Antimicrobial Susceptibility Profiles of Acinetobacter baumannii Strains, Isolated from Clinical Cases of Companion Animals in Greece

Simple Summary Acinetobacter baumannii Complex is a worldwide distributed group of species responsible for several challenges in treating nosocomial infections in humans. The emergence of highly resistant strains, even to last-resort antibiotics, constitutes a severe threat, especially for hospitalized patients. In veterinary medicine, its role has not been comprehensively investigated yet. However, many recent studies indicate its ability to cause infections in multiple animal species, primarily pets. In this study, we obtained a significant number of A. baumannii isolates from canine and feline clinical samples during 2.5 years, in Greece. Data regarding the isolates’ sample origin, type of infection and resistance profile were collected and compared. High resistance rates against several antibiotics were detected, including agents of paramount clinical importance, such as carbapenems. This study indicates the emergence of Acinetobacter baumannii Complex bacteria as pathogens for companion animals, the prevalence of strains with acquired resistance to many of antibiotics, and the danger of circulation of these strains between animals, humans, and veterinary equipment and facilities. Abstract Acinetobacter baumannii–calcoaceticus (Abc) Complex bacteria are troublesome nosocomial pathogens in human medicine, especially during the last 30 years. Recent research in veterinary medicine also supports its emergence as an animal pathogen. However, relevant data are limited. In this study, we obtained 41 A. baumannii isolates from clinical samples of canine and feline origin collected in veterinary clinics in Greece between 2020 and 2023. Biochemical identification, antimicrobial susceptibility testing, molecular identification and statistical analysis were performed. Most of the samples were of soft tissue and urine origin, while polymicrobial infections were recorded in 29 cases. Minocycline was the most effective in vitro antibiotic, whereas high resistance rates were detected for almost all the agents tested. Notably, 20 isolates were carbapenem resistant and 19 extensively drug resistant (XDR). This is the first report of canine and feline infections caused by Abc in Greece. The results create concerns regarding the capability of the respective bacteria to cause difficult-to-treat infections in pets and persist in veterinary facilities through hospitalized animals, contaminated equipment, and surfaces. Moreover, the prevalence of highly resistant strains in companion animals constitutes a public health issue since they could act as a reservoir, contributing to the spread of epidemic clones in a community.


Introduction
Bacteria of the genus Acinetobacter are members of the Moraxellaceae family, which were comprehensively described and recognized approximately 60 years ago [1,2].They are Gram-negative, non-fastidious, strictly aerobic, catalase-positive, oxidase-negative bacteria [3], regularly associated with nosocomial infections in humans [4].
The majority of clinical infections caused by Acinetobacter spp.are attributed to a specific group of species, including A. baumannii, A. nosocomialis, A. pittii, A. calcoaceticus, A. seifertii, and A. dijkshoorniae.These species are usually referred to as Acinetobacter baumannii-calcoaceticus (Abc) complex, since they are closely related and their discrimination through phenotypic and biochemical characteristics is insufficient [3,4].Acinetobacter baumannii is of major importance in human medicine over the last three decades [4].It is unsurprisingly included in the ESKAPE bacteria (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacter spp.), listed as one of the most challenging pathogens for health care professionals [5].
Bacteria of the Abc Complex are intrinsically resistant to a variety of agents, such as aminopenicillins (even combined with the β-lactamase inhibitor clavulanic acid), firstand second-generation cephalosporins, including cephamycins (cefoxitin and cefotetan), chloramphenicol and fosfomycin [6][7][8].Furthermore, the acquisition of resistance against other classes of antibiotics is frequent, dramatically reducing the available treatment options in the armamentarium of health scientists [4].For example, the worldwide emergence of carbapenem-resistant strains in the 21st century significantly limited the therapeutic strategies.Further, these strains were regularly resistant to several antibiotics, possibly due to the acquisition of mobile genetic elements containing numerous antibiotic resistance genes (ARGs) [3,5].
The objective of this study was to demonstrate the emergence of Abc infections in companion animals and the distribution of highly resistant isolates in canine and feline populations in Greece.Moreover, it indicates the danger of persistent circulating relevant strains in veterinary facilities and between animals and their owners.

Isolation and Selection of the Bacterial Strains
The bacteria included in this study were obtained from canine and feline clinical samples received during October 2020-May 2023.These samples had been collected in veterinary clinics throughout Greece during routine veterinary practices.Isolation, phenotypic and biochemical identification as well as routine susceptibility testing were initially performed.The identification of the isolates belonging to the Abc complex included criteria provided by literature [14], such as Gram-negative, catalase-positive, oxidase-negative coccobacilli, non-hemolytic, opaque circular colonies on sheep blood agar (Figure S1), growth on MacConkey Agar (pink-grey opaque colonies (Figure S2), inhibition of growth in anaerobic conditions, Tryptic Sugar Iron (TSI) Agar profile: Alkaline slant/Alkaline butt/Gas (−)/H2S (−).Additional biochemical tests were performed by a commercial identification kit (Microgen GNA-ID System, Microgen Bioproducts Ltd.-Figure S3), evaluating the following assays: negative test for mannitol fermentation, indole production, nitrate reduction, and urease test, positive for glucose and citrate utilization.Isolates identified as members of the Acinetobacter baumannii-calcoaceticus complex were subsequently inoculated into a general-purpose culture medium (Tryptic Soy Agar) in order to achieve a pure culture, collected and maintained in Brain-Heart Infusion Broth supplemented with 20% glycerol at −80 • C.

Antimicrobial Susceptibility Testing
The identification of the resistance profiles of the isolates was initially performed through the disk diffusion method (Figure S4) and consequently confirmed, by the minimum inhibitory concentration (MIC) method (VITEK ® 2, bioMérieux, Lyon, France).Antibiotics were selected from the respective table of agents that should be considered for testing against Acinetobacter spp., of the relevant CLSI document [15], focusing on agents that could be utilized in veterinary medicine.Therefore, during antimicrobial susceptibility testing (AST), piperacillin + tazobactam, imipenem, amikacin, gentamicin, tobramycin, ciprofloxacin, sulfamethoxazole + trimethoprim and minocycline were evaluated with both techniques.Doxycycline ampicillin + sulbactam, ceftazidime, cefepime and enrofloxacin were tested only by the disc diffusion method, while meropenem, colistin, and levofloxacin were examined only by MIC.
For the disc diffusion method, a colony of each isolate was inoculated into saline, and the suspension was subsequently compared to a McFarland standard tube, to achieve the desirable turbidity (0.5 McFarland).The suspension was then vortexed, and a quantity was inoculated on the surface of Mueller-Hinton agar plates using a sterile swab.Susceptibility discs were added, and the plates were incubated at 35 • C for 16-18 h.
The contents of the disks, the zone diameter and the MIC breakpoints, as specified by the CLSI document, are presented in Table 1.

PCR for Detection of the bla OXA-51-like Gene
All isolates were subjected to PCR, to detect the bla OXA-51-like gene, which is intrinsic in Acinetobacter baumannii [16].
Whole genomic DNA extraction was performed, from all the selected isolates, using a commercial spin-column kit (IndiSpin Pathogen Kit, INDICAL BIOSCIENCE GmbH).The procedures were carried out according to the manufacturer's instructions.To perform PCR, the following primers were used: OXA-51-likeF 5 -TAA TGC TTT GAT CGG CCT TG-3 and OXA-51-likeR 5 -TGG ATT GCA CTT CAT CTT GG-3 , as previously described [16].Briefly, for the reaction, a 25 µL mix was created for each strain, by adding 12.5 µL of Xpert Fast Mastermix (2X) with dye (GRiSP Research Solutions, Porto, Portugal), 2 µL (10 pmols) of each primer, 0,5 µL of bacterial DNA and 8µL of PCR-grade water.the conditions were the following: 95 • C for 1 min, and then 40 cycles at 95 • C for 15 s (denaturation), at 60 • C for 15 s (annealing), and at 72 • C for 3 sec (elongation), followed by a final extension at 72 • C for 3 min.DNA products were identified after electrophoresis in 0.5 Tris-borate-EDTA using 1.5% agarose gel stained with ethidium bromide solution.

Origin of the Isolates
A group of 41 isolates was obtained during the period mentioned above.Samples were received from 14 different veterinary clinics in four cities throughout the country (Athens, Thessaloniki, Serres, Larissa).All of them were identified as members of the Abc complex by the conventional biochemical tests.The bionumbers obtained by the VITEK ® 2 compact were 02(V a )1010(V b )03500(V c V d V e ) (V a = 0 or 4, V b = 1 or 3, V c = 2 or 3, V d = 1 or 5, V e = 0 or 2), providing an excellent identification of Acinetobacter baumannii with a 99% probability for all 41 strains.Data regarding the origin of the samples, animal species, and site of infection are presented in Table 2. Canine and feline strains were mainly associated with soft tissue (mostly wounds and skin abscesses, 61.5%) and urine samples (53.3%), respectively, showing different distribution (chi 2 = 12.48, df = 5, p = 0.028).A few bacteria originated from different sites of infection, such as the ear canal, nasal cavity, pleural effusions, and blood.
The characteristics of the infected animals and the bacterial species, which were co-isolated in the same samples, are presented in Table 3.
Approximately two-thirds (65.9%) of the infected animals (27/41) were males.However, the sample size was relatively small for a safe assumption.The average age of the infected animals was approximately 6.5 (SD = 3.8) years old.Regarding dogs, the mean age was 7.3 (SD = 3.8) years old, while the average age in cats was relatively lower, at approximately 5,2 (SD = 3.6) years old.
In contrast, Acinetobacter baumannii was the only bacterial species obtained in 9/12 urine samples (75.0%).MDR: Multi-drug resistant-an isolate that exhibits a resistant phenotype against antibiotics of three or more different classes, according to the formerly proposed criteria [17]; MRSA: methicillin-resistant Staphylococcus aureus; MRSP: methicillin-resistant Staphylococcus pseudintermedius; NA: not available; ND: not detected; SDR: single-drug resistant-an isolate that exhibits a resistant phenotype against antibiotics of one or two different classes, not counting the intrinsic resistance mechanisms of each species. 1 The isolates included in this section were obtained from the same sample with the selected bacteria.

Antimicrobial Susceptibility Testing
Detailed results of the susceptibility testing of the isolates by disc diffusion, are available in Table S1 (Supplementary File).Data regarding the prevalence of resistance for each antibiotic, by the disc diffusion method, are presented in Table 4.All strains (41/41) exhibited a quinolone-resistant phenotype, while significant resistance rates were also detected for gentamicin (75.6%), doxycycline (68.3%) and sulfamethoxazole-trimethoprim (63.4%).On the other hand, minocycline was the most effective antibiotic in vitro, as only 12.2% (n = 5) of the isolates were resistant.No statistically significant differences were detected on resistances between canine and feline samples.The results of the MIC test were in accordance with Table 4, with a few exceptions.Initially, all isolates were susceptible to colistin and resistant to levofloxacin.All three strains which were intermediate to imipenem by the disc diffusion test (A18, A26, A28) were susceptible to both imipenem and meropenem with an MIC = 2 µg/mL by VITEK ® 2.
Several profiles of antibiotic resistance were documented.Each specific resistance pattern and the isolates that demonstrated the respective phenotype by the disc diffusion method are listed in Table 5.
Additionally, the isolates are not proportionately distributed among the documented resistance patterns, since the grand majority of them are either susceptible to most drugs or XDR.
Particularly, all bacteria exhibiting an imipenem-resistant phenotype are MDR or XDR.The variation in resistance rates for each one of the antibiotics tested between carbapenemresistant and non-resistant isolate, is presented in Table 6.It is clearly observed that imipenem-resistant bacteria exhibit significantly higher resistance rates for all antibiotics (except quinolones, where the rate is 100% for both groups).The group size is relatively small; however, a considerable indication is provided that carbapenem resistance in the included strains is regularly co-current with resistance mechanisms against several other antibacterial agents.

Detection of the bla OXA-51-like Gene
All isolates (41/41) were positive for the presence of the bla OXA-51-like gene (Figure S5), in confirmation of the biochemical identification tests.

Discussion
This study indicates the presence of Acinetobacter baumannii as an upcoming pathogen for companion animals in Greece.To our knowledge, this is the first report of clinical cases in companion animals caused by Abc infections in the country.
At the same time, due to some specific properties this species possesses, such as its ability to acquire resistance against a great number of antibiotics, its tolerance in variable environments and conditions, and its capability of adherence and biofilm formation in biotic and abiotic surfaces, an important public health issue arises.Further, the XDR phenotype of a significant number of stains creates concerns about the distribution of respective bacteria through companion animals and between them and their environment.
Samples were mainly obtained from soft tissue and urinary tract infections (UTIs).This is in accordance with several recent studies of Acinetobacter spp.from companion animals [9,12,[18][19][20][21][22].Moreover, most isolates (29/41) were related to polymicrobial infections.Commonly co-current bacterial species included MDR Enterobacteriaceae and methicillinresistant Staphylococci.Further, Abc bacteria have been regularly isolated from humans polymicrobial infections [23,24].In a recent review, Pseudomonas aeruginosa was the species most commonly associated with respective cases, followed by S. aureus, K. pneumoniae, and other Enterobacterales.Thus, a possible beneficial interaction between these pathogens was suggested [24].Our study provides evidence of polymicrobial Abc infections in dogs and cats.This could be troublesome for veterinarians concerning the virulence of the combined pathogens, the available therapeutic options and possible treatment complications.
In reference to the resistance profiles of the isolates, the rate against fluoroquinolones in this study was 100%.This is in accordance with recent relevant studies [5], indicating the wide distribution of the associated mechanisms among Abc complex populations.
Moreover, a high resistance rate was detected for doxycycline, while minocycline was the most effective agent tested.Doxycycline is widely used in companion animals, and therefore, the prevalence of resistant isolates under the pressure of regular administration is anticipated.Furthermore, minocycline is able to overcome several tetracycline-resistance mechanisms [5,25].
Regarding aminoglycosides, extremely high rates were documented for gentamicin, in contrast with tobramycin and amikacin, which were relatively more effective.This fact could be explained by the wide usage of gentamicin in veterinary medicine [26].Further, these results are in accordance with previous studies [12,18].
Sulfamethoxazole-trimethoprim is a treatment option for Acinetobacter spp.infections, suitable for veterinary medicine.However, approximately two-thirds of the isolates in this study (26/41) were resistant.Comparable rates have been detected in several veterinary studies, especially in CR isolates [12,18,20,21].
Resistance to carbapenems is definitely of major importance.There are variable previous references of carbapenemase encoding ARGs in Acinetobacter spp.obtained from canine and feline specimens [12,18,20,22,[27][28][29][30][31].Most of them are associated with the bla OXA-23 gene, while other respective ARGs are only sporadically detected in pets (bla OXA-58 , bla NDM-1 , bla IMP-1 ) [9].The prevalence of carbapenem-resistant phenotypes is significant in this study, creating concerns about the distribution of respective strains in the community.Further, Greece exhibits exceptionally high rates of carbapenem-resistant Acinetobacter baumannii (CRAB) over the last years, especially in hospital-acquired strains [32].In contrast Abc strains producing class B carbapenemases (bla NDM ) have also been detected in the country [33].Furthermore, most of the imipenem-resistant isolates are XDR (19/20).This fact indicates that carbapenemase-encoding genes are regularly co-current with ARGs against antibiotics of different classes, such as aminoglycosides, tetracyclines, and folate pathway inhibitors.Relevant results have been provided by variable studies [9].In Abc isolates of human origin, extensive research has been accomplished, revealing the ability of this bacterium to accumulate resistance determinants through the horizontal transfer of mobile genetic elements [4,34].However, complete interpretation in our case should be carried out only by molecular investigation of these isolates' resistome.
Dealing with infections from Acinetobacter spp. in pets is undoubtedly a challenge.Current research data are limited; thus therapeutic strategies are based mostly on human studies [9].Furthermore, evaluation of the AST is based on human clinical breakpoints (Table 1) since no specific breakpoints exist for veterinary medicine [35].Additionally, as it is clearly indicated in this study, MDR and XDR strains usually demonstrate resistance against most of the agents available for usage in animals (aminoglycosides, carbapenems, β-lactams, tetracyclines, and folate pathway inhibitors) [9].Concerning that the only effective agents against these strains are rather critically important for human medicine (as a last resort treating options, like colistin, polymyxin B, and tigecycline) and therefore disapproved for animals [36], a Gordian knot arises for veterinarians.
Minocycline was the most effective agent, exhibiting in vitro activity, even against XDR strains.It is also a suitable alternative for methicillin-resistant Staphylococci [37], which in some cases co-existed with the A. baumannii isolates (polymicrobial infections).However, there are limitations in its usage regarding the site of infection and the presence of more bacterial species, especially Gram-negative.
In the future, assessing the potentialities of novel treatment approaches is inevitable.Evaluation of possible synergistic effects of currently available antibiotics or alternative, non-antibiotic treatments (bacteriophages, antimicrobial peptides, vaccines or nanoparticles) [38], could constitute available options for veterinarians.
Limited data are available about to the association of Abc isolates of animal origin and hospital-acquired infections.In this study, isolates were persistently obtained from samples originating from veterinary clinics for over 2.5 years (Table S1).This persistence creates concerns regarding their ability to survive and spread inside veterinary facilities.Further, as it was formerly noted, Acinetobacter spp. is able to survive for long periods on both biotic and abiotic surfaces, and thus, its eradication from the hospital environment is often extremely challenging [39].
About companion animals, preceding hospitalization has been associated with infections in several studies [11,[19][20][21]40,41].Distribution of highly resistant clones among veterinary clinics has also been detected [13].Moreover, the presence of contaminated medical equipment in cases of outbreaks indicates the danger of infection during hospitalization [11,41].Therefore, it is suggested that proper surveillance and preventive measures should be urgently established after the isolation of MDR or XDR Abc strains from an animal clinical sample (Figure S6) [11,[42][43][44].
In reference to future research perspectives, an extensive epidemiological study would be essential to identify possible predisposing factors that enhance the involvement of highly resistant Abc strains in animal infections.Moreover, adopting regular and rigorous environmental sampling in veterinary facilities could provide useful information regarding the possible dissemination and persistence of «endemic» bacteria and the effectiveness of eradication measures.Finally, molecular identification and investigation of Abc isolates could provide sufficient data about their clonality and properties of clinical interest, such as adherence and biofilm production ability, acquired ARGs and virulence factors.

Conclusions
Bacteria of the Abc complex, and more specifically A. baumannii strains, are possible emerging pathogens for companion animals in Greece since they have been regularly isolated from infection sites in recent years.Among them, several strains exhibit MDR and XDR phenotypes, and the subsequent lack of treatment options constitutes a headache for veterinarians.The prevalence of relevant strains in pets demonstrates their wide distribution in the community and illustrates the danger of further dissemination among animals, humans, and veterinary facilities.Proper prevention and surveillance measures should be established, and further research should be accomplished to comprehend the phenomenon and restrain its advance sufficiently.

Table 1 .
Antibacterial agents, disc content and breakpoints used in this study.

Table 2 .
Site of infection and origin of the samples included in this study.

Table 3 .
Characteristics of the samples, the infected animals and the co-current bacteria.

Table 4 .
Resistance rates of the Abc bacteria included in this study by the disc diffusion method.

Table 5 .
Resistance profiles of the isolates.

Table 6 .
Resistant rates in carbapenem-resistant and non-resistant bacteria.