Predominance of Acinetobacter pseudolwoffii among Acinetobacter species in domestic animals in the Czech Republic

The aim of this study was to map the spectrum of microorganisms belonging to the genus Acinetobacter in domestic animals with a specific focus on the prevalence of Acinetobacter pseudolwoffii. Additionally, the susceptibility of isolates to antimicrobial agents was determined. In the period from January 1, 2014, to August 31, 2015, a total of 9 544 samples originating from gross lesions and pathological processes of animals exhibiting clinical symptoms of the disease were examined across 41 districts in the Czech Republic. The examinations were carried out using culture methods involving meat-peptone blood agar and Endo agar under aerobic conditions at a temperature of 37 ± 1 °C for 18–24 hours. Isolates were confirmed using molecular phenotypic method MALDI–TOF MS with the MBT Compass Library Revision L 2020 covering 3 239 species/entries (9 607 MSP) from Bruker Daltonics company. Out of the 108 isolates (prevalence 1.13%), 14 species of Acinetobacter spp. were identified, with 5 isolates remaining unclassified as species. A. pseudolwoffii was the predominant species isolated in 25 cases (prevalence 0.26%). Antimicrobial susceptibility was determined for 12 antimicrobials by the disc diffusion method, with A. pseudolwoffii isolates exhibiting the lowest susceptibility to ceftazidime (32%) and co-trimoxazole (60%).

Acinetobacter spp.comprising 76 species (Euzeby 2023) are ubiquitous microorganisms found in soil, water, and clinical environments.In humans and animals, they are primarily commensals colonis-ing the skin and gut of patients and hospital staff, and contaminating hospital equipment (Bergogne-Berezin et al. 2008).Some species can cause local (e.g.wound infections) and systemic diseases https://doi.org/10.17221/65/2023-VETMED(Nemec 1999;Almasaudi 2018).In human medicine, the Acinetobacter calcoaceticus/baumannii complex (ACB complex) is of major epidemiological importance (Nemec et al. 2015).Less common species, such as Acinetobacter parvus (Nemec et al. 2003), Acinetobacter guillouiae (Nemec et al. 2010), and Acinetobacter modestus (Nemec et al. 2016) have been isolated from various human cultures.Acinetobacter spp.are commonly isolated from animals, including birds and fish (Almasaudi 2018) and can lead to various diseases, sometimes with fatal consequences (Francey et al. 2000).In the Czech Republic Acinetobacter pittii, Acinetobacter calcoaceticus, Acinetobacter towneri, Acinetobacter johnsonii, Acinetobacter lwoffii and other unspecified species of the genus Acinetobacter have been isolated from pathological processes and gross lesions in horses with a prevalence ranging from 0.2% to 2.2% (Bzdil et al. 2018).The isolation of Acinetobacter pseudolwoffii strains was first described by Nemec et al. (2019) in ruminants, horses, guinea pigs, humans, and environmental samples.The increase in resistance in Acinetobacter spp.and the emergence of multi-drug resistant strains in human medicine is worrying (Kurcik-Trajkovska 2009).Multi-drug resistance was also recorded in animals (Jokisalo et al. 2010).A detailed description of the susceptibility of Acinetobacter spp.isolates obtained from gross lesions and processes in horses in the Czech Republic is provided by Bzdil et al. (2018).In dogs and cats, strains of A. baumannii resistant even to carbapenems were found (Gentilini et al. 2018).The objective of this study was to map the spectrum of species in the genus Acinetobacter in a wide range of samples collected from animals with clinical signs of disease and to describe the susceptibility of the isolates to antimicrobial agents.

MATERIAL AND METHODS
Over the 20-month period from January 1, 2014, to August 31, 2015, a total of 9 544 clinical samples were collected from pathological processes and lesions in animals displaying disease symptoms originating from farms in 41 districts of the Czech Republic.Veterinarians at 13 clinics and veterinary hospitals and 18 private veterinarians were instructed to collect the samples from a wide variety of animals, including dogs, cats, cattle, sheep, goats, pigs, equids, i.e. horses and donkeys, waterfowl, fowl, exotic birds, rabbits, guinea pigs, mice, rats (both kept as pets), hamsters, snakes, turtles, tortoises, lizards and even bees (bee brood).However, no clinical samples from fish were included in this examination.
The collected samples included swabs or irrigations from eyes and ears, swabs and scrapings from the skin, swabs from the respiratory system, sputum and bronchioalveolar lavages, swabs from the digestive system, faeces, urine, swabs from the mucous membranes of the urogenital system, mammary gland secretions, milk, blood, swabs from the heart and blood vessels, as well as swabs and punctures from the musculoskeletal, lymphatic and nervous systems.Table 1 presents a summary of the quantities and types of clinical materials collected.Liquid and slurry materials were collected in sterile plastic containers with a volume of 60-200 ml, or in sterile plastic tubes with a 10 ml capacity and a screw cap (Dispolab Ltd., Brno, Czech Republic).Swabs were taken using the Transbak swab system containing Amies agar with activated carbon (Dispolab Ltd., Brno, Czech Republic).After collection, the samples were kept at a temperature of +4 °C to +6 °C and transported to the laboratory within 24 h, where they were immediately processed.A culture examination was performed on meat-peptone blood agar (MPBA) and Endo agar (EA), both from Trios s.r.o.(Prague, Czech Republic).The inoculated plates were incubated aerobically at a temperature of 37 ± 1 °C for 18-24 hours.Suspect strains were isolated and confirmed by MALDI-TOF MS using a Microflex LT System spectrometer (Bruker Daltonics GmbH, Bremen, Germany) and evaluated with the MBT Compass Library Revision L 2020 covering 3 239 species/entries (9 607 MSP) (Bruker Daltonics GmbH, Bremen, Germany).Identification scores (ID) within the range of 2.300 to 3.000 were evaluated as highly probable for species identification, 2.000 to 2.299 as secure genus identification and probable species identification, 1.700 to 1.999 as probable genus identification, and values ≤ 1.699 as unreliable identification.The identification of A. baumannii was confirmed by the multiplex PCR method through the detection of bla OXA-51 and bla OXA-51-like genes encoding natural carbapenemases, which are specific to this species (Turton et al. 2006).Pure cultures were tested for susceptibility to antimicrobial substances using the disc diffusion method on Mueller-Hinton https://doi.org/10.17221/65/2023-VETMEDagar (MH) (Trios s.r.o., Prague, Czech Republic) and discs (Oxoid Ltd., Basingstoke, UK).Of the antimicrobial substances, imipenem (10 µg), meropenem (10 µg), tobramycin (10 µg), netilmicin (30 µg), ofloxacin (5 µg), amikacin (30 µg), doxycycline (30 µg), ampicillin/sulbactam (20 µg), gentamicin (10 µg), ceftazidime (30 µg), co-trimoxazole (25 µg) and piperacillin (100 µg) were tested.Tests were assessed after 16-18 h of incubation at 35 ± 1 °C.Interpretation of values was performed according to CLSI (2020) standards.Reference values for Pseudomonas aeruginosa were used to assess susceptibility to netilmicin and ofloxacin.The quality of the media and discs was validated by reference strains of Escherichia coli (ATCC 25922), P. aeruginosa (ATCC27853), and Staphylococcus aureus (ATCC 25923).

RESULTS AND DISCUSSION
Out of 9 544 clinical samples, 108 Acinetobacter isolates were obtained during the observed period (prevalence 1.13%).A total of 14 Acinetobacter species were identified: Acinetobacter baumannii, Acinetobacter calcoaceticus, Acinetobacter gandensis, Acinetobacter guillouiae, Acinetobacter indicus, Acinetobacter johnsonii, Acinetobacter lwoffii, Acinetobacter modestus, Acinetobacter parvus, Acinetobacter pittii, Acinetobacter pseudol-woffii, Acinetobacter radioresistens, Acinetobacter schindleri, and Acinetobacter ursingii.Five isolates were not classified as species.The species A. gandensis and A. modestus have not yet been reported in animals or in products of animal origin in the literature.In our study, no Acinetobacter spp.isolates were found in sick pigs during the observed period.This could be attributed to the smaller number of samples collected from these animals.In addition, samples from the respiratory system mainly originated from the lungs, where the probability of finding Acinetobacter spp.tends to be lower.Samples from the digestive tract were frequently overgrown with saprophytic microflora making the isolation of individual Acinetobacter spp.problematic.
In terms of organs and organ systems, Acinetobacter spp.isolates were not found in the samples from the musculoskeletal, lymphatic, and nervous systems of the animals.Of the species we observed, A. pseudolwoffii was predominant (n = 25) with a prevalence of 0.26%.The prevalence of this species cannot be compared with other data because no similar studies exist currently.The second and third positions were held by the species A. lwoffii (n = 21) and A. pittii (n = 19) with prevalences of 0.22 and 0.2%, respectively.Despite the relatively high number of detected A. lwoffii and A. pittii isolates in our study, their prevalence is comparatively lower or similar to that reported in other studies https://doi.org/10.17221/65/2023-VETMED(Bzdil et al. 2018; Samkange et al. 2022).The finding of A. pseudolwoffii isolates was first described by Nemec et al. (2019) in nasal swabs from a cow, a calf, a goat, and a horse, in a rectal swab from a guinea pig, and in the faeces of a sheep.In our study, A. pseudolwoffii was also most often detected in domestic ruminants, such as cattle, sheep and goats (n = 17, prevalence 0.27%), in solipeds such as horses (n = 4, prevalence 2.58%), domestic rodents, such as rabbits and guinea pigs (n = 2, prevalence 1.18%) and also in domestic carnivores such as dogs and cats (n = 2, prevalence 0.09%).
Table 2 shows detailed information and a comparison with the occurrence of A. pseudolwoffii and other Acinetobacter species in domestic animals.
Literature sources so far mention the isolation  (Francey et al. 2000;Kuzi et al. 2016;Kimura et al. 2017), horses (Jokisalo et al. 2010;Bzdil et al. 2018) and other animals (Almasaudi 2018).In terms of organs and organ systems, A. pseudolwoffii was most often isolated from the respiratory tract (n = 18, prevalence 3.31%), from the diges-tive tract (n = 4, prevalence 0.28%), from the eye (n = 1, prevalence 0.43%) and from the ear and skin (both n = 1, prevalence 0.17%).Details of the numbers of Acinetobacter spp.isolates and their prevalence in relation to organs and organ systems are provided in Table 3.The specific clinical diagnoses in animals with findings of A. pseudolwoffii are shown in Table 4. Infections of the respiratory https://doi.org/10.17221/65/2023-VETMEDtract and eye caused by Acinetobacter spp., both in animals and in humans, are confirmed by literature sources (Francey et al. 2000;Jokisalo et al. 2010;Almasaudi 2018).For example, Almasaudi (2018) mentions wound infections in human patients caused by Acinetobacter spp. in his study.Differences in prevalence among individual species and groups of animals as well as among individual organs and organ systems were found in the present study.The varying frequency of findings of individual Acinetobacter species could indicate different degrees of affinity to particular animal groups and species, as well as to different organs and organ systems.The variation could be attributed to the specific biochemistry and microclimatic conditions within each organ as well as the diverse geographic, climatic, dietary, and social factors unique to each animal, including humans.
Further studies in different countries and regions worldwide are needed to confirm or refute this assumption.Antimicrobial susceptibility tests were performed on all 25 of our A. pseudolwoffii isolates and 83 other detected Acinetobacter spp.isolates.A. pseudolwoffii was susceptible in all cases to imipenem, meropenem, tobramycin, amikacin and netilmicin.For ceftazidime, only 32% of the isolates demonstrated susceptibility, while 60% were susceptible to co-trimoxazole, 80% to piperacillin,

Table 1 .
Number of veterinary clinical samples examined in the period from January 1, 2014 to August 31, 2015

Table 2 .
Number of Acinetobacter spp.isolates from individual animal groups and their prevalence (%) in the period from January 1, 2014 to August 31, 2015

Table 3 .
Number of Acinetobacter spp.isolates from individual organs and organ systems and their prevalence (%) in the period from January 1, 2014 to August 31, 2015

Table 4 .
Findings of Acinetobacter pseudolwoffii in individual groups, species and categories of animals, diagnoses and occurrence of multi-drug resistant strains ADC gene is responsible for resistance to ceftazidime, which is described in isolates of Acinetobacter spp.obtained from humans.This gene has been

Table 5 .
Susceptibility of Acinetobacter spp.isolates from animals to antimicrobials -susceptible/examined isolates (% susceptible) in the period from January 1, 2014 to August 31, 2015