of the serosa.
During the study, it was found that S. suis was predominantly isolated from the lungs (37.01%) and brain (27.50%), and, to a lesser extent, from the surface of the serosa (9.39%).The samples from the genitourinary tract had the highest proportion of SDSE isolation (32.50%), followed by the brain (27.50%) and lungs (15.00%).
Other Streptococcus spp.were isolated from various sources (heart and kidney), including the skin (33.33%), nose, organs, and raw semen (22.22% each).S. suis was the most isolated species.
After analyzing the data presented in Table 3, it was evident that several resistance patterns have emerged.
Among these isolates, twenty-two can be classified as multiresistant as they are resistant to three families of antibiotics.Seventy-four, ninety-one, and forty strains were resistant to, respectively, four, five, or six families of antibiotics.The most common patterns were found in the tetracyclines, macrolides, aminoglycosides, tetracyclines, and lincosamides groups.Aminoglycosides, Macrolides, Amphenicols, Sulphonamides 5 9.

Discussion
This retrospective study aimed to gather data on the isolation of streptococci from pig farms and expand the information about the antibiotic resistance of streptococcal species with potential zoonotic transmission.We lacked sufficient information about other diseases and previous antimicrobial treatments used in the pig farms from which the samples were collected.However, we believe that this study should be published because there is no data available on other streptococcal species found in pig farms in Romania with intensive rearing systems.The isolation of species such as S. canis highlights some unknown aspects, and therefore, we consider it important to bring it to attention.
Streptococcus suis is a significant concern in the pig industry and is particularly dangerous to piglets who have recently been weaned.It is an emerging zoonotic agent.It is important to note that S. suis can affect individuals near infected pigs, such as those who work in slaughterhouses or factories that process pork-meat products [19][20][21][22].
In a recent study conducted in Romania by Doma et al. (2021), it was found that the most common cause of pig mortality was S. suis (68-70%), followed by E. coli (30-31%) [23].
Although S. suis usually occurs more often in the upper respiratory tract than in the lower respiratory tract, S. suis has been detected more frequently in lung samples than in nasal swabs [20,24,25].We noticed the same thing in our study; the highest percentage of strains (37.01%) was isolated from the lung and only 5.14% of the strains were isolated from the noses.S. suis was also detected quite frequently in the brain (20.57%) followed by serosal surfaces (9.71%), parenchymal organs (8.23%), joints (6.28%), the genitourinary tract (5.71%), raw semen (2.85%), and skin (1.14%).
All the strains of S. suis that we isolated in pigs were from the pigs with clinical signs of disease.These pigs were also positive for other pathogens like PRRS (Porcine Respiratory and Reproductive Syndrome), PCV 2 (Porcine Circovirus 2), Mycoplasma hyopneumoniae, and Influenza A. These infections could have resulted in bacterial overgrowth, which may have made it easier for S. suis to invade.In our study, S. suis was the only species that we found in all the sites we examined, which included the CNS, upper and lower respiratory tracts, skin, mucous membranes, joints, serosal surfaces, and raw semen.
After being considered non-pathogenic for many years, SDSE is now recognized as an important bacterial pathogen [26,27].Recent epidemiological studies have shown an increasing number of invasive infections with SDSE in humans, often among immunocompromised patients and elderly patients with underlying co-morbidities, which suggests that this species is likely to become even more clinically important in the near future [28,29].
A previous study Stanton et al. [33] showed that SDSE can produce aggressive skin lesions.In our study, we isolated S. canis and S. pluranimalium, in addition to SDSE, from pig skin.
It is particularly important to elucidate the possibility that SDSE crosses the interspecies barrier between pigs and humans through comprehensive epidemiological approaches [26,34].Since the strains of streptococci isolated from pigs may be zoonotic pathogens, it is important to study their genotypes and antimicrobial resistance phenotypes to prepare for a potential public health hazard [35,36].It has been found that SDSE is a significant pathogen in pigs that can cause various health issues such as neurological signs, reproductive and respiratory disorders, and skin lesions.These findings are consistent with those obtained by Oh et al. [4].
In 2020, a study conducted in Korea revealed that SDSE strains from pigs exhibited elevated minimum inhibitory concentration (MIC) values for macrolides, tetracyclines, and fluoroquinolones.These antibiotics are commonly prescribed to individuals infected with SDSE as a substitute for beta-lactams [4].Consequently, further research with a more extensive sample of strains is necessary to ascertain the likelihood of SDSE transmission from animals to humans across various populations in Romania.
S. porcinus is the etiological agent of streptococcal lymphadenitis in growing pigs; it also causes throat abscesses, and has sometimes been isolated from pneumonia and sows' endometritis [37].However, the isolation of S. porcinus from the lower respiratory tract is still debatable as some authors consider it to be either a primary lung agent or a secondary lung agent [33,35].In contrast to previous reports, we could not isolate S. porcinus from CNS, skin, liver, spleen, or joints [8,38,39].
In our study, S. hyovaginalis was isolated from the lungs of three specimens, from the noses of two specimens, and the CNS in seven specimens, but could not be isolated from genitourinary tract samples.Previous reports have shown that S. hyovaginalis can be isolated exclusively from sow genitourinary tract specimens but did not isolate bacteria from the upper and/or lower respiratory tracts [25].
S. pluranimalium is a species of streptococci whose involvement is less well known in the etiology of some human and animal infections [40].Thus, some authors describe it as a new human pathogen [10,[41][42][43] with major implications for human health, and therefore, we consider it particularly important to know more about the prevalence of this bacterium in pig herds in Romania, as this report is the first in Romania describing the isolation of S. pluranimalium from pigs.
S. pluranimalium and S. canis were not isolated from the CNS, genitourinary tract, lungs, and joints, but were recovered from nasal cavities, skin, raw semen, and parenchymatous organs, which may indicate that they are species that can colonize the skin and apparent mucous membranes and that their presence in raw semen and the liver and spleen may only be accidental contamination.
Originally, in 1986, Streptococcus canis was thought to be a canine and bovine pathogen exclusively but has since been isolated from a range of wild and domestic mammals: cats, rabbits, rats, foxes, mink, raccoons, seals, sea lions, otters, and badgers, as well as humans [25,34,44,45].Along with other authors, we consider that S. canis may be capable of causing disease in several species, including pigs, making it one of the streptococcal pathogens with the widest host range [1,[46][47][48][49].The main hosts of this streptococcus species appear to be dogs and cats but we recovered from the nasal cavities, skin, and raw semen of pigs.Does the recovery of this species from pigs raise several questions about whether it is a species that can colonize skin and mucous membranes similar to what happens in dogs and cats?Or is it just accidental isolation?
In the context that our samples came from, pig farms with drastic biosecurity measures (no dogs on the farm), we can hypothesize that the transmission vector of S. canis could be humans.
Although in recent years the zoonotic potential of this bacteria has been widely accepted, scientific evidence remains limited [50,51].In a retrospective study conducted at the University Hospital of Bordeaux between 1997 and 2002, S. canis was confirmed in 1% (n = 80/6404) of all Streptococcus-positive samples sent for culture [45].Knowledge of the epidemiology of S. canis in both veterinary and human medicine is based on relatively few studies and information, and it is somewhat unclear how and to what extent transmission occurs between different animal species, as well as which risk factors predispose humans to S. canis infection [45,46].
It is also very important to note that more than one Streptococcus spp.isolate was recovered from animals with clinical signs in the pig herds from which the samples were taken.This may support the hypothesis that certain species not known to be primary disease agents are indeed an underestimated health risk for pigs and usually go undetected.
We could not demonstrate a causal association between clinical symptoms and the recovery of different Streptococcus spp.; further experiments and studies on a larger number of samples and information are needed for this purpose.
In Romania, antimicrobials are widely used in the animal husbandry sector, leading to widespread MDR in pig flocks; with this being very well highlighted both in this study and in other studies conducted by other researchers in our country.Thus, S. suis strains showed resistance to 50% (13/26) of the antimicrobial substances tested.
In 2022, the percentage of antimicrobial resistance to S. pneumoniae in humans in Romania was 48.3% according to the European Centre for Disease Prevention and Control [52].
The quality and safety of pork in these areas are questionable and it is believed to be the main source of human infection.Some authors suggest that exposure to pigs at work or consuming raw pork products increases the risk of S. suis infection [57][58][59][60][61].
According to research conducted by O'Dea in Australia [62], a significant proportion of isolates were found to be resistant to both tetracycline (99.30%) and erythromycin (83.80%).The study also observed low levels of resistance to florfenicol (14.90%), penicillin G (8.10%), ampicillin (0.70%), and trimethoprim/sulfamethoxazole (0.70%).Interestingly, none of the isolates were found to be resistant to enrofloxacin.However, the global trend of increasing antimicrobial resistance among streptococcal species is becoming increasingly problematic.
In this study, all isolated strains were resistant to at least one class of antibiotics, and a fairly high percentage were resistant to three or more classes of drugs, which indicated substantial multidrug resistance (MDR).Since 1980, there has been an increasing resistance of streptococci to antimicrobials commonly used in the pig industry, such as the tetracycline and macrolide group, around the world [50,60,[63][64][65].
In Romania, there is currently no systematic collection of data or comparisons on the antimicrobial resistance of S. suis from pig farms to different antimicrobials.However, there are extensive global reports of the high resistance of these bacteria [24,51,[64][65][66][67].
Our results also corroborate the results of Yongkiettrakul et al. (2019) [57] and Hernandez-Garcia et al. (2017) [68], who reported that the strains of streptococci isolated in their research became resistant to all classes of antibiotics used in pigs.
In the current context of reducing the use of antimicrobials in animals, the importance of different species of streptococci as zoonotic pathogens is increasing.Streptococci represent a threat to human health due to the lack of effective vaccines on the market; vaccines that could reduce the number of streptococci in pigs and therefore reduce the risk of human disease [69].More and more frequently, the idea is emerging that, in addition to reducing the use of antibiotics on farms, biosecurity and improved health management must also be considered [70].
Therefore, these atypical Streptococcus spp.isolated from pigs may pose a greater underestimated health risk to pigs but also to humans and should be correctly identified by more complex and sensitive veterinary diagnostic methods.

Clinical Samples
This is a retrospective study based on data collected by the Faculty of Veterinary Medicine Timisoara.The data comprises 4783 samples.These samples were collected over five years, from January 2018 to January 2023.Samples were taken from pigs displaying various clinical symptoms such as respiratory problems, nervous disorders, skin lesions, arthritis, pneumonia, genitourinary tract infections, and septicemia.All samples were collected by veterinarians and sent to the Faculty of Veterinary Medicine Timisoara-Department of Infectious Diseases and Preventive Medicine for routine diagnostic purposes.
A total of 267 samples of Streptococcus spp.were obtained from pigs of all age categories (sows, gilts, fattening pigs, weaned pigs, and suckling piglets) out of 4783 samples examined (Table 4).The samples came from 40 pig farms in the western and north-western parts of Romania, from the counties with the highest density of pigs in the country.The farms are intensive, closed systems, which are breeding, farrow-to-finish and fattening farms.Semen samples were taken from a semen center.

Bacterial Isolation and Identification
In this study, we describe the detection of Streptococcus (S.) suis, S. dysgalactiae subsp.equisimilis (SDSE), S. hyovaginalis, S. porcinus, and other streptococcus species (S. canis, S. pluranimalium) isolated from various types of samples.Antibiotic resistance paternity was also performed for strains of Streptococcus isolates.
The isolates with alpha-hemolytic colonies were further identified by conventional biochemical tests and API 20 Strep (bioMerieux SA, Marcy l'Etoile, France).Subsequently, the colonies were confirmed to be Streptococcus spp.by the matrix-assisted laser desorptionionization-time of flight mass spectrometry (MALDI-TOF MS).

MALDI-TOF MS Bacterial Identification
The ethanol/formic acid protocol was used to prepare MALDI-TOF MS samples.The bacterial protein suspension had a volume of 1 µL and was transferred to a MALDI target plate (Bruker, Daltonik, Germany).Then, 1 µL of matrix (10 mg α cyano-4-hydroxycinnamic acid ml-1 in 50% acetonitrile 2.5% trifluoroacetic acid) was added to the bacterial suspension.Bacterial mass spectra were acquired using the Microflex™ mass spectrometer (Bruker, Daltonik).For MALDI-TOF MS identification, the spectra captured were loaded into MALDI BioTyper™ 3.0 and compared to the manufacturer's library.Standard Bruker interpretation criteria were applied.Scores ≥ 2.0 were accepted for species assignment, and scores ≥ 1.7 but ≤ 2.0 were used for genus identification [43].

Antimicrobial Susceptibility Testing
Agar disk diffusion was used to perform susceptibility testing on a total of 267 isolates, following the recommendations given by the CLSI documents M100 (Clinical and Laboratory Standards Institute, 2023) [71].Antimicrobial substances were selected based on their interpretative criteria for certain species of Streptococcus spp., as well as their importance for both porcine and human health.
Each strain was subcultured on Columbia blood agar (5% sheep blood) and incubated overnight at a temperature of 37 • C with 5% CO 2 .Antimicrobial susceptibility was tested using the disc diffusion method on a Mueller-Hinton plate containing 5% sheep blood and according to the standards of Clinical Laboratory Standards Institute (CLSI) (VET01-A and VET01-S) [71,72].To prepare an inoculum for each strain, an initial concentration of 1 × 10 8 CFU/mL was used to inoculate the Mueller-Hinton plate before the application of antimicrobial discs (BIO-RAD, Marnes-La-Coquette, France).The plates were then incubated for 24 h at 35 • C.After bacterial growth, the inhibition zone diameters were measured.
The reference strain Streptococcus pneumoniae ATCC 49619 was used as a quality control according to CLSI recommendations.
Multidrug resistance (MDR) is defined as resistance to at least one agent from three or more antimicrobial categories, as classified by Magiorakos et al. [73].

Conclusions
To our knowledge, this is the first study in Romania on the isolation of streptococcal species other than S. suis from different pig pathologies.
Our results highlight the presence of atypical Streptococcus spp. in pig herds, which may represent a high and underestimated health risk for pigs but also for humans and should be correctly identified by more complex and sensitive veterinary diagnostic methods (MALDI-TOF MS).
We believe that further large-scale studies should investigate the impact of SDSE and S. porcinus on the pathogenesis of reproductive disorders in sows, in particular.
Nearly half of the isolated strains showed resistance to four or more groups of antibiotics.The use of antibiotics in animals resulted in a selection of resistant bacteria in animals.To what extent antibiotic use and resistance in animals also influence the occurrence of resistance in humans remains an important area of debate.It is desirable that the use of antibiotics continues to decrease, and eventually for it to become a rare occurrence in pig farming.However, this will require additional efforts and a focus on better husbandry, biosecurity, and management.Ultimately, this reduction will lead to a decrease in resistance selection, which will benefit both animal and human health, global food safety, and food security.

Figure 1 .
Figure 1.Distribution of the Streptococcus spp.isolated from various organs of pigs.

Figure 1 .
Figure 1.Distribution of the Streptococcus spp.isolated from various organs of pigs.

Table 1 .
Total number of isolated Streptococcus spp.from different sources.

Table 2 .
Antimicrobial sensitivity of isolated S. suis strains.

Table 3 .
The antimicrobial sensitivity patterns of the strains isolated from pigs.

Table 4 .
Number of Streptococcus spp.strains isolated from each age group.