ANTIMICROBIAL RESISTANCE MECHANISMS OF BACTERIA WITHIN THE GENUS ENTEROCOCCUS ISOLATED FROM ANIMALS

Za ovo istraživanje prikupljeno je 100 izolata bakterija roda Enterococcus porijeklom iz kliničkih uzoraka domaćih životinja, većinom pasa (66) i mačaka (22). Najveći broj izolata izdvojen je iz uzoraka urina (34) i obrisaka zvukovoda (16). U daljnje istraživanje uključeni su izolati identificirani kao Enterococcus faecium (21 izolat) i Enterococcus faecalis (62 izolata). Kirby-Bauer disk-difuzijskim postupkom istražila se osjetljivost izolata navedenih vrsta na sljedeće antimikrobne lijekove: penicilin, ampicilin, vankomicin, enrofloksacin, ciprofloksacin, nitrofurantoin, rifampicin, tetraciklin, kloramfenikol, streptomicin i gentamicin. Streptomicin i gentamicin korišteni su za detekciju izolata s rezistencijom visokog stupnja na aminoglikozide. Najveći broj izolata bio je rezistentan na enrofloksacin i rifampicin (83 %), a najmanje izolata bilo je rezistentno na kloramfenikol (12 %). Rezistencija na vankomicin bila je prisutna u 23 % izolata, a 17 % posjedovalo je rezistenciju visokog stupnja na aminoglikozide. Vrsta E. faecium bila je rezistentna na više skupina lijekova od E. faecalis, a značajna razlika dokazana je u broju rezistentnih izolata na penicilin, ampicilin, ciprofloksacin, nitrofurantoin i tetraciklin. Kod svih izolata rezistentnih na vankomicin i tetraciklin te onih s rezistencijom visokog stupnja na aminoglikozide, istražen je mehanizam rezistencije. Kod tri od 19 izolata rezistentnih na vankomicin dokazana je prisutnost vanA i vanB gena. Gen tetM bio je prisutan u 49 od 50 tetraciklin rezistentnih izolata, a gen tetL u njih 16. Izolati s rezistencijom visokog stupnja na gentamicin bili su pozitivni na gene aph(3’)-IIIa i aac(6')-Ie-aph(2'')-Ia, dok su izolati s rezistencijom visokog stupnja na streptomicin bili pozitivni na gene aph(3’)-IIIa i ant(6)-Ia.Introduction: Bacteria belonging to the genus Enterococcus are extremely hardy and widely distributed in a variety of environmental habitats such as soil, sediments, beach sand, water, aquatic and terrestrial vegetation. In addition, enterococci are common inhabitants of the gastrointestinal tract of many animals, from insects to humans, as well as common opportunistic pathogens. Enterococci cause a number of infections in humans and animals; including urinary tract infections, mastitis, endocarditis, meningitis, intra-abdominal and wound infections. Enterococcus faecium and Enterococcus faecalis are the most prevalent species found in clinical samples, but also food and environmental specimens. Enterococci are intrinsically resistant to several commonly used antimicrobial drugs. At the same time, they have a great capacity to acquire new resistance mechanisms that can lead to selection of multidrug-resistant isolates that can only be partially treated with currently available antimicrobial agents. In the last three decades, vancomycin-resistant strains have become particularly prominent in human medicine, becoming one of the leading causes of nosocomial infections. The World Health Organization has included E. faecium to the list of bacteria for which new antibiotics are urgently needed. Enterococci are also becoming more common causative agents among the infections in animals, particularly in recurrent urinary tract infections. Treatment of enterococcal infections can be challenging and complicated by the multidrug-resistant isolates and biofilm formation. There are several different therapeutic strategies available in human medicine, depending on the complexity of the infection or the species causing it; however, ideal and effective therapy does not exist. Fewer drugs are available in veterinary medicine, and optimal therapeutic strategies for patients with enterococcal infections are undefined. As it seems, enterococci can develop resistance to any drug or combination of drugs used against them. Consequently, inappropriate and excessive use of antimicrobials leads to further selection of resistant bacterial strains. Transfer of resistant enterococci between animals and humans has already been observed, as well as the presence of enterococcal genes for multidrug resistance in different gut bacteria in humans. Considering the close contact between humans and animals, surveillance and control measures are crucial to prevent transmission of resistant enterococci from animals to humans. Since there are no new antimicrobials available, special attention should be paid to protect public health within one health continuum. The aim of this study is to investigate the prevalence and antimicrobial susceptibility of Enterococcus species isolated from various clinical samples of domestic animals in Croatia. Secondly, to determine the mechanisms of resistance in isolates resistant to vancomycin, tetracycline or aminoglycosides. Material and methods: One hundred Enterococcus isolates were isolated in the bacteriological laboratory at the Department for Microbiology and Infectious Diseases with Clinic at the Faculty of Veterinary Medicine, University of Zagreb in the period from January 2016 until the end of September 2021. Isolates were recovered from various domestic animal samples, mostly from urine and ear swabs, followed by vagina, wound, nose and skin swabs. Most of the samples were from dogs and cats, and several samples were from pet rodents and farm animals (horses, cows, sheep, and goat). Identification of the genus Enterococcus was carried out according to the procedure described by Markey et al. (2013). All isolates were tested in reaction with hydrogen peroxide, to demonstrate the absence of catalase. Furthermore, to distinguish members of the genus enterococcus from other gram-positive catalase-negative cocci, isolates were cultured on kanamycin-aesculin agar. Grey colonies with black surrounding area were considered to be enterococci. Multiplex PCR method with primers for species specific ddl genes was used to identifie Enterococcus faecium and Enterococcus faecalis. Only isolates identified as E. faecalis and E. faecium were selected for further research. Antimicrobial susceptibility of both species was determined by Kirby-Bauer diskdiffusion assay for eleven antimicrobials: penicillin, ampicillin, vancomycin, nitrofurantoin, rifampicin, tetracycline, chloramphenicol, enrofloxacin, ciprofloxacin, streptomycin and gentamicin. Streptomycin (300 μg) and gentamicin (120 μg) were used to test high-level aminoglycoside resistance. E. faecalis ATCC® 29212 was included in the study as a control strain. Inhibition zone diameters were interpreted according to criteria recommended in the Clinical and Laboratory Standards Institute document (CLSI, 2015). Detection of mechanisms responsible for vancomycin, tetracyclin and aminoglycoside resistance was performed using molecular methods. Multiplex PCR was used for testing the vancomycin-resistant and tetracycline-resistant isolates. For detection of resistance mechanisms, the following genes were examined: vanA and vanB, as well as tetL and tetM in the case of vancomycin and tetracycline, respectively. In aminoglycoside-resistant isolates PCR screening for genes encoding aminoglycoside-modifying enzymes aph(3’)-IIIa, aac(6')- Ie-aph(2'')-Ia and ant(6)-Ia was carried out. For statistical analyses STATISTICA v.13.5 (Statistica, Inc., 2020) program was used. Fisher exact test was used to compare the differences between E. faecium and E. faecalis isolates, including differences in frequency of the species among isolates, in susceptibility to antimicrobial agents and in number of multidrug and high-level aminoglycoside resistant isolates. Results: Isolates were mostly recovered from urine (34%) and ear swab (16%) samples and the majority were from dogs (66%) and cats (22%). Among 100 Enterococcus spp. isolates, 62 isolates were identified as Enterococcus faecalis and 21 as Enterococcus faecium. E. faecalis was the predominant species (p<0,05) of the total number of tested isolates, as well as in those recovered from dogs. Based on disk-diffusion assay results for both species cumulative, highest resistance was observed to enrofloxacin and rifampicin (83%), followed by ciprofloxacin and tetracycline (60%). Resistance to vancomycin was 23%, and the lowest resistance was to chloramphenicol (12%). Multidrug resistance is defined as acquired non-susceptibility to at least one agent in three or more antimicrobial drug classes. It was found in 60% of enterococci isolates. Highlevel streptomycin resistance was present in 17%, and high-level gentamicin resistance in 8% of isolates. Two isolates had high-level resistance to both streptomycin and gentamicin. In total, high-level aminoglycoside resistance was present in 20% of the isolates. Comparing the susceptibility of the species, E. faecium showed the highest resistance to enrofloxacin and ciprofloxacin (90%), followed by tetracycline (85%) and penicillin, ampicillin, and rifampicin (81%). The lowest resistance was to chloramphenicol and vancomycin (5%). Multidrug resistance was found in 90% of the E. faecium isolates. Highlevel streptomycin resistance was present in 24%, and high-level gentamicin resistance in 5% of the isolates. One isolate had high-level resistance to both streptomycin and gentamicin. E. faecalis isolates showed highest resistance to rifampicin (84%) and enrofloxacin (80%) followed by tetracycline (52%) and ciprofloxacin (50%). Resistance to vancomycin was present in 29% of the isolates. Lowest resistance was to nitrofurantoin (2%) and there was no isolate resistant to ampicillin or penicillin. Multidrug resistance was observed in 69% of the E. faecalis isolates. High-level streptomycin resistance was present in 15%, and high-level gentamicin resistance in 10% of isolates. One isolate had high-level resistance to both streptomycin and gentamicin. E. faecium isolates were resistant to more classes of antimicrobials than E. faecalis. E. faecium isolates were significantly more resistant to penicillin, ampicillin, ciprofloxacin, nitrofurantoin and tetracycline, however, E. faecalis isolates were significantly more resistant to vancomycin (Fisher exact test, p < 0, 05). Considering the high number of urine samples positive to enterococci growth, susceptibility of those isolates was revised separately. The highest resistance was observed to rifampicin (84%) and enrofloxacin (78%), followed by ciprofloxacin and tetracycline (68%). Resistance to vancomycin was present in 23% of the urine isolates. The lowest resistance of the isolates was to chloramphenicol (19%). Multidrug resistance was found in 72% of enterococci isolates from urine. High-level streptomycin resistance was present in 16%, and high-level gentamicin resistance in 9% of isolates. Comparing the susceptibility of the species, E. faecium isolates from urine were resistant to more antimicrobials than E. faecium isolates from other specimens. Eleven out of twelve E. faecium isolates were resistant to penicillin, ampicillin, enrofloxacin, ciprofloxacin, rifampicin and tetracycline (92%). One isolate was resistant to vancomycin (8%), and no isolates were resistant to chloramphenicol. Multidrug resistance was found in 92% of the E. faecium isolates. High-level streptomycin resistance was present in 17%, and high-level gentamicin resistance in 8% of isolates. In total, high-level aminoglycoside resistance was present in 17% of the E. faecium isolates. Among E. faecalis isolates from urine, highest resistance was observed to rifampicin (80%) and enrofloxacin (70%). Resistance to vancomycin was present in 35% of the isolates. There were no resistant isolates to penicillin, ampicillin and nitrofurantoin. Multidrug resistance was found in 60% of the E. faecalis isolates. High-level streptomycin resistance was present in 15%, and high-level gentamicin resistance in 10% of isolates. E. faecium isolates from urine samples were resistant to more classes of antimicrobials than E. faecalis. There was significantly more resistant isolates of E. faecium to penicillin, ampicillin, ciprofloxacin, nitrofurantoin and tetracycline (Fisher exact test, p < 0, 05). Among 19 isolates resistant to vancomycin, PCR revealed the presence of the acquired resistance gene in three isolates. Two enterococci isolates were positive for vanA and one for vanB gene. Among 50 isolates resistant to tetracycline, PCR revealed the presence of tetM gene in 49 isolates and tetL gene in 16 isolates. All isolates that were carrying tetL, were also carrying tetM gene. Acquired resistance genes were not detected in one isolate. All isolates with high-level gentamicin resistance, were positive to aph(3’)-IIIa and aac(6')-Ie-aph(2'')-Ia genes. Six isolates with high-level streptomycin resistance were positive to ant(6)-Ia gene, and two of them also had aph(3’)-IIIa. Acquired resistance genes were not detected in three isolates. One isolate which was phenotypically resistant to both gentamicin and streptomycin had all three genes present. Conclusions: The vast majority of the Enterococcus spp. bacteria isolated from animal samples in Croatia belong to two species, Enterococcus faecium and Enterococcus faecalis. Enterococcus faecalis is the dominant species among enterococci isolated from animal samples in Croatia. Resistance of enterococci to penicillins is present only in isolates of the species Enterococcus faecium. Due to the possibility of transmission of resistant enterococci from animals to human, there is a risk of spreading penicillin-resistant strains of Enterococcus faecium to the human community. Since penicillins are the primary therapy for enterococcal infections, the spread of such isolates represents a public health problem. Synergistic therapy of enterococcal infections with penicillin and aminoglycosides remains the primary choice in the treatment of animals with enterococcal infections because penicillin resistance with high-level aminoglycoside resistance is not common in enterococci of animal origin. Despite its concentrating properties, enrofloxacin is not a good choice for the treatment of urinary tract infections caused by enterococci because the vast majority of the isolates tested are resistant to enrofloxacin. Kirby-Bauer disk-diffusion method is not a reliable method for assessing the susceptibility of Enterococcus spp. isolates to vancomycin. For the purpose of more precise determination of the mechanism responsible for a highlevel streptomycin resistance, the presence of two genes, ant(6)-Ia and ant(3’’)-Ia, should be examined by molecular methods