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
|Abstract (english)|| |
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
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
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
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.