Multidrug-Resistant Acinetobacter baumannii in Veterinary Clinics, Germany

An increase in prevalence of multidrug-resistant Acinetobacter spp. in hospitalized animals was observed at the Justus-Liebig-University (Germany). Genotypic analysis of 56 isolates during 2000–2008 showed 3 clusters that corresponded to European clones I–III. Results indicate spread of genotypically related strains within and among veterinary clinics in Germany.


The Study
The Institute for Hygiene and Infectious Diseases of Animals in Giessen receives samples for investigation from other veterinary departments of the university (mainly referral clinics) and from external veterinary clinics throughout Germany.During 2000-2008, Acinetobacter spp.were obtained from 137 hospitalized animals.From these animals, 56 isolates were selected for further characterization.The selection was made to refl ect the diversity in epidemiologic origin of the collection regarding date of isolation, animal species, specimen, and veterinary clinic (82% from Giessen) (online Appendix Table, www.cdc.gov/EID/17/9/101931-appT.htm).Only isolates with possible clinical signifi cance were included as inferred from the fact that they were the only or the dominating agent within the sample.Furthermore, according to data from the diagnostic laboratory, the selected isolates were highly resistant.
Confi rmatory susceptibility testing of isolates was conducted by using the Clinical Laboratory Standards Institute broth dilution method (9) (Table ).For precise species identifi cation, amplifi ed ribosomal DNA restriction analysis was performed.By this method, the 16S rDNA sequence was amplifi ed by using PCR, followed by restriction of the amplifi ed fragment by 5 restriction enzymes: CfoI, AluI, MboI, RsaI, and MspI.The combination of electrophoretic patterns of the respective enzymes was compared with a library of profi les (10).
Fifty-two isolates were identifi ed as belonging to A. baumannii and 3 to A. pittii (Acinetobacter gen.sp. 3) (11); 1 with a yet undescribed profi le remained unclassifi ed.Amplifi ed fragment length polymorphism (AFLP) DNA fi ngerprint analysis was performed as described for confi rmative species identifi cation, for strain typing, and for clone identifi cation (4,12,13).Briefl y, EcoRI and MseI were used to generate restriction fragments that were selectively amplifi ed by using a Cy-5-labeled Eco-A and an Mse-C primer.Amplifi cation products were separated by electrophoresis and subjected to cluster analysis with the BioNumerics software package 5.1 (Applied Maths, St-Martens-Latem, Belgium).For species identifi cation, isolates were compared with reference strains of all described Acinetobacter species included in the Leiden University Medical Center AFLP database (Leiden, the Netherlands).Isolates with profi les >50% similar were considered to belong to the same species (1).
To assess the type diversity of the organisms, isolates were typed by pulsed-fi eld gel electrophoresis (PFGE) (14) and by AFLP analysis.For PFGE, DNA was digested with the restriction endonuclease ApaI.Digitized profi les were analyzed with the BioNumerics software.For AFLP typing, a subset of 27 isolates was analyzed (online Appendix Table ).The profi les obtained were compared with each other and with those of the Leiden database, including those of the European clones I-III.A similarity cutoff level >80% was used to delineate members of the same clone and >90% to delineate organisms related at the strain level (4,12,13).
For PFGE, at a similarity level of 86%, 3 major clusters (A, B, and C) and 6 unique isolates were distinguished (Figure 1).Within major cluster C, 2 main subclusters (C1 and C6) and 4 single profi les (C2-C5) were observed at 97% similarity (online Appendix Table ; Figure 1).Despite some band differences, the patterns in major cluster C were strikingly similar.The maximum number of band differences in subcluster C1 was 3, which indicates that the organisms were genetically closely related.In subcluster C6, only minor differences in size of the fragments were observed (Figure 1).
For AFLP, we investigated a subset of 27 isolates, including at least 1 isolate of each of the 16 different PFGE profi les and the 3 isolates nontypeable by PFGE.Seventeen AFLP types were distinguished at the 90% similarity cutoff level for strain delineation.Identifi cation by AFLP showed full agreement with amplifi ed ribosomal DNA restriction analysis species identifi cation (online Appendix Table ).Comparison of isolates to those of the Leiden AFLP database grouped isolates with AFLP profi le 8 (corresponding PFGE profi les A1, A2) with isolates of European clone I, those with profi les 10-16 (corresponding PFGE profi le C1-C6) with clone II, and with profi le 7 (corresponding PFGE profi les B1, B2) with clone III (online Appendix Table ).Examples are shown in Figure 2.

Conclusions
The occurrence of PFGE type C in different animals admitted to 3 different clinical wards of the Justus-Liebig-University Giessen over 9 years might indicate endemic occurrence of these organisms on these wards.Survival in the hospital environment (15), patient-to-patient transfer, and transfer from 1 animal clinic to another may have contributed to their persistence and spread.Because veterinarians, stockmen, and students rotate between the various clinics and departments, transmission by hands or equipment should be considered.Frequent transport of colonized animals to and from shared examination rooms, e.g., for computer-assisted tomography, might also have contributed to the chain of spread.Because type C isolates also were found in samples from animal clinics throughout Germany (online Appendix Table ), limited genetic variation in animal strains of A. baumannii also is possible.
AFLP data were, further to comparative typing of the animal isolates, also used to assess the relatedness of the  isolates in our study to those of the widespread European clones I-III that represent genetically related but not identical strains that are frequently multidrug resistant and associated with epidemic spread in human clinics (1,(4)(5)(6).
Although not all strains were characterized by AFLP, we conclude by inductive generalization of results that the fi ndings apply to all isolates of the PFGE types from which the organisms were selected.Thus, a large proportion of the animal A. baumannii isolates were genetically congruent with the European clone I, II, or III.Occurrence of such isolates in ill, hospitalized animals of various species might indicate that, as in human medicine, A. baumannii is an emerging opportunistic pathogen in veterinary medicine.The occurrence of clones I-III in animals and humans also raises concern about whether the organisms can spread from animals to humans or whether the animals have acquired the organisms from humans.The occurrence of genotypically related, antimicrobial drug-resistant A. baumannii strains in hospitalized animals suggests that these organisms are most likely nosocomial  pathogens for animals.If so, veterinary clinics face a great challenge regarding prevention, control, and treatment of infections with these organisms, similar to situations in human hospitals.Finally, the possibility of spread from humans to animals or vice versa requires special attention.

Figure 1 .
Figure 1.Computer-assisted cluster analysis of pulsed-fi eld gel electrophoresis fi ngerprints of 53 Acinetobacter baumannii and 2 Acinetobacter spp.pittii isolates.COL 20820 was used as the reference standard for normalization of the digitized gels (14).

Figure 2 .
Figure 2. Amplifi ed fragment length polymorphism analysis of 9 animal Acinetobacter baumannii isolates belonging the major pulsed-fi eld gel electrophoresis types and 9 reference strains of the European clones I-III from the Leiden University Medical Center collection.*Reference strains of European clone I; †reference strains of European clone III; ‡reference strains of European clone II.

Table .
Resistance profiles of 56 animal Acinetobacter spp.isolates for 19 antimicrobial agents, obtained by CLSI broth microdilution test *