Isolation of a Multidrug-Resistant vanA -Positive Enterococcus faecium Strain from a Canine Clinical Sample in Greece

: An Enterococcus faecium strain was obtained from a paraprostatic cyst of a 17-year-old dog in Greece. Antibiotic susceptibility testing (AST) was accomplished by disc diffusion and MIC methods, and the isolate demonstrated a multidrug-resistant (MDR) phenotype against a great variety of antibiotics, such as β -Lactams, Quinolones, Macrolides, Tetracyclines, Rifampin, Nitrofurantoin, and surprisingly, Glycopeptides, Fosfomycin and Gentamicin (high-level). Molecular screening for Vancomycin resistance genes was carried out, and a vanA gene cluster was identiﬁed. To our knowledge, this is the ﬁrst report of a vanA -positive E. faecium strain isolated from a companion animal in Greece. Importantly, this strain was related with the presence of paraprostatic cysts, a pathological condition requiring treatment. The presence of a highly resistant isolate in a canine clinical sample and the consequent need for treatment constitutes a new challenge for veterinarians due to the lack of available treatment options. Our ﬁndings indicate the occurrence of respective bacteria in companion animals, which could act as a reservoir of epidemic MDR strains or relevant mobile genetic elements (MGE) in the community, constituting a threat for public health.


Introduction
Enterococci are Gram-positive facultative anaerobic cocci that were classified as group D Streptococci until the 1980s [1]. They can be easily obtained from a wide variety of hosts [2]. There are at least 58 recognized species so far, with E. faecium and E. faecalis being more regularly associated with clinical infections [1]. These species are included in ESKAPE organisms (Enterococcus spp., Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacter spp.) which, notably, have been demonstrated by World Health Organization (WHO) as a rising threat for public health due to multidrug resistance and the challenging nosocomial infections they cause [3].
A variety of factors contribute to the acquisition of resistance in Enterococci. Concerning the antibiotics used in veterinary medicine, the most important aspects are described in Table 1.

B-lactams
• Production of PBPs 2 that demonstrate a lower binding affinity to agents of this class, such as Ampicillin and Cephalosporins.
[ [20][21][22][23] Aminoglycosides Enzymatic inactivation of Gentamicin, Streptomycin, or both of them, mediated by acquired ARGs, confers high-level aminoglycoside resistance (HLAR) to Enterococci, while they are intrinsically resistant against the other agents of this class, escaping their bactericidal action by variable procedures. [20,24,25] Tetracyclines • Ribosomal protection encoded by genes tet(M), tet(O) and tet(S) results in resistance against all the available agents of this class in veterinary medicine (Tetracycline, Doxycycline and Minocycline). • Efflux proteins encoded by specific genes, such as tet(K) and tet (L) confer resistance against Tetracycline. [26]

Quinolones
Mutations of the target genes of the antibiotics, gyrA and parC, confer high-level acquired resistance, while Enterococci express low levels of resistance to Quinolones intrinsically. [20,27] Rifampin Mutations of the rpoB gene and consequently substitutions in the β-subunit of the RNA polymerase, which is the target of this agent. [28] Macrolides Production of a methyltransferase that alternates the 23S rRNA subunit, inhibiting the binding of the antibiotic, and is mediated by erm genes (and specifically ermB). [29] Limited data exist on the detection of vancomycin-resistant Enterococci strains of canine origin worldwide. In this study, we report the first case of a vanA-positive E. faecium strain with MDR phenotype obtained from the paraprostatic cyst of a 17-year-old dog in Greece, and we discuss the challenges faced by veterinarians when dealing with MDR strains and rising public health concerns.

Origin of the Isolate
A 17-year-old, male mongrel dog was admitted to a veterinary clinic in Thessaloniki, Greece, in January 2023. The dog had an open fracture in the radius as a result of a car accident, which had occurred 25 days earlier (Supplementary File, Figure S1a). In the intervening period, the dog received an antibiotic treatment consisting of marbofloxacin and clindamycin for about 20 days. During the clinical examination, paraprostatic cysts were also detected and demonstrated using diagnostic imaging (Supplementary File, Figure  S1b). Drainage of the cysts was carried out, samples were received and sent for investigation. Aerobic and anaerobic cultures were accomplished after inoculation on sheep blood agar and MacConkey agar and a 24 h incubation at 37 • C; Enterococcus spp. were isolated. The strain was initially identified by phenotypic and biochemical tests: Gram-positive cocci with characteristic colonial appearance (small colonies of approximately 1 mm with gamma-hemolysis), oxidase-and catalase-negative, aesculin-hydrolysis positive, no growth on MacConkey agar, and no sorbitol fermentation. Results were confirmed using the VITEK 2 biochemical identification system (Biomerieux, Supplementary File, Figure S2).

Antibiotic Susceptibility Testing
The disk diffusion method was used to evaluate susceptibility or resistance to a variety of antibiotics routinely tested in clinical samples of companion animals. Briefly, a colony of the strain was added to saline, and the resulting suspension was compared to a McFarland standard tube in order to achieve a 0.5 McFarland turbidity. The suspension was vortexed and, subsequently, a sterile swab was used to inoculate a quantity of it on the surface of Mueller-Hinton agar plates. Susceptibility discs were added, and the plates were incubated at 35 • C for 16-18 h. For the evaluation of Vancomycin zone diameter, a 24 h incubation period was essential. Due to the multidrug-resistant phenotype of the Enterococcus isolate, additional antibacterial agents were added to the antibiotic susceptibility test (AST). Consequently, the minimum inhibitory concentration (MIC) method was also evaluated (VITEK2, Biomerieux), including some antibacterial agents strictly used in human medicine, to confirm the previous results and to identify the isolate's resistance profile. The contents of the disks, the zone diameter, and the MIC breakpoints, as specified by the CLSI documents [30,31], are available in Table 2.  [30]. 1 Test for detection of high-level aminoglycoside resistance. 2 Any Growth = resistant. 3 Breakpoints for E. faecalis were used due to lack of respective breakpoints for E. faecium.

Multiplex PCR
The isolate was identified as E. faecium. Moreover, the vanA gene cluster was detected, confirming the Glycopeptide-resistant phenotype (Figure 1). None of the other antibiotic resistance genes (ARGs) included in the test were identified (vanB, vanC1-C2, vanD, vanE, vanG).

Multiplex PCR
The isolate was identified as E. faecium. Moreover, the vanA gene cluster was detected, confirming the Glycopeptide-resistant phenotype (Figure 1). None of the other antibiotic resistance genes (ARGs) included in the test were identified (vanB, vanC1-C2, vanD, vanE, vanG).

The Importance of Glycopeptide Resistance in a Canine Clinical Isolate
There are limited data about Vancomycin-resistant Enterococci in companion animals worldwide. To our knowledge, in Greece, this is:  The first report of a VREf isolate from a companion animal.  The first report of a VREf isolate causing an infection in any animal.

The Importance of Glycopeptide Resistance in a Canine Clinical Isolate
There are limited data about Vancomycin-resistant Enterococci in companion animals worldwide. To our knowledge, in Greece, this is:

•
The first report of a VREf isolate from a companion animal. • The first report of a VREf isolate causing an infection in any animal.
Additionally, this was the first Enterococcus spp. strain detected by the research team, among approximately 1072 isolates from clinical samples of companion animals, during the last five years, demonstrating Glycopeptide resistance, when tested by disc diffusion method.
Furthermore, the vanA-mediated high-level Glycopeptide-resistance of the strain, requires greater attention due to the co-current phenotypic resistances which were detected. This MDR profile is of major significance for two reasons.
Initially, there was a lack of available agents routinely used in veterinary practice for an effective treatment. For example, the respective CLSI document for veterinary isolates [31], in the breakpoints tables for Enterococcus spp., includes agents against which this isolate is phenotypically resistant (Penicillin, Ampicillin, Erythromycin, Rifampin, Vancomycin, Tetracycline, Doxycycline, Minocycline, Nitrofurantoin), with the exception of Chloramphenicol. Regarding Phenicols, even though they have been used in the past against Vancomycin-resistant Enterococci [34,35], their use is not regular nowadays (especially in human medicine) due to side effects (myellosupression, aplastic anaemia) and emerging resistance [36,37]. The identified high-level Gentamicin resistance is an additional notable aspect, as it is not usually observed in high rates among Enterococci of canine origin, even MDR strains or VRE [6,8,[38][39][40][41].
Moreover, the colonization of companion animals with respective MDR strains creates concerns regarding the transmission of these bacteria to their owners due to their accommodation in household environments.

Possible Factors Enhancing the Prevalence of VRE in a Companion Animal
Several causes related to the generic prevalence of such stains in the community, host affecting factors and bacterial adjustment properties could provoke the colonization of a dog by VRE.
The use of the glycopeptide Avoparcin as a growth promoter in food-producing animals, until its prohibition (1997 in EU), was related with the emergence of VRE in animals worldwide [55]. Since more than 25 years have passed though, the effect of its use is hopefully not a significant current factor for the VREf prevalence.
Prior exposure to several antibiotics has been described to provoke VRE colonization of human patients in several studies. Vancomycin, Cephalosporins, Aminoglycosides, Carbapenems, and Antianaerobic Agents, such as clindamycin and metronidazole, are some of the main associated agents [56][57][58][59][60]. Moreover, co-selection of resistance and a genetic linkage between Vancomycin and Macrolides has been identified for E. faecium in livestock animals [61,62]. As Cephalosporines, Aminoglycosides, Macrolides, Metronidazole, and Clindamycin are agents widely used in companion animals, the danger of VRE colonization of dogs through a co-selection reinforced by other antibiotics is significant.
Horizontal transfer of MGE and spread of epidemic clones enhance the prevalence of MDR Enterococci worldwide. The identification of a variety of unique strains is supported by the hypothesis that Vancomycin resistance could have initially emerged more by horizontal spread of MGE carrying the vanA and, perhaps, vanB gene cluster, among enterococci, rather than by transmission of a few major clones [63][64][65]. However, the spread of specific related clones with nosocomial infections has occurred in the last few years, and many of them are well characterized [66][67][68]. Companion animals could become a factor in a circulation of such strains in a community, as is in some cases VRE isolates from dogs demonstrating similar genetic lineages to hospital-acquired infections in humans [11,38,43].
Moreover, Enterococci possess the ability to develop resistance by facilitating survival in the environment of the gastrointestinal track; therefore, through intestinal colonization, the rise and spread of a multidrug-resistant clone among different hosts becomes possible, indicating a serious challenge for public health [69,70].
In accordance with all these, the gastrointestinal colonization of the dog by the VREf isolate in this study is possible, as Enterococci are species commonly detected in canine flora [6,7,10] and the host's own flora is usually the source of infection in prostatic and paraprostatic tissues [71,72]. The prior long-lasting antibiotic treatment could be a reinforcing factor, as the isolate is resistant to both Quinolones and Clindamycin and, therefore, its prevalence had been possibly enhanced by these agents. Finally, the presence of a mobile genetic element that could mediate an MDR phenotype to additional strains or the spreading of a specific hospital-associated MDR strain, as the animal is colonized, is definitely a matter of concern, indicating the need of surveillance in case of similar events.

Previous Research and Relevant Data in Greece
In the literature regarding VRE in Greece, data are mainly associated with human medicine and food-producing animals. In studies related to hospital environment, Vancomycin-resistant isolates were mostly identified as E. faecium with vanA-type resistance [73][74][75][76]. Furthermore, a link has been detected between VRE colonization and exposure to agents such as Vancomycin, Piperacillin-Tazobactam, Carbapenems, Antianaerobic Agents and Quinolones [75,77], while the duration of treatment with the respective antibiotics was an additional factor [77].
In reference to livestock animals, a 21.1% resistance rate to Vancomycin was detected in Enterococci isolated from raw pork meat from 2004 to 2007 [78]. Pigs, hospital and urban wastewater were screened for VRE in 2005-2006. VanA-positive E. faecium was dominant among the isolates, and a genetic diversity between Enterococci of different origins was identified [79]. In another study, samples from broilers and poultry slaughterers were collected during 2005-2008, and 130 VRE were recovered. The majority of these isolates were E. faecium harboring vanA gene, whilst no relationship was identified between poultry and the respective human-VREf clinical isolates originated from two hospitals in Greece [33].
Concluding, vanA-positive E. faecium seems to be the prevalent glycopeptide-resistant Enterococcus spp. encountered in the country and the main threat for public health. Mobile genetic elements are, rather, the cause of spreading of resistance, as genetic diversity is present among hospital-acquired and community strains. Finally, the induction of VREf colonization of hosts, is possibly related with prolonged usage of specific antibiotics.
The findings of our study are in accordance with these data, as a vanA-type VREf was isolated, the presence of a plasmid-mediated resistance is suspected, and a prior long-lasting treatment with clindamycin and marbofloxacin had occurred.
A noteworthy fact is that in the studies that referred to both human and animal samples, HLGR (which is identified in the isolate from this study) was related with hospital/human-associated strains, as it was rarely observed in samples of animal origin, and a significant statistical difference was detected [33,79].

Fosfomycin Resistance
A specific mention should be carried out for the Fosfomycin-resistant phenotype of this isolate, as it is an agent infrequently used in dogs, and, to our knowledge, the dog from this study had never received the antibiotic.
Fosfomycin has potentialities as an alternative agent against VRE, alone or combined with other agents [80][81][82][83][84]. In previous studies searching Fosfomycin resistance in VRE, it was mediated by the fosB gene (one or multiple copies) located in transferable plasmids, in all the isolates tested. A physical link between the fosB and Vancomycin ARGs (vanA or vanM) was detected, emphasizing the need of Fosfomycin-resistance surveillance in VRE [85][86][87][88]. Moreover, an amino acid substitution on the agent's active site of MurA protein has been detected in VREf expressing high-level Fosfomycin resistance [89]. The Fosfomycin-resistant phenotype in this isolate indicates a possible occurrence of one of the previously described mechanisms. The presence of a plasmid co-conferring Fosfomycin and glycopeptide resistance would definitely be a more significant issue and should be further investigated.

Conclusions
The isolation of a VREf co-expressing resistance to a wide spectrum of antibiotics from a canine sample is undoubtedly a matter of concern. This highly resistant phenotype is rarely encountered in community strains, whereas it is more common in hospital-associated ones. Moreover, the site of the sample, a contaminated paraprostatic cyst, is indicative of the isolate's origin from the host's own flora. A possible colonization of companion animals by similar strains raises an issue for public health, enhancing the prevalence and the circulation of MDR epidemic strains and respective MGEs between pets, owners, and their environment. Variable factors could contribute to this spreading, such as the prolonged and excessive usage of antibacterial agents in human and veterinary medicine. Surveillance measures are essential for the accomplishment of a comprehensive investigation of these factors, which could provide us the appropriate preventive actions.