Imported Pet Reptiles and Their “Blind Passengers”—In-Depth Characterization of 80 Acinetobacter Species Isolates

Abstract

Reptiles are popular pet animals and important food sources, but the trade of this vertebrate class is—besides welfare and conservation—under debate due to zoonotic microbiota. Ninety-two shipments of live reptiles were sampled during border inspections at Europe’s most relevant transshipment point for the live animal trade. Acinetobacter spp. represented one significant fraction of potentially MDR bacteria that were further analyzed following non-selective isolation or selective enrichment from feces, urinate, or skin samples. Taxonomic positions of respective isolates were confirmed by MALDI-TOF MS and whole-genome sequencing analysis (GBDP, dDDH, ANIb, and rMLST). The majority of the 80 isolates represented established species; however, a proportion of potentially novel taxa was found. Antimicrobial properties and genome-resistance gene screening revealed novel and existing resistance mechanisms. Acinetobacter spp. strains were most often resistant to 6–10 substance groups (n = 63) in vitro. Resistance to fluorchinolones (n = 4) and colistin (n = 7), but not to carbapenems, was noted, and novel oxacillinase variants (n = 39) were detected among other genes. Phylogenetic analysis (MLST) assigned few isolates to the known STs (25, 46, 49, 220, and 249) and to a number of novel STs. No correlation was found to indicate that MDR Acinetobacter spp. in reptiles were associated with harvesting mode, e.g., captive-bred, wild-caught, or farmed in natural ecosystems. The community of Acinetobacter spp. in healthy reptiles turned out to be highly variable, with many isolates displaying a MDR phenotype or genotype.

1. Introduction

The genus Acinetobacter is highly diverse [1] and comprises, at the time of writing, approximately 73 validly published species (https://lpsn.dsmz.de/genus/acinetobacter (accessed on 8 February 2022)). Members of this genus are Gram-stain negative cocci or cocco-bacilli that are non-motile, non-spore-forming, strictly aerobic, cytochrome-oxidase negative, and catalase-positive bacteria [2]. Despite one of the genus’ major characteristics of being non-fermentative bacteria, members have been found to survive under different environmental conditions without oxygen for prolonged periods of several weeks. This is also true for A. baumannii [3], one of the most important nosocomial microorganisms that forms a species complex with the emerging opportunistic pathogens A. calcoaceticus, A. pittii, A. nosocomialis, and some other species [4] that are responsible for healthcare-associated outbreaks and severe infections, particularly in critically ill patients with impaired immunity [5,6]. Acinetobacter spp. are notorious for the accumulation of antibiotic resistance genes, which represents—together with other multiple drug resistant Gram-negative (MRGN) bacteria—an ever-increasing problem for global public health [6]. Furthermore, A. baumannii is referred to as an “ESKAPE pathogen”, an acronym that further includes Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Enterobacter spp., as potentially life-threatening, multiple-drug resistant (MDR), and virulent nosocomial pathogens [7]. A high number of Acinetobacter spp. has originally been described from human clinical specimens [8]. However, other sources from the environment include activated sludge [9], wetlands [10], forest soil [11], seawater [12], dumpsites [13], manure and anaerobic digestates of biogas plants [3], wastewater [14], and freshwater [15].

From a veterinary perspective, Acinetobacter spp. are also frequently isolated from companion [16,17,18], zoo [19], wildlife animals [20,21] and livestock [22,23,24,25,26], and their role as nosocomial MRGNs has also been evaluated as critically important in animal clinics and patients suffering from, e.g., skin, wound, and systemic infections [27]. Direct zoonotic transmission chains seem to be rare, but companion animals represent a potential source for spillover in this regard [16,28,29,30]. Furthermore, Acinetobacter spp., particularly those colonizing livestock, were discussed as being adapted to certain animal reservoirs or as representing a potential source for food contamination, and can thus contribute to the colonization among humans and the contamination of their surroundings in the context of One Health issues [22,23,31,32,33,34].

Eventually, several studies also found Acinetobacter spp. in free-ranging, as well as in ex-situ-bred, members of the vertebrate class of reptiles [35,36], which might have implications with respect to their roles as important food sources in some countries [37] and as popular pet animals [38,39]. Possible spread events of MDR between reptiles and humans seem to be, at the least, probable since identical clones of ESBL-producing Enterobacteriaceae have recently been found in geckos living in close proximity to patients in a hospital in Ghana [40]. Our group has previously published preliminary findings on mcr-1 positive Escherichia coli isolated from imported reptiles during border inspections [41]. We hypothesized that intemperance and amount of antimicrobial use in third countries might have an influence also on MDR microbiota in reptiles, especially when they are farmed in or near natural ecosystems. We here provide results on MDR of the fraction of Acinetobacter spp. that were isolated in a cross-sectional study evaluating 92 shipments of live reptiles, representing 160 batches of single species. Importations and samplings were carried out at the Frankfurt Airport, Germany, which represents the most relevant transshipment point for the trade of live animals worldwide [42].

2. Materials and Methods

2.1. Sample Collection

Between July 2013 and May 2014, samples from imported reptiles were drawn at the Frankfurt Airport, Germany. The sampling was carried out in cooperation with authorized veterinarians from the Border Control Post of the Frankfurt Airport during import control at the Frankfurt Animal Lounge directly after the arrival of the animals.

The investigated reptiles were assigned to the orders Squamata (lizards and snakes) and Testudines (turtles). The different shipments contained either one or several species that were transported in separate boxes, according to the guidelines of the International Air Transport Association (IATA). A total of 92 shipments were included, and each animal species per shipment was considered as one sample batch (n = 160), from which one or more samples were obtained (n = 183). Specimens collected were remnants of ecdysis (shed skin), feces (90% of the specimens), urinate (separate or mixed; n = 164) or swabs from the skin surface. The 92 shipments came from 23 different countries in Africa (Egypt, Madagascar, Mozambique, Uganda, South Africa, and the United Republic of Tanzania); North America (Canada and USA); Central and South America (Brazil, Ecuador, El Salvador, Guatemala, Guyana, Colombia, Nicaragua, and Panama); Asia (China, Japan, Turkey, Uzbekistan, and Vietnam), and Europe (Macedonia and Ukraine). The majority of shipments came from the USA (38.0%), Vietnam (10.9%), Uzbekistan (9.8%), Ukraine, and Canada (5.4% each).

The reptiles from this study could be assigned to different natural sources (Supplementary Table S1). A major proportion represented obviously captive-bred species (CB (33.8%); e.g., Pogona vitticeps from the USA). In a few cases, a clear assignment remained uncertain because captive breeding seemed principally possible, but not economical in large quantities and in the light of nearby, wild, autochthonous or allochthonous (i.e., neozoon) populations (presumably CB (3.2%); e.g., declared CB Sceloporus malachiticus from the USA that naturally occur in nearby Central America). Based on our experience, we assigned the remaining species as wild-caught (WC (28.3%); e.g., Physignathus cocincinus from Vietnam), farm-bred (FB (19.8%)); also including ranched species (reared in a controlled environment, taken as gravid females, eggs, or juveniles from the wild) that according to the CITES glossary [43] would “otherwise have had a low probability of surviving to adulthood”, and releasing a proportion back into the wild [26]. A minority of samples were obtained from species that could not unequivocally be assigned to one of the former groups and represented wild-caught or farm-bred/captive-bred (WC/FB (14.0%) or WC/CB (1.1%)) reptiles.

2.2. Bacterial Isolates and DNA Extraction

All samples were stored in sterile, fecal sample tubes at 4–7 °C until they were transported to the laboratory. Within no more than 48 h after sampling, fecal and urinate samples were directly streaked on blood agar (Merck, Darmstadt, Germany), supplemented with 5% sheep blood (SBA), water-blue-metachrome-yellow-lactose agar (Gassner), (Oxoid, Wesel, Germany), and on MacConkey agar (Oxoid) containing 1 mg/L cefotaxime (Sigma-Aldrich/Merck). Skin samples were re-suspended in 0.9% NaCl prior to cultivation, and 100 µL aliquots were streaked onto the same media. The plates were cultured for 24 h at 37 °C. In addition, all samples were cultivated in 3 mL, standard I nutrient broth (Roth GmbH + Co. KG, Karlsruhe, Germany), supplemented with a 10 µg meropenem disc (Mast Group Ltd., Reinfeld, Germany) for 24 h at 37 °C. In case of visible growth (at least slight turbidity of the broth), 50 µL of the solution was streaked onto SBA and Gassner agar and incubated for 24 h at 37 °C. Each sample was additionally inoculated into 5 mL of nutrient broth supplemented with bovine serum (cultivated at 37 °C for 24 h) as a kind of enrichment culture. Morphologically different colonies were again subcultured on SBA and Gassner agar, and presumptive Gram-negative bacteria, including putative Acinetobacter spp., were stored at −80 °C in a liquid nutrient broth containing 20% glycerol before they were used for further analysis. DNA was extracted with a “Master Pure™ DNA Purification Kit” (Biozym Scientific GmbH, Hessisch Oldendorf, Germany).

2.3. Matrix-Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS)

To identify Acinetobacter spp. among the presumptive Gram-negative isolates MALDI-TOF MS was used with isolates grown on SBA for 24 h. The workflow was performed as recently described [44]. Biomass was transferred to steel targets using the direct transfer protocol according to the manufacturer’s instructions (MALDI Biotyper; Bruker Daltonik, Bremen, Germany). Analysis was performed on a microflex LT mass spectrometer (Biotyper version 3.3.1.0 with MBT compass software). The database used (DB 9045 plus user-derived spectra of A. pseudolwoffii and A. stercoris-type strains) comprised 40 species and 154 strains of Acinetobacter spp. in total. The MALDI Biotyper real-time classification (RTC) software calculates log scores based on similarities between the observed results and stored database entries. Correct species-level identifications were assumed when the first- and second-best matches indicated the same species with log scores of >2.0, or when the best match indicated a species with a log score of >2.0, and the second-best match indicated a different species with a log score of <2.0 and consistency category A. The identification was done in duplicate to verify the original findings. Isolates identified as at least Acinetobacter spp. were stored at −80 °C for further investigations.

2.4. Antimicrobial Susceptibility Testing

All 80 non-duplicate Acinetobacter spp. isolates were screened for reduced susceptibility to different antimicrobial substances. Briefly, antimicrobial susceptibility testing was carried out using the broth microdilution susceptibility testing method. A commercially available panel layout for livestock (Micronaut/Bruker according to the guidelines of the working group for antimicrobial resistance of the German Veterinary Society, (DVG) was used. In this layout, 16 different antimicrobial substances (in µg/mL: amoxicillin/clavulanic acid (2/1–16/8), ampicillin (0.25–16), ceftiofur (0.125–4), cephalothin (1–16), colistin (0.5–2), enrofloxacin (0.016–1), erythromycin (0.125–4), florfenicol (1–8), gentamicin (0.125–8), penicillin G (0.063–8), spectinomycin (4–64), tetracycline (0.25–8), tiamulin (0.25–32), tilmicosin (0.5–16), trimethoprim/sulfamethoxazole (0.25/4.75–1/19), and tulathromycin (1–64))) were employed. Results were interpreted for human Acinetobacter species according to European Committee on Antimicrobial Susceptibility Testing (EUCAST, version 11.0) for broth dilution susceptibility testing and when clinical breakpoints were available.

2.5. Whole-Genome Sequencing Analysis and Bacterial Species Confirmation

All isolates were subjected to whole-genome sequencing. Sequencing libraries were prepared using the Nextera XT Library Preparation Kit (Illumina GmbH, Munich, Germany) for a 250 bp paired-end sequencing run on an Illumina MiSeq sequencer (Illumina Inc., San Diego, CA, USA) with a minimum coverage of 100-fold. FASTQ files were quality trimmed before they were assembled de novo and annotated using SPAdes v.3.15.1 (http://cab.spbu.ru/software/spades/ (accessed on 10 September 2021)) and RAST v.2.0 (http://rast.nmpdr.org/ (accessed on 10 September 2021)).

Draft genome sequence data of 80 Acinetobacter spp. were uploaded to the Type (Strain) Genome Server (TYGS), a free bioinformatics platform available under https://tygs.dsmz.de (accessed on 8 February 2022), for a whole-genome-based taxonomic analysis [45]. The analysis also made use of recently introduced methodological updates and features [46]. Information on nomenclature, synonymy, and associated taxonomic literature was provided by the TYGS’s sister database, the List of Prokaryotic names with Standing in Nomenclature (LPSN, available at https://lpsn.dsmz.de (accessed on 8 February 2022)) [46]. The results were provided by the TYGS on 9 February 2022. The TYGS analysis was subdivided into the following steps:

Determination of the closest type strain genomes was done in two complementary ways: First, all user genomes were compared against all type strain genomes available in the TYGS database via the MASH algorithm, a fast approximation of intergenomic relatedness [47], and the 10 type strains with the smallest MASH distances were chosen per user genome. Second, an additional set of 10, closely related type strains was determined via the 16S rRNA gene sequences. These were extracted from the user genomes using RNAmmer [48], and each sequence was subsequently BLASTed [49] against the 16S rRNA gene sequence of each of the 15,873 type strains available in the TYGS database (as of 9 February 2022. This was used as a proxy to find the best 50 matching type strains (according to the bitscore) for each user genome, and to subsequently calculate precise distances using the Genome BLAST Distance Phylogeny approach (GBDP) under the algorithm “coverage” and distance formula (d5) [50]. These distances were finally used to determine the 10 closest type strain genomes for each of the user genomes.

For the phylogenomic inference, all pairwise comparisons among the set of genomes were conducted using GBDP and accurate intergenomic distances inferred under the algorithm “trimming” and distance formula (d5) [50]. A total of 100 distance replicates were calculated for each one. Digital DNA–DNA hybridization (dDDH) values and confidence intervals were calculated using the recommended settings of the GGDC 3.0 [46,50]. The resulting intergenomic distances were used to infer a balanced minimum evolution tree with branch support via FASTME 2.1.6.1, including SPR postprocessing [51]. Branch support was inferred from 100 pseudo-bootstrap replicates each. The trees were rooted at the midpoint [52] and visualized with PhyD3 [53].

The type-based species clustering using a 70% dDDH radius around each of the 79 type strains was performed as previously described. Draft genomes of the 80 Acinetobacter species were additionally uploaded to JSPeciesWS (Ribocon GmbH, Bremen, Germany, Version 3.9.0), an online platform for a BLAST-based measurement of the average nucleotide identity (ANIb), which contained 14,976 type strain genomes and a total of 55,565 genomes at the date of analysis (21 February 2022) [54]. Finally, we performed a ribosomal multilocus sequence typing (rMLST) analysis using the PubMLST.org website [55].

2.6. Antimicrobial Resistance Gene Screening

The online platform tool ResFinder version 4.1, provided by the Center of Genomic and Epidemiology [56], was used to identify resistance genes based on whole-genome sequence data. In addition, we used the Resistance Gene Identifier (RGI) software of the Comprehensive Antibiotic Resistance Database to predict resistance genes [57].

2.7. Detection of Oxacillinase Genes and Phylogenetic Analysis

Nucleotide and amino acid sequences of intrinsic oxacillinases were used to query the NCBI nucleotide, genome, and protein databases using BLAST, implemented in Geneious (version 8.1.9, Biomatter Ltd., Auckland, New Zealand). A total of 970 bla_OXA sequences (available on 30 December 2021) were aligned with bla_OXA sequences from the reptile A. baumannii isolates. A maximum likelihood phylogeny of bla_OXA genes was estimated using FastTree. A translation of the nucleotide alignment was used to identify all OXAs that were different from currently known variants. Novel bla_OXA genes were submitted to GenBank.

2.8. Multilocus Sequence Typing of Acinetobacter Species and Assignment of A. baumannii Isolates to International Clones

Multilocus sequence types (STs) according to the MLST scheme developed by the Pasteur Institute [58] were deduced from whole-genome sequence data using PubMLST (https://pubmlst.org/bigsdb?db=pubmlst_abaumannii_seqdef (accessed on 1 October 2021)). This typing tool allows for the definition of STs for A. baumannii and for other Acinetobacter species. In the case that novel alleles and allele profiles were identified, they were submitted to the database curator to designate novel allele and ST numbers. International clones (IC) IC1 to IC3 were identified by multiplex-PCR [59]. In addition, a whole-genome-based comparison of all A. baumannii isolates from reptiles with representative genomes of IC1 to IC9 was performed to categorize the strains under study into the different clonal groups. In the case that isolates could not be assigned to an IC, their genomes were submitted to BacWGSTdb 2.0, a repository for bacterial whole-genome sequence typing and source tracking [60]. The tool “single genome analysis” was used to identify close isolates from the database based on a SNP strategy using a threshold of 1000 SNPs. The first three genomes that matched with our isolates were included in an SNP-based genome comparison.

3. Results

3.1. Sample Collection

Out of 160 sample batches, 183 specimens (162 fecal, 15 skin, 3 urinate, and 3 mixed fecal/urinate) were acquired. Based on MALDI-TOF MS analysis data, presumptive Acinetobacter spp. were present in 45.7% of 92 shipments, in 57 (35.6%) of 160 batches/sample units, and in 58 (31.7%) of 183 specimens. In accordance with the high number of shipments from the USA and some other countries, most of the 80 Acinetobacter spp. isolates were obtained from samples from the USA (46.25%); Ukraine (10.0%); Canada (8.75%); and Vietnam (7.5%); but also from South Africa (5.0%); El Salvador, Madagascar, and Nicaragua (3.75% each); Uzbekistan (2.5%); and Brazil, Ecuador, Japan, Colombia, Mozambique, the United Republic of Tanzania, and Uganda (1.25% each). Acinetobacter spp. isolates were retrieved from Squamata (77.5%) (83.9% lizards and 16.1% snakes) and from Testudines (22.5%) (100% turtles and tortoises).

The reptiles from which the 80 Acinetobacter spp. were isolated represented both, obviously CB species, which accounted for 42.5% of the isolates (

Table 1. Animal origin, species designation, multilocus sequence types, and OXA types of 80 Acinetobacter spp. isolated in this study.

Strain ID	Species *	STPast **	OXA Type **	Sample			Animal ****
				Shipment	Batch	Type ***	Species	Country	Origin
IHIT27741	Ar	2046	813	17	21	S	Jackson’s chameleon	USA	CB
IHIT27742	Ar	2053	23	46	69	F	Horsfield’s tortoise	UZB	FB
IHIT27743	Ar	2073	809	73	125	F	Fat-tail gecko	USA	CB
IHIT27744	Ar	2085	23	85	151	F	Horsfield’s tortoise	UKR	FB
IHIT33475	Aind	2061	neg.	60	100	F	Leopard tortoise	TZA	FB
IHIT35877	Ab	727	65	2	5	S	Sand monitor	USA	CB
IHIT35878	Ab	1211	65	18	23	S	Green basilisk	USA	FB
IHIT35879	Ab	866	385	21	27	F	Common leopard gecko	CAN	CB
IHIT35880	Ab	1290	342	23	30	F	Eastern collared lizard	USA	WC
IHIT35881	Ab	294	343	27	40	F	Central bearded dragon	USA	CB
IHIT35882	Ab	25	64	33	50	F	Ball python	CAN	CB
IHIT35884	Aseif	1291	neg.	37	54	S	Rough green snake	USA	WC
IHIT35885	Ab	311	799	38	55	F	Armored pricklenape	VNM	WC
IHIT35886	Ab	1111	91	39	62	F	Chinese water dragon	VNM	WC
IHIT35887	Ab	25	64	42	64	F	Rainbow boa	USA	CB
IHIT35888	Ab	1292	132	44	67	F	Madagascar day gecko	UKR	CB
IHIT35889	Ab	1293	104	50	76	F	Indigo snake	USA	pr. CB
IHIT35890	Ab	1294	383	50	77	F	Fat-tail gecko	USA	CB
IHIT35891	Ab	294	343	51	80	S	Rough green snake	USA	WC
IHIT35892	Ab	1295	69	52	81	F	Common green iguana	SLV	FB
IHIT35893	Ab	1212	69	58	95	F	Saw-scaled curly-tail	USA	WC/FB
IHIT35894	Ab	1296	#	70	118	F	Boa constrictor	USA	CB
IHIT35895	Ab	46	104	72	122	F	Common green iguana	SLV	FB
IHIT35896	Ab	1297	91	75	131	F	Green spiny lizard	USA	pr. CB
IHIT35897	Ab	1298	800	75	135	S	Savannah monitor	USA	CB
IHIT35898	Ab	1299	106	82	145	F	Sand monitor	CAN	CB
IHIT35899	Ab	1300	314	83	146	F	Common leopard gecko	CAN	CB
IHIT35900	Ab	1301	749	84	148	F	Common leopard gecko	USA	CB
IHIT39733	Ab	866	385	21	28	F	Crested gecko	CAN	CB
IHIT44648	Ap	2038	978	1	1	F	Green pricklenape	VNM	WC
IHIT44649	Alac-like	2047	979	18	25	F	Green spiny lizard	USA	pr. CB
IHIT44650	Alac	2048	980	27	40	F	Central bearded dragon	USA	CB
IHIT44651	Atown	2049	neg.	35	52	F	Red-footed tortoise	COL	WC/FB
IHIT44652	Ager	2050	981	39	60	F	Eastern garden lizard	VNM	WC
IHIT44653	Anoso	1269	neg.	39	60	F	Eastern garden lizard	VNM	WC
IHIT44654	Ap	2039	506	44	66	F	Central bearded dragon	UKR	CB
IHIT44655	Aber	2051	301	44	66	F	Central bearded dragon	UKR	CB
IHIT44656	Aviv	2052	982	44	66	F	Central bearded dragon	UKR	CB
IHIT44657	Aber	2054	983	48	72	F	Yellow mud turtle	USA	FB
IHIT44658	Acalc	2055	329	50	77	F	Fat-tail gecko	USA	CB
IHIT44659	Acour	2056	984	51	80	S	Rough green snake	USA	WC
IHIT44660	Ap-like	2057	985	51	80	S	Rough green snake	USA	WC
IHIT44661	Ap-like	2040	986	52	81	F	Common green iguana	SLV	FB
IHIT44662	Aolei	2058	987	53	82	F	Red-footed tortoise	BRA	WC/FB
IHIT44663	Ap	2041	988	58	93	F	Hispaniolan masked curly-tail	USA	WC/FB
IHIT44664	Alac-like	2059	989	58	95	F	Saw-scaled curly-tail	USA	WC/FB
IHIT44665	Atan	2060	neg.	58	97	F	Striped mud turtle	USA	FB
IHIT44666	Acalc	2062	990	64	110	F	East African black mud turtle	MOZ	WC
IHIT44667	Aschin	2063	991	65	111	F	Leopard tortoise	ECU	FB
IHIT44668	Agem	2064	992	66	113	F	Yellow-headed gecko	NIC	WC
IHIT44669	Aviv	2065	993	66	113	F	Yellow-headed gecko	NIC	WC
IHIT44670	Avar	2066	neg.	67	114	F	Horsfield’s tortoise	UZB	FB
IHIT44671	Aviv	2067	994	68	115	F	Jackson’s chameleon	UGA	CB
IHIT44672	Acour	2068	995	69	116	F	Cuvier’s Madagascar swift	MDG	WC
IHIT44673	Acour	2069	996	69	116	F	Cuvier’s Madagascar swift	MDG	WC
IHIT44674	Aviv	2070	997	69	117	F	Southeastern girdled lizard	MDG	WC
IHIT44675	Acour	2071	998	71	119	F	Rough green snake	USA	WC
IHIT44676	Ager-like	2072	999	73	124	F	Red corn snake	USA	CB
IHIT44677	Ap	2042	1000	73	125	F	Fat-tail gecko	USA	CB
IHIT44678	Aber	2074	257	73	126	F	New Caledonia giant gecko	USA	CB
IHIT44679	Ap	2043	502	73	126	F	New Caledonia giant gecko	USA	CB
IHIT44680	Ap	2044	502	74	128	F	Leopard tortoise	ZAF	FB
IHIT44681	Aber	2075	1001	74	128	F	Leopard tortoise	ZAF	FB
IHIT44682	Aber	2075	1001	74	128	F	Leopard tortoise	ZAF	FB
IHIT44683	Ap	2045	1002	74	128	F	Leopard tortoise	ZAF	FB
IHIT44684	Aolei	2076	1003	75	129	F	Cuban giant anole	USA	WC/FB
IHIT44685	Aseif	2077	neg.	75	130	F	Argentine black and white tegu	USA	CB
IHIT44686	Atan-like	n.d.	neg	75	130	F	Argentine black and white tegu	USA	CB
IHIT44687	Anoso	2078	neg	75	130	F	Argentine black and white tegu	USA	CB
IHIT44688	Acour	2079	1004	75	131	F	Green spiny lizard	USA	pr. CB
IHIT44689	Ap	220	820	76	136	F	Shingleback lizard	JPN	CB
IHIT44690	Aber	2080	1052	78	138	F	Chinese water dragon	VNM	WC
IHIT44691	Ap	220	820	79	142	F	Painted wood turtle	NIC	WC
IHIT44692	Aschin	2081	1005	80	143	F	Painted wood turtle	USA	pr. CB
IHIT44693	Aj-like	2082	1006	82	145	F	Sand monitor	CAN	CB
IHIT44694	Aolei	2083	1007	82	145	F	Sand monitor	CAN	CB
IHIT44695	Alac-like	2084	1008	84	148	F	Common leopard gecko	USA	CB
IHIT44696	Asol	2086	neg	87	154	F	Horsfield’s tortoise	UKR	FB
IHIT44697	Acalc	2087	1009	87	154	F	Horsfield’s tortoise	UKR	FB
IHIT44698	Alac	2088	1010	91	159	F	Central bearded dragon	UKR	CB

* Ab = A. baumannii, Aber = A. bereziniae, Acalc = A. calcoaceticus, Acour = A. courvalinii, Agem = A. geminorum, Ager = A. gerneri, Aind = A. indicus, Aj = A. johsonii, Alac = A. lactucae, Anoso = A. nosocomialis, Aolei = A. oleivorans, Ap = A. pittii, Ar = A. radioresistens, Aschin = A. schindleri, Aseif = A. seifertii, Asol = A. soli, Atan = A. tandoii, Atown = A. towneri, Avari = A. variabilis, and Aviv = A. vivianii. ** Sample type: F = feces; S = skin. *** Novel multilocus sequence types and novel OXA protein variants are underlined. **** CB = captive-bred, FB = farm-bred, WC = wild-caught, and pr. = presumably.

). Contrarily, 23.8% of the isolates were obtained from WC animals, followed by FB reptiles (21.3%). A small proportion of 10 isolates (12.5%) was gained from wild-caught or farm-bred/captive-bred (WC/FB or WC/CB) reptiles.

3.2. Species Identification Based on MALDI-TOF MS Analysis and Whole-Genome Sequence Analysis

Using MALDI-TOF MS, 60 of the 80 isolates from this study could be assigned to the following species with unequivocal quality results (Supplementary Table S2): A. baumannii (n = 23), A. bereziniae (n = 4), A. calcoaceticus (n = 5), A. courvalinii (n = 1), A. lactucae (syn. A. dijkshoorniae) (n = 3), A. indicus (n = 1), A. johnsonii (n = 1), A. nosocomialis (n = 3), A. pittii (n = 12), A. radioresistens (n = 4), A. schindleri (n = 1), A. seifertii (n = 1), and A. towneri (n = 1). Twenty strains gave unreliable results with respect to a species-specific identification.

As a countercheck to MALDI-TOF MS analysis, species IDs were investigated by different sequence-based analysis tools. The results are summarized in Supplementary Table S2. The species determination and dDDH values resulting from TYGS analysis revealed 62 isolates that were clearly assigned on a species level, while 18 isolates were identified as potentially new species (d4 value < 70.0%). The species clusters resulting from this analysis are additionally listed in Supplementary Table S3, whereas the taxonomic identification of the query strains is found in Supplementary Table S4. The clustering of non-baumannii Acinetobacter spp. isolates yielded 81 species clusters, and the provided query strains were assigned to 30 of these. The taxonomic placement of 57 non-baumannii Acinetobacter spp. isolates is shown in Supplementary Figure S1.

Based on ANIb analysis, 70 isolates were reliably assigned to a species level, while 10 isolates revealed identity values below the threshold of 95%. Lastly, when using the rMLST species ID tool (https://pubmlst.org/species-id (accessed on 8 February 2022)), 62 isolates fulfilled the criteria for a clear species identification, while the genomes of 18 isolates where either below the cutoff value (≤95%) or showed percentages of similarity to reference strains of two different Acinetobacter species.

Overall, congruent results by the different methods were only produced for 43 isolates (53.75%), including all 23 A. baumannii isolates. In the case that an isolate revealed congruent results with at least two of the methods, we assumed that as a reliable species identification. This was the case for 67 isolates (83.75%), in alphabetical order: A. baumannii (n = 23), A. bereziniae (n = 6), A. calcoaceticus (n = 2), A. courvalinii (n = 2), “A. geminorum” (n = 1; not validly published), A. gerneri (n = 1), A. indicus (n = 1), A. lactucae (n = 2), A. nosocomialis (n = 2), A. oleivorans (n = 2), A. pittii (n = 9), A. radioresistens (n = 4), A. schindleri (n = 2), A. seifertii (n = 2), A. soli (n = 1), A. tandoii (n = 1), A. towneri (n = 1), A. variabilis (n = 1), and A. vivianii (n = 4). Another 13 isolates showed incongruent results in at least three of the methods applied and were therefore defined as A. lactucae-like (n = 3), A. pittii-like (n = 2), A. calcoaceticus-like (n = 2), A. courvalinii-like (n = 3), A. gerneri-like (n = 1), A. tandoii-like (n = 1), and A. johnsonii-like (n = 1) (Table 1).

3.3. Phenotypic Antimicrobial Resistance

Using the broth microdilution method in a commercial veterinary layout that includes 16 antimicrobial substances from 10 different substance groups, MIC values and—where available—clinical breakpoints (according to EUCAST v. 11.0) were obtained for all isolates in order to screen for MDR patterns. Most of the 80 Acinetobacter isolates under study showed high MIC values (in µg mL⁻¹) as expected for penicillin (>8; n = 73), ampicillin (≥4; n = 73), amoxicillin/clavulanic acid (≥4/2; n = 71), cephalothin (≥16; n = 78), and ceftiofur (≥4; n = 72) (Supplementary Table S5). Mainly high MIC values were also found for florfenicol (≥4; n = 71), erythromycin (≥4; n = 74), tilmicosin (≥16; n = 74), tulathromycin (≥32; n = 68), tiamulin (≥32; n = 68), and spectinomycin (≥64; n = 62). Contrarily, the majority of strains displayed low MIC values for gentamicin (<4; n = 79), fluorchinolones (enrofloxacin <1; n = 75), colistin (≤2; n = 73), tetracycline (≤4; n = 66), and trimethoprim/sulfamethoxazole (≤2/38; n = 70). Irrespective of intrinsic resistances but rather based on high MIC values, Acinetobacter spp. strains were most often resistant to six substance groups (n = 49). A rather uncommon phenotype was observed in 9 strains with resistance to only 1–4 substance groups, the majority of which were characterized by low MIC values against penicillins (IHIT33475 (IHIT is an acronym for the German term for the Institute of Hygiene and Infectious Diseases of Animals), IHIT44651, IHIT44670, IHIT44680, and IHIT44681). A total of 14 strains were resistant against 7, 8, and 10 substance groups with A. bereziniae IHIT44655 (bearded dragon, Ukraine) showing the highest resistance phenotype. With respect to critically important antimicrobials, in vitro resistance to fluorchinolones and colistin are of particular interest. Whereas one of each strain of A. towneri (IHIT44651), A. bereziniae (IHIT44655), A. schindleri (IHIT44667), and A. pittii (IHIT44689) were resistant to enrofloxacin, three strains of A. bereziniae (IHIT44655, bearded dragon, Ukraine; IHIT44657, Turtle, USA; and IHIT44690, water dragon, Vietnam), one A. gerneri (IHIT44652, sand monitor, Vietnam), one A. courvalinii (IHIT44688, green spiny lizard, USA), one A. oleivorans (IHIT44694, sand monitor, Canada), and one A. johnsonii-like isolate (IHIT44693, sand monitor, Canada) showed colistin resistance in vitro.

3.4. Antimicrobial Resistance Genes

Among the 80 Acinetobacter spp. isolates under study, 9 (11.3%) carried tet(39) and 6 strains (7.5%) were positive for tet(B). A novel tet(X)-variant (tet(X5)-like) was identified in an A. towneri (IHIT44651) that was isolated from a red-footed tortoise from Colombia (Supplementary Table S5). Sulfonamide gene sul2 was displayed by 11 strains (13.8%). Several genes encoding aminoglycoside-modifying enzymes were identified, however in low frequencies: aac(6‘)-Ir (7.5%), strA, strB and aph(3‘)-III (2.5% each), aadB, aac(3)-IIa, aac(6‘)-Ia, aph(3‘)-IX, and aph(3‘)-X (1.25% each). One strain of A. schindleri (IHIT44667) possessed the florfenicol resistance gene floR, whereas all of the A. baumannii isolates and nearly half (49.1%) of the 57 of non-baumannii isolates had the chromosomally encoded ADC cephalosporinase.

3.5. Intrinsic Oxacillinase Genes and Novel OXA Protein Variants

The diversity of the bla_OXA-like gene sequences from reptile Acinetobacter spp. isolates was high, not only between the different species, but also among isolates of the same bacterial species. A total of 70 strains (87.5%) carried an intrinsic oxacillinase gene with 19 known and 39 novel protein variants, designated as OXA-799, OXA-800, OXA-809, OXA-813, OXA-820, OXA-978 to OXA-1010, and OXA-1052 (Table 1, Supplementary Table S7). An additional OXA-allele variant with 99.3% amino acid similarity to OXA-715 of the OXA-51 family was found to be non-functional due to the introduction of an internal stop codon at position 42 (A. baumannii isolate IHIT35894). Based on a comparison of OXA amino acid sequences from reptile Acinetobacter spp. isolates and sequences available in the database, we identified the most-closely related OXA-alleles. They were included in a phylogenetic tree that was created based on aligned amino acid sequences (Figure 1). As expected, OXA-alleles basically clustered with OXA-type families and their variants that are intrinsic in the different Acinetobacter species, such as the OXA-51-like variant in A. baumannii, the OXA-272-like variant in A. pittii, or the OXA-23-like variant in A. radioresistens. For A. baumannii, it has been shown that the clonal affiliation of an isolate basically correlates well with specific variants of the bla_OXA-51-like gene [61]. Here, some of the OXA-variants did not follow the phylogeny of A. baumannii. For example, OXA-65 occurred in ST727 (IHIT35877; shipment 2/batch 5; sand monitor; USA) and ST1211 (IHIT35878; shipment 18/batch 23; green basilisk, USA), two sequence types that differ in all alleles of the seven-gene MLST^Past scheme. Also, other OXA-types, such as OXA-69 (ST1212 and ST1295), OXA-91 (ST1111 and ST1297), and OXA-104 (ST46 and ST1293) were identified in different STs that varied by 4 to 7 MLST alleles. This incongruence was also seen for some of the other Acinetobacter species, as shown in Supplementary Table S6.

3.6. MLST and International Clones

The 23 A. baumannii isolates belonged to 20 distinct STs according to the Pasteur scheme; there were 13 novel STs (Table 1, Supplementary Table S6). Two strains, isolated from a ball python from Canada (IHIT35882; shipment 33/batch 50) and a rainbow boa from the USA (IHIT35887, shipment 42/batch 64) belonged to the worldwide-distributed ST25, which belongs to IC7. Based on cgMLST analysis using the BacWGSTdb online tool and its data deposits, IHIT35887 particularly revealed a close relationship, differing by 62 to 139 alleles to ST25 isolates from other sources, such as IHIT38008 (dog, urine, Germany, 2018), AB24 (human, pus, Malaysia, 2012), 4300STDY7045893 (human, Thailand, 2016), and NM3 (human, sputum, United Arab Emirates, 2008). The second isolate, IHIT35882, revealed the lowest number of different alleles (136–266) to A. baumannii OCU_Ac2 (blood culture, hospitalized patient, Japan, 2014), OIFC143 (human, USA, 2003), IHIT38008 (urine, dog, Germany, 2018), and MRSN14237 (wound, human, Honduras, 2012).

Based on the comparison of the core genome of reptile A. baumannii isolates and representative A. baumannii isolates of IC1–IC9, other ICs (other than IC7) could not be assigned to the reptile isolates (Figure 2). The majority of non-IC1–IC9 isolates were singletons for which no related genome could be found in the database (BacWGSTdb). However, ST294 isolates IHIT35881 (central bearded dragon, USA, shipment 27/batch 40) and IHIT35891 (rough green snake, USA, shipment 51/batch 80) clustered close to ST294 isolate PG20180064, which was isolated from a mouse in Canada in 2018) and to IHIT32296, an OXA-72-producing strain recently published by our group (grey parrot, Luxembourg, 2016) [63]. Also, ST46 isolate IHIT35895 (common green iguana, El Salvador) clustered together with genomes from the database, e.g., ST46 isolates 57185_12EESBL (biogas plant, Germany, 2012), R20 (human, USA, 2016), and ST149 isolates SP816 and BA22685 (both isolated from humans, India, 2019).

Non-baumannii Acinetobacter spp. isolates were mostly assigned to novel STs. Only A. pittii isolates IHIT44689 (yellow-headed gecko, Japan) and IHIT44691 (painted wood turtle, Nicaragua), belonged to the previously known ST220 that has been described for human clinical isolates in different countries, including carbapenemase-producing isolates in Japan [64] and Thailand [65].

4. Discussion

Although antimicrobial treatments are usually not carried out in wildlife animals, a growing number of reports have been published on MDR bacteria in wild and game animals. The spillover of antimicrobials and MDR bacteria into ecosystems and direct or indirect contact with MDR-shedders also seems to play a pivotal role in the acquisition of resistance genes in wildlife. In this regard, we hypothesized that poor hygienic and environmental conditions in habitats and breeding facilities might be correlated with increased rates of MDR bacteria and their global distribution in imported reptiles [66]. Very few studies have sampled reptiles directly at the point of entry in order to prevent bias of the detected microbiota from inland sources. Therefore, the aim of the current study was to evaluate a fraction of Acinetobacter spp. in healthy reptiles during importation at border inspection. Results on other microbiota from the same sampling have been reported [41] and will be published elsewhere. The Frankfurt Airport in Germany, is regarded as one of the most important hubs in animal importation, worldwide. In 2019, 306 shipments declared as ‘reptiles’ were registered for import into the European Union, and 93 shipments were sent in transit with a total number of 968,192 live reptiles imported from 21 third-party countries [67]. Although the importation of wild-caught animals has been widely banned, a considerable number of the imported species under study seemed to represent specimens that had been collected from natural resources (Supplementary Table S1), while the majority of species had been bred or raised in breeding facilities for the pet market and as laboratory animals. In the present study, 92 shipments containing live reptiles were tested for the presence of MDR bacteria, and the dataset of Acinetobacter spp. isolates has been further elucidated. Generally, Acinetobacter spp. are regarded as non-pathogenic bacteria in reptiles; some infections seem to be closely related to immune compromise [36]. This bacterial group may possess a highly diverse array of beta-lactamases that hydrolyze and confer resistance to penicillins, cephalosporins, and carbapenems [5,44]. This was in agreement with our study population since Acinetobacter spp. isolated from pet reptiles displayed in more than 60% of the isolates the chromosomally encoded ADC cephalosporinase. In addition, 87.5% of the isolates contained highly diverse intrinsic oxacillinase genes with 37 novel variants. There are several indications that typing of clinical isolates of A. baumannii belonging to ICs by MLST correlates well with specific OXA-51-like variants [21,61]. We have identified some OXA-51-like variants in the reptile A. baumannii isolates, such as OXA-64, OXA-65, and OXA-69, that are commonly found in the well-described clinical A. baumannii international clonal lineages IC7, IC5, and IC1. However, only the OXA-64 isolates clustered well with IC7 reference genomes, while we found no significant similarity between OXA-65 (IHIT35877 and IHIT35877; both from lizards from the USA) and OXA-69 isolates (IHIT35892 and IHIT35893; lizards, El Salvador and USA) and any of the clinical A. baumannii IC lineages (Figure 2). This, together with findings from others [21,25], indicates that MLST types and OXA-51-like variants are not strictly correlated. Notably, both studies observed this convergent evolution of bla_OXA-51-like genes among A. baumannii isolates from non-human sources, namely white storks in Poland and Germany, and livestock and food samples in Lebanon. We recommend further investigating the extent to which OXA proteins from animal sources may act as indicators of the clonal affiliation of A. baumannii isolates.

Almost 20% of the reptile isolates simultaneously displayed reduced susceptibility or resistance to tetracyclines, which are encoded by two major groups of tet genes. The first group mediates energy-dependent efflux pumps for tetracyclines (tet(A), tet(B), tet(H), and tet(39)) and was represented by 21 isolates, whereas the second group confers resistance by ribosomal protection (tet(M)). Acinetobacter towneri isolate IHIT44651 (red-footed tortoise, Colombia) contained a tet(X3)-like gene associated with tigecycline resistance. However, the isolate demonstrated low MIC for tigecycline as determined by a MIC test strip (MIC 0.38 mg/L; Liofilchem Diagnostica). Other resistance patterns included aminoglycoside resistance genes in 18 strains, sulfonamide resistance in 11 strains, and a florfenicol resistance gene in A. schindleri (strain IHIT44667). The goal of the current study was to assess the presence of Acinetobacter species and other Gram-negative bacterial species (data not shown) of supposed pet reptiles during the process of importation. The magnitude and variability of resistance, assessed solely in one bacterial genus, Acinetobacter, suggests that imported reptiles are an underestimated source of resistant bacteria. In this regard, the vast majority (78.8%) of the study population showed phenotypical resistance to 3 (n = 2), 4 (n = 1), 5 (n = 7), 6 (n = 49), 7 (n = 1), 8 (n = 3), and 10 (n = 1) antimicrobial drug classes. Contrarily, resistance gene screening revealed evidence for only 0 (n = 3), 1 (n = 43), 2 (n = 20), 3 (n = 12), 4 (n = 1), and 5 (n = 1) groups of specific resistance genes, which can best be explained by as-yet-undetermined, intrinsic resistance mechanisms. Eventually, strains IHIT33475 and IHIT44676 both contained at least one tet resistance gene that was seemingly not expressed in vitro. None of the strains from the present study was found with phenotypical or molecular evidence for carbapenem resistance.

The SNP-based phylogeny, as well as the assessment of the MDR genotype, revealed several relationships to the resistant Acinetobacter spp. that have been isolated from other clinical sources. The two A. baumannii strains, IHIT35882 and IHIT35887 from a ball python (Canada) and a rainbow boa (USA), respectively, turned out to belong to ST25 (IC7), which represents a clonal lineage of clinically relevant strains with an MDR phenotype (including carbapenem resistance) that emerged on different continents over the last decade and was also reported in animals, including pets and wild birds [21,68,69,70,71]. One of our isolates was affiliated with a group of human clinical isolates which belonged to ST46 and related STs and which were not linked to a distinct ST. It remains to be seen if the worldwide, increasing number of A. baumannii genomes that is becoming available from different sources, including clinical, non-clinical, animal, and human sources, will lead to the establishment of novel A. baumannii ICs and a more complex understanding of the molecular epidemiology of this important pathogen.

From a zoonotic perspective, imported reptiles are almost exclusively sold as pets and—contrarily to the situation in some of their home countries—are not utilized as food sources following importation. However, zoonotic bacteria, especially Salmonella [72], are well-known pathogens that frequently occur in reptiles and might—under inadequate hygienic conditions—be transferred to humans. The same situation must also be anticipated with MDR bacteria, and a spillover of these bacterial genes might occur via wastewater and terrarium soil, for example. Studies on findings of MDR bacteria in reptiles have increased in recent years. Among the reasons for conducting this kind of research were proofs for anthropogenic changes, including conservation issues, ecosystem health status, and environmental pollution [66,73,74]; veterinary aspects [75,76]; and possible zoonotic infections [40]. Most studies have been conducted to assess the health situation of reptiles in their ecosystems or when injured reptiles suffered from infection. Deems et al. [77] have analyzed a number of Acinetobacter species from the feces of wild painted turtles (Chrysemys picta). Although the authors also could not find strains displaying phenotypical carbapenem resistance, they demonstrated the capability of biodiesel degradation and biofilm formation in one A. oleivorans strain that possessed a putative type 6 gene cluster. However, as with other animal and herbal goods, the global trade does play a role in the dissemination of resistant bacteria in reptiles. Since imported live reptiles are not consumed as food items and are known to harbor a wide variety of possible zoonotic microorganisms, public health issues should focus on proper hygienic precautions in order to prevent human infections and the spread of MDR bacteria. The sole prohibition of reptile importation would most likely not prevent the spread since respective bacteria will be introduced by other routes, including humans themselves. Another study focused on investigating meat microbiota from recently imported reptiles and amphibians that are sold in specialty markets in Canada [37]. The authors found evidence for a further, highly virulent, NDM-1-producing Acinetobacter sp. that was isolated from a dried turtle carapace, suggesting that future studies should focus on the full diversity of genotypes as well as a comparison with captive-bred, pet reptiles.

Interestingly, reptile β-defensins, toxin components, and peptides with antimicrobial and antibiofilm activity against A. baumannii and other MDR bacteria have been described as a component of the innate immune system in different species, which might represent promising treatment options, even in human infections, especially when outperforming human homologues [78,79,80,81,82,83]. Reptiles might also cope with facultative pathogenic bacterial loads by a higher activity of antimicrobial molecules that could, for instance, be found in snakes and water monitors inhabiting polluted environments [84].

In conclusion, the results from the present study have shown that imported, healthy pet reptiles represent another mosaic stone in the distribution pattern of Acinetobacter spp. Although the sole presence of these widely distributed bacteria in animal samples is not surprising, their discovery in every other shipment and the expression of a MDR phenotype in over 78% of the isolates should however, address future awareness on the fate of these lineages. With regard to the initial hypothesis, we could not confirm a higher load with MDR bacteria in reptiles from ‘antimicrobially polluted’ environments alone, but the strains with the highest resistance properties also seemed to be equally distributed in the group of supposed captive-bred species. Fortunately, the vast majority of Acinetobacter spp. isolated as “blind passengers” from pet reptiles proved susceptible to critically important antimicrobials, and there is currently no suggestion that reptile isolates represent a serious public health issue.