Acinetobacter baumannii’s lifestyle includes soil-dwelling colonization of decaying plant material and airborne spread

Domain: biology
Published: 2026-03-10
Editorial outcome: major_revision

Abstract
Acinetobacter baumannii is a Gram-negative nosocomial pathogen that is notorious for its rapid development of antibiotic resistance. However, its ecology and evolution outside hospital settings remain poorly defined. Here, we demonstrate that the natural lifestyle of A. baumannii includes soil-dwelling and airborne dissemination, which helps explaining its adaptability and tolerance to desiccation, radiation and antibiotics, and thus its predisposition to establish within hospitals. Starting from white stork nestlings previously discovered as a reservoir, we studied food chains and associated environments and identified soil and decaying plants as habitats. We demonstrate that sterilized plant material is rapidly colonized by airborne A. baumannii. A set of 401 genomes were sequenced and compared to publicly available genomes, revealing numerous links between wildlife isolates and hospital strains, and disclosing intercontinental dispersal. Our pan-genome estimate of the species (~51,000 gene families) more than doubles that of previous studies. Our data further suggest massive radiation of the species early after its emergence, possibly fostered by human activity since the Neolithic. Now, it is possible to study the ecology and evolution of A. baumannii in nature at an unprecedented temporal and spatial resolution and to elucidate the adaptive evolution of environmental bacteria towards multidrug-resistant opportunistic pathogens.

Introduction
The Gram-negative Acinetobacter baumannii is notorious for its potential to act as an opportunistic pathogen in hospitals, facilitated by an exceptional resistance to desiccation stress and intrinsic as well as acquired resistance to antibiotics and disinfectants. The genus Acinetobacter currently comprises of around 100 described or tentative species1,2, the majority of which consist of environmental species, or commensals of humans and animal that rarely cause any disease. Some species like A. lwoffii and A. johnsonii, which are regularly found on human skin, mucosa and within the gut of humans and animals, appear to play an essential role in the development of immune tolerance3–6. In contrast, the species most frequently involved in hospital infections, e.g., A. baumannii, A. pittii, A. nosocomialis, and A. seifertii do not represent commensals of humans and animals, and their natural habitats are still poorly described7–9. Although the zoonotic potential of A. baumannii is evident from previous work10–14, the pathogen is not generally accepted as zoonotic, e.g., not listed as zoonotic pathogen by public health agencies such as the Centers for Disease Control and Prevention (CDC), Public Health England (PHE) or the European Center for Disease Prevention and Control (ECDC)15–17. As a hallmark, the killing of more than 400 individuals of a flock of sheep by a multidrug-resistant (MDR) and hypervirulent strain related to international clone 2 (IC2) illustrates the threat due to reverse zoonosis18. The relevancy to One Health is further underlined by the reservoir of antimicrobial resistance (AMR) genes found in environmental Acinetobacter spp., as well as other bacteria, that can be mobilized into pathogenic Acinetobacter spp. via horizontal gene transfer19–21. Moreover, the release of MDR A. baumannii from wastewater into the environment has been well-described22,23.

Here, our intention was to improve the understanding of the ecology of A. baumannii in natural habitats as we expect this knowledge to contribute to our understanding of the ecology of these bacteria in the hospital setting thus fostering better prevention and control measures. Moreover, from the hitherto available genome data it was impossible to retrace the evolution of the species because of the strong bias toward the study of MDR isolates and those of the predominating IC2 in particular24. Hence, starting from the association of diverse A. baumannii with white stork nestlings as recently described12, we further explored the ecology of the bacteria. Preliminary data pointed to an only transient colonization of the nestlings and indicated the feed to be the true source. Following these potential paths, we came up with particular soil habitats being highly conducive to A. baumannii. Consequently, it was possible to establish a large collection of wildlife and environmental A. baumannii isolates and to develop a genomics-based view on the ecology and evolution of the bacteria which is presented here.

Results
Update on the prevalence of A. baumannii in white stork nestlings
Previously, we had presented data from 661 white stork nestlings sampled between 2013 and 2016 in Poland which were found to be colonized with A. baumannii in the choana region at a prevalence of 25%12. We continued the systematic study of this environment and, based on 1319 white stork nestlings sampled between 2013 and 2018 across multiple regions of Poland, the average colonization rate was identified as 27.6% (Fig. 1; and Suppl. Table 1). White stork nestlings were also sampled on a smaller scale in Germany in 2015 and the positive rate of choana samples reached 34.5% (n = 29). Choana samplings in Spain near Ciudad Real in 2015 revealed no positives from 57 white stork nestlings, but a positive rate of 7.8% (n = 64) was detected among white stork nestlings sampled in 2019 near Madrid (Suppl. Table 1). Interestingly, this included for the first time the isolation of A. nosocomialis (n = 2) from white stork samples, indicating differences in the ecology of bacteria and/or storks in Spain compared to Poland.

Cloacal sampling of white stork nestlings was conducted on 747 individuals in different regions of Poland throughout the study period and revealed an overall positive rate of 8%, however with great variability depending on the region and year of sampling (Suppl. Table 1). Cloacal sampling of 64 white stork nestlings in Spain in 2019 yielded no A. baumannii isolates. Thus, choana sampling yielded higher isolation rates of A. baumannii compared to cloacal sampling.

Notably, significantly increased white blood cell counts in colonized nestlings argue for an infection by A. baumannii rather than mere colonization (Suppl. Table 2, Suppl. Note 1). The probability of isolating A. baumannii from white stork nestlings decreases with the age of the chicks (Suppl. Figure 1).

Pellets from white storks
We had previously described that white storks spit pellets, consisting of indigestible remains of their food, are occasionally contaminated with A. baumannii and might therefore be of interest for studying the ecology of both bacteria and storks12. We collected pellets from selected nests in Loburg and neighboring villages (Germany/state of Saxony-Anhalt) during March to August throughout the years 2015 and 2016. Interestingly, pellet samples proved positive for A. baumannii not earlier than the end of May in 2015 and the end of April in 2016, respectively (Suppl. Figure 2). In 2015, positive samples were identified until migratory departure of the storks in August, with an overall rate of 14.8% positive pellets (13 out of 88), while in 2016 only a few positive samples were found from end of April until June (overall 7.1% positive pellets, 6 out of 85). Sporadically collected egg shells and feathers were also a source of A. baumannii in few cases, and a stork chick found dead was also sampled positive in 2016. Pellets collected from stork colonies in Spain during the winter season of 2014/2015 were negative irrespective of their particular feeding grounds (landfills or nature, Suppl. Figure 3). Overall, our data suggest that white storks arriving from their wintering grounds do not appear to be stably colonized by A. baumannii. Rather, they seem to acquire these bacteria after arrival in the summer quarters depending on the progression of the season, likely via the food chain.

It is worth mentioning, that pH values in pellets contaminated with A. baumannii were found to be as low as pH 2 in some cases and it was proven that the bacteria were not only localized at the surface of the pellets but also inside (Suppl. Figure 4). Consequently, although under laboratory conditions A. baumannii cannot thrive at pH 2, it can survive such harsh conditions in a natural setting.

Diversity of strains in individual nests
To challenge our hypothesis on an only transient carriage/colonization of parental white storks with A. baumannii we analyzed the diversity of strains isolated from nestlings within individual nests. As a proxy of diversity, we made use of the intrinsic blaOXA-51-like gene of A. baumannii encoding the class D oxacillinase OXA-51, the protein variants of which are indexed and previously proven useful as phylogenetic marker12,14,25,26. We sequenced the blaOXA-51-like gene of all isolates collected from white stork nestlings within a single year in a selected region of Poland and visualized the diversity on a map (Fig. 2). The diversity found within individual nests was striking, with up to 5 nestlings each carrying a distinguishable strain. These findings are inconsistent with a vertical transmission from stably colonized parental storks and support our hypothesis of a transient colonization/carriage via food intake from a diverse A. baumannii population.

Tracing the food sources
The predominant prey of white storks consists of small vertebrates, especially rodents and shrews, as well as arthropods and earthworms27,28. In line with the literature, the dominant contribution of rodents and arthropods to the diet of white storks could be directly estimated from pellets of white storks collected in Poland and Germany (Suppl. Figure 5), although analysis of the remains of earthworms from pellets, while possible29, is time-consuming. To assess the potential role of beetles and other insects as sources of A. baumannii, we compared the content of pellets tested positive for A. baumannii with that of pellets tested negative and found no difference in the fraction of insect matter (Suppl. Figure 6). Moreover, dominant arthropods found in pellets such as the ground beetles Zabrus tenebrioides and Pterostichus melanarius (Suppl. Figure 7) collected from nature, albeit at low numbers (n = 13), were also found negative for A. baumannii. We also had the opportunity to sample grasshoppers collected in great quantities by an individual, deceased white stork, which was dissected and found gorged with grasshoppers, however all of the swallowed grasshoppers (>50) tested negative for A. baumannii. Moreover, pellets from Spanish storks, containing high amounts of crab and grasshopper remains (Suppl. Figure 3), also tested negative. Altogether, we found no evidence of a contribution of arthropods captured by white storks to the acquisition of A. baumannii.

Next, we examined the potential role of rodents and shrews as a source of A. baumannii. We collected cat-captured rodents and shrews throughout Germany (states of Saxony-Anhalt, Hesse and Thuringia) in the period from 2014 to 2017 and took samples from the trachea and rectum. We found an average colonization rate of 2% for rodents (n = 154) and 4.7% for shrews (n = 64). Interestingly, all individuals colonized with A. baumannii were captured between August and September (Suppl. Fig. 8). Next, we sampled wild, captive and laboratory rats (Rattus norvegicus) from Germany30–32, as well as other rodents of the genera Microtus, Myodes, and Apodemus captured in Germany33,34 in the period 2007 to 2017. No rats and other rodents were found positive (n = 491 and n = 532).

Earthworms were collected in Germany in the state of Saxony-Anhalt between August 2016 and September 2020. Out of 618 individual earthworms in total, we isolated A. baumannii from 22 earthworms (3.6%) provisionally assigned to the genera Lumbricus, Octolasium, Dendrobaena and Eisenia (Suppl. Tab. 1). Of these positive samples, 19 were collected from May to September and only three isolates dated from March, April and November, respectively (1% positive of n = 75 earthworms collected between October 21st and March 21st). The overall positive rate of A. baumannii isolated from garden compost earthworms collected in Wernigerode (Germany) was 4.2% (14/331), and 4.3% (4/94) for a specific site at the bank of the river Holtemme (Germany), where we had recently also isolated A. nosocomialis from soil35.

Other birds and their prey
We reasoned that studying other birds with an overlapping prey spectrum compared to white storks could help to indicate specificities of the relationship to A. baumannii. Consequently, we collected pellets, feathers and egg shells from a breeding colony of gray heron (Ardea cinerea) in Germany. With the exception of one egg shell sample collected in June from which we could isolate A. baumannii (Suppl. Fig. 9), all samples (n = 35) were negative. The detailed analysis of the pellets indicated their diet to consist of a significant portion of small mammals and insects (Suppl. Fig. 9). Further, we collected pellets (n = 101) from kestrel (Falco tinnunculus) between March and October 2015 and 2016, and found three isolates (3%) of A. baumannii, each collected between August and September (Suppl. Fig. 10). Occasionally discovered owls’ pellets (n = 6), putatively from Tyto alba and Asio otus, collected in August 2015 in Germany, were also positive for A. baumannii in two cases. Moreover, we took cloacal samples from nestlings of the black stork (Ciconia nigra), a species which, in contrast to white stork, is not synanthropic and is preferentially breeding and foraging in forests. Notwithstanding, anthropogenic waste was found in 26% of occupied nests36. The diet of black stork nestlings in Poland is known to be dominated by fish and amphibians37. Not a single one of 64 cloacal samples collected in Poland from black stork nestlings over a period of four years was found positive for A. baumannii. Taken together, gray heron, kestrel and black stork apparently differ from white stork regarding carriage of A. baumannii although this might be influenced by differences in sampling depth and sampling types.

Compost, soil, rhizosphere and the forest paradox
Following up on a possible role of earthworms in the transmission of A. baumannii to white storks, we started collecting soil samples. Initially, we selected compost soil which is wet and rich in decomposing material, providing an environment from which earthworms could be easily collected. As we realized early on that compost soil represented an excellent source for A. baumannii, we utilized a boring rod to study the profile within one meter depth of the compost (Suppl. Fig. 11). Continued sampling of the soil originating from a single compost across a period of seven months yielded 86 isolates containing no less than 20 different variants of the OXA-51 protein family (60/91 samples positive, 66%). Concomitant sampling of earthworms within the same compost yielded 10 different variants of the OXA-51 protein from 7/44 (16%) positive earthworms. We observed a remarkable dynamic of isolated lineages and of the depth-depending colonization over time. The compost, which was continuously used for the deposition of vegetable waste and egg shells during this period, was not permanently colonized throughout all layers and there was a shift of the dominant lineages isolated over time. Continued sampling in subsequent years revealed that isolation rates declined (i) during winter and (ii) within 8-10 weeks after stopping to replenish the compost with fresh material.

Due to the anthropogenic influence on the compost and the ambiguity of the origin of A. baumannii throughout this setting, we next attempted to identify soil habitats in more pristine environments. Searching for wet habitats rich in decomposing material where earthworms can thrive, we identified a site at the bank of the river Holtemme near Wernigerode, Germany, where we successfully isolated A. baumannii from earthworms, soil samples and the rhizosphere of different plants (Urtica spp., Impatiens spp., different grass species etc.). This site at the river bank with an area of only ~0.1 m2 turned out to be a hotspot for the isolation of A. baumannii and yielded 22 isolates within 3 months representing 18 different variants of OXA-51 (Suppl. Fig. 12). Moreover, samples collected at this specific site yielded 10 isolates on a single day, each harboring a different OXA-51 variant. Noteworthy, extensive horizontal gene transfer (HGT) was detected between these co-colonizing lineages (Suppl. Fig. 13). Strikingly, this site has remained a hotspot over the years even though the river bank has been remodeled several times due to flooding. None of the many additional sites were peatedly sampled over the years along the river Holtemme showed any comparably high isolation rate.

Concomitantly, we had collected soil and earthworm samples from a forest area south of Wernigerode, representing both deciduous and coniferous forests of various compositions at an altitude between 300 and 450 m above sea level. Again, we chose wet habitats with decomposing plant material, mostly alongside creeks and ponds, however, of 269 samples only two were positive for A. baumannii (0.7%) (Suppl. Fig. 14). To prove the deduced principle that A. baumannii preferentially colonize alongside creeks but not within forest areas, we selected a creek not previously sampled which has its source within the forest and which, after leaving the forest, flows through grassland before reaching the first village. We were unable to isolate A. baumannii from any sample collected inside or along the edge of the forest although the creek’s banks were rich in decomposing material. The first positive sample was instead collected at a distance of approx. 300 m to the edge of the woods, but positive samples remained rare until the creek had passed the first village (Suppl. Fig. 15). After passage of this village, we discovered two hotspots, which repeatedly collected positive samples (6/12 positive samples (50%) and 7/12 positive samples (58%), respectively). Collectively, forests possibly do not offer soil habitats supportive of A. baumannii, not even alongside creeks, whereas A. baumannii apparently can thrive alongside creeks and rivers outside of forests even if they are lined with trees. Potentially, there are hotspots of A. baumannii where they can occur in striking diversity outside forests.

Diversity in terms of OXA-51 variants collected from Poland and Germany
Currently, there exist 381 assigned variants of the β-lactamase OXA-51 protein encoded by the intrinsic blaOXA-51-like gene of A. baumannii (http://www.bldb.eu/, accessed November 6th, 2024). Our collection of strains encompasses 209 variants, thus about 55% of the presently known worldwide diversity. Of the 209 variants encompassed by our collection, 122 represent previously undescribed variants which were made publicly available throughout the course of this study (Suppl. Data 1). The dominant source of novel variants was white storks. Given that some of these OXA-variants have been shown to be useful as indicators of phylogenetic relationships, our findings suggested that our collection might represent a considerable portion of the worldwide known diversity of lineages. To substantiate this hypothesis, we performed extensive sequencing and phylogenomic analyses.

Phylogenomics-based representation of the diversity of our collection
From our collection of 1,452 non-redundant A. baumannii isolates collected outside the hospital context (Suppl. Table 1), we chose at least one strain representing each OXA-variant for whole genome sequencing. Further, we selected few OXA-variants for the sequencing of larger sets of representative strains, in order to illustrate the diversity within these supposed lineages (e.g., OXA-104, -106, -126, 343, -374, -378, -431), and in addition, selected several strains representing each of the OXA-variants associated with the international clones (ICs) 1 to 8 (OXA-51/STPas 15, OXA-64/ST25, OXA-65/ST79, OXA-66/ST2, OXA-68/ST10, OXA-69/ST1, OXA-71/ST3, OXA-90/ST78)26. Moreover, due to the higher diversity of carbapenem-susceptible clinical isolates compared to carbapenem-resistant ones8, 15 carbapenem-susceptible human clinical isolates from Germany were selected according to their encoding OXA-variant, some previously described39. Finally, the collection was supplemented by a diverse set of 21 isolates covering veterinarian und human clinical isolates from a previous study40. Altogether, our study provides 401 novel genomes, of which 229 originated from white stork nestling isolates, 77 from soil and plant root samples, 2 from air samples above compost, 59 from diverse animal materials including earthworms, rodents and shrews, pellets from diverse bird species, egg shells, feathers and nesting materials, 7 from veterinarian samples, and 27 from human clinical samples. For comparison to the previously available diversity within the species A. baumannii, a generic protocol selected 413 additional genomes available within public databases (Suppl. Data 2).

The pan-genome of this collection includes 50,989 gene families, given a threshold of 90% amino acid sequence identity (Table 1). The phylogenomic tree based on the core genome set of 1,728 conserved genes representing 1.328 megabasepairs (Mbp) of A. baumannii genomic sequences is illustrated in Fig. 3. Especially remarkable is the share of deeply branching lineages, suggesting massive radiation early in evolution of the species. Most of the isolates differ by 18,000 to 23,000 single nucleotide polymorphisms (SNPs) between each other (Suppl. Data 3). While only a few OXA-variants represented multiple times in this study are apparently monophyletic, such as OXA-431 or OXA-68, most OXA-variants show phylogenetic clustering (see interactive microreact project at (https://microreact.org/project/sNy69Jjwpj1qBKGFrkGSMz-acinetobacterproject04nov2024) and Suppl. Fig. 16). In contrast, few OXA-variants show broad scattering, in particular the prototypic OXA-51 and some other OXA-variants (OXA-65, OXA-69, OXA-71) found associated with ICs26 (Suppl. Fig. 16). Another remarkable observation is that the collection of wildlife and environmental strains from Poland (n = 235) and Germany (n = 159), respectively, each covers a considerable portion of the worldwide known diversity (Fig. 4), pointing to an efficient mechanism of global spread. Moreover, human, animal and environmental isolates likewise spread all-over the phylogenetical tree (Fig. 5). Of particular interest are the many clades represented by environmental, animal and human clinical isolates, underpinning the relevancy of the One Health concept in regard to A. baumannii. This also includes representatives of ICs, namely IC8 (Suppl. Fig. 17). Additionally, IC4, IC5, IC6 and IC7 are now also represented by at least one isolate from wild animals, livestock or the environment. Distances between human clinical isolates and their related isolates from environmental or wildlife sources cover a broad range, approaching distances of 27 SNPs within the core genome in the case of a human clinical isolate from Canada related to a white stork isolate from Poland (both OXA-64), 35 SNPs between a white stork isolate from Poland and a human clinical isolate from Thailand (both OXA-433), or 48 SNPs between a human clinical isolate from Japan and another white stork isolate from Poland (both OXA-762) (Suppl. Data 3, Suppl. Tab. 3). These results called for a molecular clock analysis to allow for temporal placement of the genetic and geographical distances. Accordingly, we performed BEAST analyses41 on the OXA-126 clade (n = 34), revealing a mutation rate of 1.1345 × 10-6 (+/- 3.2 × 10−7) per site per year corresponding to ~1.5 SNPs per year within the core genome (range 1.08-1.93 SNPs per year). However, given the weak temporal signal of our dataset covering only 9 years of sampling, it is important to note that the molecular clock rate reported for IC1 is only slightly different with 1.5 × 10-6 substitutions per site per year41 (Suppl. Data 4). The most distant isolates within the OXA-126 clade, 13-291-1C and 19-Pos71-1, differ by 12,354 SNPs corresponding to 8236 years (range ~6400–11,440 years), pointing to a strong and constant selection pressure stabilizing the OXA-126 variant within the natural context. Applying this molecular clock on the species level, the most recent common ancestor of all members of the species A. baumannii likely existed only around 15,000 years ago. Accordingly, when applied to the closest relatives found between clinical and environmental/wildlife isolates in our study, those intercontinental pairs of isolates mentioned above share their most recent common ancestor (MRCA) only 18-32 years before present (and only 14-24 years if applying the IC1 rate). Further relationships of particular interest are listed in Suppl. Table 3.

Natural endowment with resistance genes and insertion sequence (IS) elements
Altogether, intercontinental spread between North America, Europe and Asia within a few years cannot be explained by the migratory activity of white storks which migrate between Europe and Africa. Rather, human activity, or natural mechanisms of airborne spread, need to be considered. Hence, we analyzed the genomes regarding their endowment with AMR genes and IS elements as indicators of anthropogenic selection pressure (Suppl. Data 2; Suppl. Fig. 18-19). Globally, the newly sequenced isolates harbor an intrinsic blaOXA-51-like gene (96% of the collection), a majority of which also harbor the blaADC gene in one of 43 variants (Fig. 3). Nickel resistance gene nreB and efflux pump gene amvA are also highly abundant. Moreover, most isolates also harbor the ant(3”)-IIa aminoglycoside and the abaF fosfomycin resistance genes, suggesting these also to be part of the intrinsic genomic endowment of the species. Only 12 isolates of our collection exhibit AMR gene counts above 3 (Suppl. Data 2). Six of these are human clinical isolates representing STPas 2, 106, 1357, 2402, and 2409, four are animal isolates of veterinary and livestock origin representing STPas 23, 241 and 866. Only two isolates of environmental and wildlife origin reach a count of 4 AMR genes (STPas 23 and 309). This indicates the comparably pristine habitats they originate from. Similarly, while IS elements are rarely enriched in environmental and wildlife-associated isolates, clinically related strains harbor an increased amount of IS elements (Suppl. Data 2, Suppl. Fig. 19). Moreover, few of our environmental or wild animal isolates harbored an interrupted comM gene, a configuration typically found in MDR lineages firmly established in the hospital42. Altogether, this suggests that our environmental and wildlife-associated isolates show little signs of anthropogenic selection pressure and indeed originate from pristine environments.

Environmental and clinical isolates differ considerably in the content of various functional gene categories
Assuming that there are additional differences between clinical and environmental isolates besides resistance genes and IS elements, we comparatively analyzed the genomes of 91 representatives of these groups regarding the contents of clusters of orthologous gene families (COG categories). These analyses revealed that genomes of clinical isolates are significantly enriched in several categories including replication, recombination and repair functions (Fig. 6).

Colonization of compost material and linkages to the fungal world
As our phylogenomic analyses suggested global spread of clonal lineages long before globalization of modern times, we considered various modes of dispersion independent of human activity. Our samplings in garden compost suggest that colonization of A. baumannii is associated with decomposition of fresh plant material, as A. baumannii vanishes after the feed of the compost is stopped. We hypothesized, this may point to attraction of A. baumannii throughout the early steps of decomposition, during which fungi are key players. We thus speculated that A. baumannii might start colonization together with fungal spores that could serve as a vehicle for local as well as global spread of A. baumannii. To prove an airborne spread of A. baumannii in a local setting we mounted open tubes in a distance of 50 cm above an active compost with the opening positioned downwards or the tube positioned vertically for 24 h. Then the tubes were closed, transported to the laboratory, filled with liquid medium and processed to cultivate A. baumannii. As this ad hoc setting instantly yielded isolates of A. baumannii from 4 out of 5 samples, we next designed experiments to study the colonization of sterilized fresh plant material deposited in the garden. The autoclaved plant material was deposited either directly in the garden in a distance of 5 to 10 m from the compost, or it was deposited in a sterilized plastic bag either open top and shielded from the soil or shielded from the top and accessible from the soil. We found that every setting (n = 4) was colonized within 2-3 weeks, indicating that colonization can occur not only via soil contact but also via the air.

Next, we tested if A. baumannii is capable of attaching to fungal spores using Aspergillus spp., which are known to play a key role during the first steps of plant decomposition and dominate within the compost setting43. We incubated spores from different Aspergillus spp. isolates with different strains of A. baumannii and observed a marked adherence of the bacteria to fungal spores (Fig. 7). Similarly, adherence to spores from different Penicillium spp. isolates was observed. Interestingly, adherence was not an ad hoc phenomenon but required 2 to 3 h of incubation to manifest and resulted in an inhibition of spore germination, a process that typically became observable after 5-6 h of incubation in our setting. Taken together, these experiments demonstrate that A. baumannii has the capacity to interact with fungal spores and to colonize new habitats via the air.

The where and when of sampling
Finally, we challenged our current model of the prevalence of A. baumannii in natural habitats. We chose to collect samples during late summer when positive rates at our hotspot sites peaked. We collected 20 samples from river banks within forests and 20 samples from river banks outside of forests across the Harz district, Germany. For both groups we chose sites we had never sampled previously and with indicators of nutrient-rich soil, such as deposition of decomposing plant material and growth of nettles (Urtica spp.), Impatiens spp. or Aegopodium podagraria. While 1 out of 20 (5%) forest samples was positive for A. baumannii, 6 out of 20 (30%) samples were positive from river banks outside of forests. The positive rate of 30% in selected soil samples thus is in the same range as that yielded from choana sampling of white stork nestlings.

Methods
We confirm that our research complies with all relevant ethical regulations.

Legal permissions
Collection of earthworms and insects was granted by the Landesamt für Umweltschutz, Sachsen-Anhalt, Halle/Saale, Germany (RL-0489-V and RL-0497-V). Collection of stork samples was granted by The Local Ethics Committee and The General and Regional Directorates for Environmental Protection in Poland: 2121/2028, 42/2018, 43/2010, 44/2015, 47/2017, 028/2019/P1, 348/2016, DOP-OZGIZ.6401.03.101.2012.km.2, WBA/11/Z/10, WPN.6401.109.2017.AP, WPN.6401.211.2017.MK, DZP-WG.6401.75.2022.WW, DL-III.6713.11.2018.ABR, WPN-II.6401.167.2015.AS.2, DZP-WG.6401.03.98.2016.km, DLP-VIII-6713-21/29762/14/RN. In accordance with the convention on biological diversity (Nagoya protocol) a certificate of compliance was issued to collect samples from Spanish storks (ABSCH-IRCC-ES-246587-1; Dirección General de Biodiversidad y Calidad Ambiental del Ministerio para la Transición Ecológica, Madrid, Spain). Small mammals were obtained according to legislation and approved by the relevant authorities. Trapping small mammals in Thuringia and trapping Norway rats in North Rhine-Westphalia was covered by permits from the district and State authorities, respectively (Thuringia: Landratsamt Unstrut-Hainich-Kreis file number 31109-16-301, Landkreis Eichsfeld file number 70.2-6-85/Kleinsäuger/Ausn.-01, Landratsamt Kyffhäuserkreis file number lll.3.3364.53.1/2016-11-01; North Rhine-Westphalia: LANUV file number 84-02.04.2015.A279).

Sample collection and processing
Agar gel medium transport swabs (COPAN 108C, HAIN Lifescience, Germany) were used for sampling of white stork nestlings (choana and cloaca), and COPAN 110C swabs were analogously used to sample dead rodents and shrew (tracheal and rectal sampling). Swabs were immediately transferred to Amies transport medium and stored at 4°C until direct plating on CHROMagarTM Acinetobacter (CHROMagar, France). CHROMagar Acinetobacter was prepared according to the manufacturer’s description without addition of the CHROMagar MDR supplement CR102. Pellet and soil samples were preincubated in minimal salt medium supplemented with 0.2% acetate for 5 h at 37°C prior to plating on CHROMagar Acinetobacter. Pellet samples for enrichment cultures were taken from the interior of pellets (~1 g) using sterile tweezers.

The compost was fed with plant remains from the garden, vegetable kitchen waste and egg shells which were deposited within a wooden frame (length/width/height: 0.8 m/0.8 m/0.5 m), the compost regime was as follows: feeding for twelve months beginning in spring (~March), afterwards rest period for 12 months without relocation and without stirring or turning, then spreading into garden, continuous sampling over the complete two years (~10 cm below surface (sampling with metal shovel and disinfection wipes); occasionally, sampling was applied with a boring rod of an effective length of 1 m (Bodenprobentechnik Peters, Germany); samples were taken every 10 cm.

Cat-captured rodents and shrews, deposited dead, were wrapped in aluminum foil or transferred into 50 ml tubes by the instructed collectors using gloves, stored at 4°C and transported to the lab within 24 h. In a biosafety level 2 laboratory, tracheal and rectal swab samples were taken under the safety cabinet while wearing gloves. Carcasses were inactivated by autoclaving.

Blood sample collection from white stork nestlings and processing
Two ml of blood was collected from each individual from the basilic vein. Due to the delicate walls of blood vessels in birds, it was decided to use a combination kit consisting of a 20 G intravenous cannula and a 10 ml syringe. The blood from the syringe was then poured into the K2-EDTA tubes to prevent the blood from clotting until laboratory analysis was conducted. Blood sample analysis included: red blood cell (RBC) number, hematocrit (PCV), and hemoglobin content (HGB). To determine the hematocrit, the blood was transferred to the capillaries, without anticoagulant, with a volume of 75 µl. The capillaries were centrifuged for 3 min at 12,000 g. The result was read on a hematocrit reader (MPW)110,111. Drabkin’s cyanomethemoglobin method112 was used to determine hemoglobin in nestlings. For this purpose, 20 µl of blood was added to a test tube containing 5 ml of Drabkin’s reagent (Stamar), thoroughly shaken and allowed to stand for 20 min. After this time, the tubes were centrifuged at 3000 RPM for 5 min to separate the membranes and cell nuclei of the hemolyzed erythrocytes. Supernatant (hemoglobin bound to Drabkin’s reagent) was poured into polystyrene cuvettes (MedLab products). Then, using a UV-VIS spectrophotometer (λ = 540 nm) (Prove 300, Merck), the absorbance of the solution against cyanomethemoglobin was measured. The RBC count was determined using a Bürker hemocytometer, with Natt and Herricks staining being used because of the presence of nuclei in the erythrocytes113. For this purpose, 20 µl of blood (dilution 1:200) was added to a test tube containing 4 ml of dye. The tubes were stoppered and continuously mixed with a roller mixer for 15 min. After this time, 20 µl of the mixture was placed in the hemocytometer chamber and allowed to stabilize for 2 min. Red blood cells were counted in 80 small squares at 400x magnification (MT5300, Meiji). The number of white blood cells was determined analogously to the number of red blood cells. The only difference was that blood cells were counted from the entire area of the Bürker counting chamber (magnification 400x (MT5300, Meiji)). Mean corpuscular hemoglobin (MCH) and mean corpuscular volume (MCV) was calculated based on RBC number, PCV, and HGB content values114.

Bacterial species identification
Species determination of isolates recovered from CHROMagar Acinetobacter was based on PCR detection of blaOXA-51-like115, partial 16S rRNA gene sequencing116, and partial rpoB sequencing using primers Ac696F and Ac1598R117. To determine blaOXA-51-like variation the coding region was fully sequenced26. New OXA-51 protein variants were assigned and deposited at GenBank (Suppl. Table 3).

Illumina sequencing
Libraries for Illumina short read sequencing were prepared from 1 ng of extracted DNA utilizing the Nextera XT DNA Library Prep Kit according to the manufacturer’s recommendations (Illumina Inc., USA). Sequencing was carried out in paired-end (2 × 300 base pairs) on a MiSeq benchtop instrument. Quality control included the removal of adapter sequences, minor contaminations and short contigs below 700 bp in length through an in-house pipeline. The whole genome shotgun project of 401 isolates has been deposited at GenBank under the BioProject accession PRJNA862736.

Genomic reconstruction and gene-marker analysis
Short-read DNA fragments were successively utilized to re-construct high-quality genomes of the isolate collection using the de novo SPAdes assembler (v3.11.1)118. To assess the quality of the genome assemblies, wrapper stats.sh of BBMap (v38.22)119 was utilized. Genomic completeness was further assessed using checkM (v1.2.2)120. On average, completeness and contamination of the genomes were 99.9% and 0.92%, respectively. The overall completeness of the genomes ranged from 86% to 100%, with contamination detected by checkM ranging from 0% to 37%. Overall, 824 isolates exhibited high completeness (>98%) and low contamination (<5%), indicating high-quality genomes for further analysis. A full overview of assembly statistics is listed in the supplementary material (Suppl. Data 5). Reconstructed genomes were then subjected to in silico MLST profiling using the mlst tool (v2.23.0) (https://github.com/tseemann/mlst) with both the ‘Oxford’ and ‘Pasteur’ schemes for A. baumannii. A total of 246 and 143 novel sequence types were identified for the Oxford and Pasteur MLST schemes respectively. A total of 346 novel isolates containing one or more novel allelic sequences were submitted to the isolate database of PubMLST (https://pubmlst.org/). Additional AMR gene profiling was conducted through the AMRFinderPlus pipeline (v3.11.26) (https://github.com/ncbi/amr), utilizing the NCBI Antimicrobial Resistance Library for AMR (dated 2023-07-05) with default values of 80% for identity and coverage, respectively. Next, IS element characterization was performed via the ABRicate software (v1.0.1) (https://github.com/tseemann/abricate), utilizing a custom database based on the ISfinder collection121 (dated 2023-07-05) with 90% identity and coverage. Finally, novel blaADC variants and the continuity of the comM gene were further investigated through a BLAST-based custom Python script. A total of 32 novel blaADC variants were identified and subsequently submitted to NCBI.

Functional Annotation
Genome assemblies were annotated using Bakta (v1.9.4)122 with default settings. The analysis included automated prediction of coding sequences (CDS), transfer RNA (tRNA), ribosomal RNA (rRNA), and pseudogenes. Functional annotations were derived from the UniProtKB and RefSeq database, as integrated within the Bakta pipeline. The CDS were extracted from the gbff output and translated using gbseqextractor (v0.0.5)123 which was then used as input for eggNOG-mapper (v2.1.12) (https://github.com/eggnogdb/eggnog-mapper) for functional prediction against the eggNOG DB (v5.0.2). We then compared the identified counts of COG categories as defined by eggNOG associated with genes per sample between environmental and clinical isolates (Fig. 6 and Suppl. Fig. 22). Hereby, 91 out of 240 clinical samples were randomly subsampled to match the amount of environmental samples evaluated within this study. One-sided Wilcoxon tests were performed on each category to assess statistical differences between both groups.

Phylogenetic characterization
Open reading frames (ORFs) predicted by Prokka (v1.13)124 were subsequently used as input for Roary (v3.12.0)125 in order to conduct pan-genome analyses. Computed core genes were subsequently extracted, aligned and concatenated using default settings. The resulting alignment was then utilized to calculate a maximum likelihood-based phylogeny with RAxML (v.8.2.10)126 using 100 bootstraps under the assumption of the GTR-gamma DNA substitution model. ClonalFrameML (v1.11)127 was then used to correct for recombination events and phylogenetic groups were identified through Bayesian Analysis of Population Structure (BAPS). Here, we utilized BAPS with hierarchical clustering as implemented in the R package RhierBAPS (v1.0.1)128. Grouping of the accessory genome was further assessed via t-distributed stochastic neighbor embedding (t-SNE), in order to cluster the data through a range of values for perplexity (p = 5, 10, 20, 50), the results of which were visualized using the microreact platform129. SNP distances between individual samples were computed through the snippy pipeline (v4.6.0) (https://github.com/tseemann/snippy).

Temporal signal analysis
Temporal signal analysis was utilized to evaluate whether the samples evolve at a specific rate over an evolutionary time period. To assess this, a root-to-tip regression of genetic divergence was performed for the OXA-126 group against their sampling dates, using the previously generated maximum likelihood tree with a GTR-gamma DNA substitution model within TempEst (v1.5.3)130. The best fitting tree contained the outgroup A. baumannii 14-9D1. Upon the removal of visible outliers, the correlation coefficient was identified to be 0.25, with an R2 value of 0.0655, indicating an overall weak temporal signal. To compensate for this, the following molecular clock analysis was based heavily on previously defined parameters for IC-141.

Molecular clock analysis
In order to assess the molecular clock of A. baumannii, a xml file was configured using BEAUTi and subsequently utilized for BEAST (v2.5.0)131 analysis. BEAST was run on the OXA-126 group, containing detailed sampling dates of 30 isolates. The run was allowed to continue for 50 million iterations, sampling from the posterior every 1000st iteration. We inferred the temporal phylogeny for OXA-126 under a strict clock setting (i.e., normally distributed rate variation over the tree). The strict clock was set to utilize a log-normal prior on the clock rate, with a mean of -5 and standard deviation (sd) of 1.2541.

Isolation of fungi and interaction studies with A. baumannii
Fungal isolates were obtained from environmental samples previously tested positive for A. baumannii using the following protocol: Samples (e.g., 1 g of soil) were resuspended in 10 ml of minimal salt medium supplemented with 0.2% acetate (see above) and 100 µl suspension before being spread on Kimmig fungal agar (132, Becton Dickinson, Heidelberg, Germany) supplemented with 80 mg/l chloramphenicol (Kimmig agar). After 2-3 days of incubation at 27°C, fungal colonies were selected and spread on Kimmig agar several times until considered pure cultures after macroscopic and microscopic inspection. The absence of bacterial contamination was verified via colony PCR using global 16S rRNA gene primers116. Provisional species determination of fungal isolates was performed by sequencing of the rRNA genes and ITS (internal transcribed spacer)133,134. Aspergillus spores were harvested after growth of fungi on Kimmig agar for 3 days at 34°C. To this end, the plate was flooded with 5 ml of sterile phosphate-buffered saline (PBS) supplemented with 0.1% Triton X-100, followed by soft panning. Subsequently, the suspension was removed with a pipette while avoiding contact with the fungal colonies, aliquoted and frozen at -80°C until further use. For seeding into 24-well plates, frozen spore suspensions were thawed, diluted by a factor of 50 into LB medium and 300 µl deposited into each well. The A. baumannii strains were cultured in LB medium overnight at 37°C, diluted 1:50 into fresh medium and further cultured until an optical density (OD) 600nm of 1 was reached. For the spore adhesion assay, the bacteria were diluted 1:200 into LB medium and 300 µl were added to the spore seeding of a well. After 2-4 h at 37°C, the medium was removed from the wells and replaced twice with fresh medium to filter any non-adherent bacteria before inverted microscopy was performed. For the spore germination inhibition assay, bacterial suspensions with an OD 600nm of 1 generated as above were diluted 1:2000 into LB medium, of which 300 µl were added to the spore seeding of a well. Microscopic evaluation of spore germination inhibition was conducted after 5–7 h at 37°C. For scanning electron microscopy (SEM), ThermanoxTM coverslips (ThermoFisher Scientific) were placed in 6-well plates, the incubation volume was reduced to 300 µl with seeding and incubation as above at 37°C and fixed after 2 h in a solution of 2.5% glutaraldehyde, 1.0% paraformaldehyde in 50 mM HEPES buffer for 24 hrs. All samples were then washed in 50 mM HEPES, dehydrated in 30, 50, 70, 90, 95, 100% ethanol, critical point dried, mounted on aluminum stubs, sputter coated with a 10 nm layer of gold-palladium and finally examined in the SEM (ZEISS 1530 Gemini, Carl Zeiss Microscopy GmbH, Germany) operating at 3 kV using the in-lens electron detector.

GIS-based analyses
All spatial data were analyzed using geographic information system (GIS) tools (Quantum GIS Software, 2010). Feeding grounds for all white stork nests were set as an area around the nest with a 3.5 and 4 km radius, respectively27. Habitat structures of all feeding grounds were determined using CORINE Land Cover 2012 types, including arable land, pastures, heterogeneous agricultural areas, forests, total area of water reservoirs and total length of rivers.

A total of 295 nests were analysed (50 from the southern part of Lubsz voivodeship near Zielona Góra city, hereafter Zielona Góra; 95 from the southern part of Greater Poland voivodeship near Leszno city, hereafter Leszno; 150 from Opole voivodeship near Opole city, hereafter Opole). From these 295 nests during breeding seasons 2015–2018, we analysed 454 broods (mean number of broods per nest was 1.5). A total number of 1080 chicks was analysed. First, we tested for spatial autocorrelation with Moran’s local indicator135. As dataset we used geographic position of 295 nests and whether we isolated or not A. baumannii in nests during four years of study. We found no spatial autocorrelation (Supplementary Table 5, Suppl. Fig. 23). The spatial autocorrelation was computed using SAM software136.

For each nest we obtained several environmental variables in a radius of 3.5 km and 4.0 km around the nest. We determined the landscape variable according to Corine land cover classification137 as proportion to total buffer area: artificial surface (CLC_1), arable land (CLC_2.1), pastures (CLC_2.3), heterogeneous agricultural area (CLC_2.4), forests (CLC_3.1), water reservoirs area (CLC_5.1.2) and water courses length (CLC_5.1.1). The GIS data were processed by Quantum GIS Software138. Before analysis, the environmental variables were log-transformed in order to minimize effects of detached observations and skewed distributions.

We checked whether there is multicollinearity between those environmental variables by using variance inflation factor. We found that there is collinearity between arable land and forest areas. There is strong negative correlation between them (Pearson correlation for radius 3.5 km: -0.72, p < 0.001 and for radius 4.0 km: -0.75, p < 0.001), so we excluded forest variable from the analysis. It should be noted when interpreting the results that high area of arable land indicates low area of forest.

Precipitation data were obtained from the Institute of Meteorology and Water Management National Research Institute, Warsaw, Poland. Yearly sum of rainfall for each nest was calculated based on assignment to the closest meteorological station within a 3.5 km radius of the nest.

We compared the environmental variables within 3.5 km buffers around nests. We then compared regions using Kruskal-Wallis tests with results shown in Supplementary Table 6. Zielona Góra and Leszno had significantly more extensive agricultural use (mostly pasture) than Opole. On the other hand, Opole had the highest proportion of arable area, followed by Leszno and Zielona Góra. The proportion of forests was highest in buffers around nests in Zielona Góra.

Our dependent variable was the proportion of the number of positive chicks to total number of chicks in the broods. We used a generalized linear model with a binomial error distribution. Because the broods measurements were repeated in different years we used a mixed model with nest ID and year of study as random factors of the study. As independent variable we used above mentioned environmental variables, precipitation and mean age of fledglings in the broods (age was estimated according to size of beak) and region (as factor variable with three levels: Zielona Góra, Leszno, Opole). We started from the global models with the interaction of region factor with all other environmental covariates. The final model was selected using backward stepwise removal of nonsignificant effects of interactions (P < 0.05)139.

For the statistical analyses we used R software140 and for mixed modeling approach we used lme4 package141. Graphics were made using the packages ggplot2 v.3.4.4142 and ggeffects v.1.3.1143.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "Summary\nThe authors conduct what might be the most comprehensive study about isolates of A. baumannii from non-human sources, which is very welcome. The authors collected 1450+ non-redundant isolates from many different sources and sequenced 401 genomes. This is a huge amount of work both in terms of data and analyses, which provides novel and interesting insights into the ecology (in particular the habitat) of A. baumannii and the importance of the non-human populations of this species. However, some important issues should be addressed in a revised version of the manuscript. There are two relevant technical aspects the authors want to pay attention to (see major comments 1 and 2), other two of background context (major comments 3 and 4) and some non-major issues (final major comment and minor comments) about the structure and tone of the manuscript.\nMajor comments\n1. There are some important technical details regarding the genomes newly sequenced. Please, run CheckM on your genomes and report the completeness and contamination values. Also, please add to Supplementary Table 4 the sequencing statistics (N50, coverage, number of genes, etc.) of the new genomes. These two aspects are relevant to have an idea of the quality of genomes sequenced by the authors. In the results, you want to add one or two sentences about the quality of the genomes sequenced (sequencing statistics, completeness and contamination).\n2. Maybe the most salient issue is with the molecular dating analysis. An essential aspect of any molecular dating analysis is to determine if the data has a temporal signal (the mol dating analysis is valid only when there’s such a signal). Did the authors check if their data has a temporal signal? They want to do that using TemEst or any similar program. Please report the value of the regression of dates of sampling against the root-to-tip distances. Secondly, why did the authors choose a strict molecular clock? A strict mol clock is not very realistic, biologically speaking. A relaxed molecular clock model is a more sensible option. Third, some information is missing regarding the setting of the substitution model. Please mention the DNA substitution model used for the analysis and other parameters (Proportion of Invariant sites, Gamma category counts, etc.). Finally, but as relevant as the first point, ideally the analysis should be run twice or more to evaluate consistency (similar values should be got for the time to the most recent ancestor (tMRCA) in both runs). Please evaluate the trace plot for the tree likelihood and check if there is good mixing in the tree likelihood. Also, please make sure the effective sampling size of tMRCA is higher than 200 in both runs. The confidence of the mol dating analysis is very important given the statement made by the authors that early adaptive radiation in A. baumannii occurred around the Neolithic. Of note, even if there is a weak or lousy temporal signal (and thus the Mol dating analysis is not reliable), I think the rest of the data and analyses in this study are very valuable.\n3. There are a couple of recent phylogenomic studies that have studied non-human A. baumannii isolates that are relevant to the subject of this paper. A recent genomic and phenotypic study found A. baumannii in agricultural surface water, urban streams, and even milk tanks (ref A below). Another recent study found a significant diversity of novel clones of A. baumannii in grass (ref B). It would be interesting to know if the OXA-51 allelic variants found in ref B are similar to the OXA-51 variants found by the authors in their soil and plant isolates. This could indicate that certain clades are preferentially found in soil/plant habitats\nRef A\nhttps://pubmed.ncbi.nlm.nih.gov/38440986/\nRef B\nhttps://pubmed.ncbi.nlm.nih.gov/37439781/\n4. Given that most molecular epidemiology studies of A. baumannii use MLST as the standard approach, please state how many new STs (either new combinations of known alleles or even new allelic variants for some loci) were found as per both Pasteur and Oxford MLST schemes. Also, considering the 125 novel OXA-51 variants (line 306 and listed in Supp Table 3) please state the source of the isolates (soil, bird, etc.) and briefly mention which source has most of the novel variants.\n5. Finally, some edits can enhance the manuscript. For one, the abstract could be more focused. Also, although the discussion is a great debate on many aspects of the ecology (and even evolution) of A. baumannii, it is a bit of a whopper. It feels rather long and some points, although very interesting, are not directly connected to the findings of the paper. For instance, the section about the comparison of the population structure of A. baumannii and P. aeruginosa. Regarding the methods, the sections “garden compost” and “Collection of rodents, rats and shrews” can be included in the first section “Sample collection and processing”.\nMinor comments\nI think the data linking A. baumannii dispersion and fungi are interesting but not conclusive. Maybe the authors want to tone down this a little bit.\nLine 63: instead of “around 400” please give the actual number.\nLines 66-68: maybe you want to mention how many different types of sources you sampled.\nLine 99: provide the actual number of genomes sequenced.\nLine 141-143: This a clear case of reverse Zoonosis, If I were you, I would say explicitly that.\nLines 262-266: how many of those OXA-51 variants were novel?\nLine 330: “unique genes” can be misleading; “gene families” or “homologous groups” are better options\nLine 624: issue is misspelled.", "reviewer_2": "The manuscript, titled \"On the ecology of Acinetobacter baumannii – jet stream rider and opportunist by nature,\" aims to describe the ecology of A. baumannii by analyzing 1,450 isolates, of which approximately 400 were genome sequenced. It is an interesting and well-written manuscript; however, several points need to be addressed before it is ready for publication.\nSome general and specific aims are:\n- My main concern is the claim that this work has resolved the ecology of A. baumannii by analysing around 1500 strains, of which approximately 400 were sequenced. Given the widespread distribution of A. baumannii species in the environment, resolving the ecology of Acinetobacter baumannii requires a geographically evenly distributed sampling approach, as there might be several additional hidden niches in the environment, including other animals, etc., that play a pivotal role as reservoirs of strains. I believe the conclusions drawn are biased towards the samples available for this study (i.e. storks and specific geographical regions). Conclusions are valid for the regions studied NOT at the global level. I am concerned because even including the publicly available GenBank genomes also does not normalise the sample biases as only a tiny fraction of the genomes available in GenBank are from environmental samples. I think to address this, the tone and working around this needs to be modified throughout the text.\n- Lines 112-115: Not sure about this section of the conclusion, \"Taken together, the intrinsic resistance endowment and potential to acquire antibiotic resistance can be explained by coevolution with antibiotic-producing fungi and other microorganisms within the soil,\" as I think it includes a lot of generalisations. For example, parts of the widespread antimicrobial genomic islands found in A. baumannii international clones have been shown to have been acquired from other clinically relevant bacteria such as Salmonella and Enterobacterales. Overall, I believe this study does not include substantial evidence to support this claim.\n- Lines 138-140: Please include the link in the reference list.\"\n- Line 158: Please clarify why the smaller scale? Was the sample size determined through statistical methods? Please clarify!\nLines 169-172: Please include some numbers (e.g. colonisation, mortality rate etc.).\n- Line 171: Did you identify increased white blood cell counts in all colonised individuals? Please clarify.\n- Line 176: In the section \"We collected pellets from selected nests\", how did you ensure that the samples are genuinely from pellets and nothing else (e.g. egg shells OR feathers)? Please clarify!\n- Line 178: This is an interesting observation, the fact that no pellets was positive earlier than May-April, could this be a correlation with the rain season in the area and that pellets might have washed away? Could you please elaborate on this?\n- Line 186: Could this be due to the seasonal conditions?\n- Line 248: Please specify which regions?\n- Line 317-318: Please include the sequence type number (ST#) representing each of the international clones. (e.g. OXA-66, ST2 etc.).\n- the blaOXA-51-like and blaADC are considered part of the core genome in A. baumannii, so can you elaborate on the sequence type(s) of those that lack these genes? And is there a correlation between the lack of both genes (i.e. when blaADC is absent blaOXA-51-like is also absent)?\n- Lines 381-389: Please include the sequence type(s) of those with AMR genes. This is important given that all the significance of A. baumannii is in the context of AMR.\n- Line 402: Please be specific here, how many isolates were recovered? Was a sufficient number of strains recovered to support the claim (i.e. airborne spread)?\n- Line 412: in all samples tested? Please clarify!\n- Line 436: This is not accurate, several publications including genome sequence data have documented the isolation of IC1 (ST1) from natural environments. Please fix this.\n- Lines 441-461: I can see a lot of merits in comparing the population structures of A. baumannii and Pseudomonas aeruginosa, however, it seems very out of place in the context of this study given the completely different evolutionary stories and global/clonal expansions that these two genera have experienced. Please remove this section.\n- The discussion is very long, the manuscript will benefit more from a more concise discussion.", "reviewer_3": "Dear Authors,\nI enjoyed and spent some time reviewing your manuscript ‘On the ecology of Acinetobacter baumannii – jet stream rider and opportunist by nature’\nYour manuscript is conspicuous, well clearly presented and participated and it contributes to greater understanding the ecology and evolution of A. baumannii in natural habitats, that to date is really poorly understood outside the hospital.\nMy compliments for the supplementary datasets that are rich in content and very useful for data visualization and interpretations.\nAll the comments and suggestions I have made are of a minor nature and I would be happy for the manuscript to be accepted for publication in Nature Communication once these have been appropriately addressed point by point as follows:\n- Why authors identified white storks as a model system to study the ecology of A. baumannii? Why not other wild or synanthropic birds?\n- Considering the huge epidemiological study, I wonder if the authors also found other pathogens of interest from the samples analyzed.\n- Regarding the Diversity in terms of OXA-51 variants collected from Poland and Germany and the Phylogenomics-based representation of the diversity, I agree with the choice of at least one strain representing each OXA variant for whole genome sequencing.\n- Very useful and well done the ‘Phylogenetic distribution of strains carrying selected variants similar to OXA-51’ illustrated in Suppl. Fig. S14 and deepened in the microreaction project\nRegarding ‘Compost, soil, rhizosphere and the forest paradox’ (line 257), really interesting the sampling of the soil originating from a single compost across a period of seven months that yielded 86 isolates with 20 different variants of the OXA-51 family, and earthworms within the same compost that yielded 10 different variants of OXA-51.\nInstead, (line 298-301) although very interesting as data, considering the low number of samples examined, I would go with more moderation in stating that A. baumannii can thrive along streams and rivers outside of forests. The decomposition of plant material by fungi would seem to lay the foundation for the proliferation of A. baumannii. You could clearly assume that decomposition of plant material by fungi would seem to appear to set the stage for proliferation of A. baumannii.\n- Line 398: Regarding the Colonization of compost material and linkages to the fungal world, the experiment is not so clear to me. How many tubes did you mounted? How many trials have you conducted? Please specify\n- Line 845: Collection of rodents, rats and shrews. How many cat-captured rodents have you collected? Particularly, can you describe in detail the capture procedure and whether there are laws and permits (especially for rats) in the country of collection (in addition to those already mentioned) about the associated biological risks.\nFIGURES\n- Figures 3, 4, 5 are obviously simply representative and at first glance difficult to understand especially because of the amount of isolates represented graphically. The link https://microreact.org clearly describes and represents this. But to make the graphic part of the figures, especially 4 and 5, more immediate and understandable, please divide each of the figures into A and B and briefly describe the individual parts before directing the reader to the link.\n- Please enlarge the central part of Fig. S1 ‘Spatial correlogram’ to make it more readable\n- I suggest putting letters A, B, C... for each Supplementary Figure consisting of two or more figures and where the legend is not prorply specified, to make them even clearer (E.g. Suppl. Fig. S18)\nHope my review could help you to improve your work.\nRegards", "reviewer_4": "In this study, Wilharm et al. investigated multiple aspects of ecology and distribution of Acinetobacter baumannii. Authors have dedicated a quite a bit of resources for this study and provide some novel insights into the ecology and evolution of A. baumannii complex. However, I find the current form of the manuscript, presentation of the results and the analyses do not justify the effort put into this study and data generated by the authors. Also, there are few concerns related to methodology of this study. Below I highlight my major concerns and comments.\n1. Structure and the organization of the manuscript: The current written style of this manuscript includes a collage of mini experiments, at times seems very fragmented.\na. The introduction itself is not giving a proper background to justify why it is important to conduct the study that authors have done, and I don’t think “we intended to unravel the ecology of these bacteria in their natural habitats” is a good argument. I would suggest re-writing a proper introduction highlighting the thought process and major gaps that authors are anticipating on filling with their work. Also, it is a bit weird to see the first citation in the introduction is the 90th citation in the reference list.\nb. Detailed results related to isolation information of the A. baumannii can go into the supplementary and just provide a summary of what was isolated.\nc. Authors need to analyze the genomes bit more thoroughly. This is the most interesting dataset of the study and there are many analyses that authors can do to identify the ecology and evolution of this species complex (see my major point 3).\nd. Supplementary: There are two supplementary files and one includes the figures and the other includes a mix of things which also have some interesting results on GIS based analyses. I think these two documents need to be merged (one of this document even have a figure 1). I also think the GIS analyses need to be highlighted in the main text results and to show whether the number of isolates have an association with the proximity to human activity. This is what I saw with your transect study, where you only isolate A. baumannii after the village (Fig S12).\ne. Discussion is extremely long and highly speculative, while making huge statements without adequate support. I think authors need to first define their objectives and make the discussion more streamlined and avoid jargon.\n2. Uneven sampling:\na. Authors have done an amazing job with sampling White storks in Germany and Poland. Then they have attempted to sample other bird species (grey-herons, kestrels and lack storks), which have a very small number of individuals compared to the white stork samples and only sampled the cloaca of the birds. This raises the question whether this uneven sampling led to the observed lack of isolation of A. baumannii from other bird species. Second, in White storks it seems like A. baumannii is isolated more from choanae and for other species authors took cloacal samples. Again, this makes me wonder if authors sampled choanae in other birds whether they would have gotten different set of results.\nb. Autoclaved compost test: This was an interesting approach but why did you only do this in a garden. I think it would make more sense to conduct this experiment both in the garden setting, semi-natural area and in the forest environment. This way you will have the opportunity to test your hypotheses related to airborne nature of dispersion of A. baumannii away from human settlements.\n3. Genome analyses: I feel like this is the most important dataset of the study and authors just looked at the surface of this dataset.\na. It would be good to show genes/gene families/SNP differences between environmental isolates and clinical isolates in a figure (it should be a main figure).\nb. Given that authors have identify clades of environmental, animal and clinical isolates I think authors should conduct positive selection analyses to investigate if there are certain genes under positive selection when A. baumannii become a hospital strain. Moreover, this will allow you to identify if there are particular genes that show converged positive selection when strains become clinical isolates across the clades.\nc. Molecular clock analysis: What Acinetobacter species did the authors used as the outgroup when doing this analysis? Also given the diversity and the geographic distribution of the genomes you have, you should conduct ancestral state reconstruction to see if you can point our a potential origin of the radiation of this species clade.\n4. Tracing the food sources: I think the proper way to do this is first to conduct metabarcoding of the pallet material. Then identify food items common in pallets with A. baumannii isolates and then collect the wild individuals (slikewhat you did with earthworms) and then see if those wild diet items carry A. baumannii.\n5. I also wonder why authors did not conduct whole microbiome sequencing of their samples to confirm that isolation actually represent the presence of A. baumannii. This is because the isolation alone might not give the full picture of the presence of A. baumannii in a given sample. Have you done any preliminary work to make sure you always identify A. baumannii from samples when the bacteria are present?\nMinor comments\nL127: Citations are weird. Please double check.\nL154: Is “average rate of 25%” mean they were isolated from 25% of samples. In that case isn’t it the prevalence?\nL164: Terminology for birds is “Cloacal” not “Rectal”.\nL233: Can you split the isolate numbers of the bacteria according to different earthworm genera?\nL254-256: I think this is an issue of sampling depths and sampling types.\nOxa-51 variants: What is your cutoff value to call a strain as a different variant? Is it 1bp difference? Also, what is the length of this segment?\nL330-331: Can you summaries the geographic distribution of your genomes? This will help readers to easily see if there are geographic biases in the isolates you used in this study.\nL413: Is the incubation temperature you used natural (e.g., environmental temperature)? Also, if the adherence of A. baumannii inhibit the germination of fungal spores, can that also impact other properties of the spores (like the dispersal ability)?\n441-461: Very weird section to start your discussion with, given that the study was not a comparative study between P. aeruginosa and A. baumannii.\n462-501: This part is also very speculative, and I struggle to find how this relate to authors findings, except for that authors show the ability of the bacteria to adhere to fungi. I think if authors want to discuss more “Evidence for interrelatedness of A. baumannii life cycle with the fungal world” they need to conduct more experiment to properly show the claims they make here.\nL509: Basidiomycota is a very broad and diverse division of fungi and don’t think using this is a good argument here.\nL541-585: I am not sure how your findings support A. baumannii uses air currents to travel 1000s of kms between continents. Maybe I missed something here?\nL559-560: This I don’t underatand. There seem to be an association with your isolates and presence of humans and then here you say “All in all, there is no evidence that the observed global pattern of distribution of environmental A. baumannii is caused by human activity”. You have data to test this!\nL660-668: This section seems like it is repeating things. Please add this section to other corresponding sections.\nL809: Did the incubation happened at room temperature?\nL822-828: Results for this methods section is not in the “Results” section. I think it is very interesting and should be in the main paper.\nFigures: Please provide better legends for the figures. The interactive link is cool but you need to provide enough information these figure legends to guide reads (figures and legends should be able to stand alone). Also please provide information on the color codes associated with phylogenies.", "reviewer_author_response": "Point-by-point response\nREVIEWER COMMENTS\nReviewer #1:\nSummary\nThe authors conduct what might be the most comprehensive study about isolates of A. baumannii\nfrom non-human sources, which is very welcome. The authors collected 1450+ non-redundant\nisolates from many different sources and sequenced 401 genomes. This is a huge amount of work\nboth in terms of data and analyses, which provides novel and interesting insights into the ecology (in\nparticular the habitat) of A. baumannii and the importance of the non-human populations of this\nspecies. However, some important issues should be addressed in a revised version of the manuscript.\nThere are two relevant technical aspects the authors want to pay attention to (see major comments\n1 and 2), other two of background context (major comments 3 and 4) and some non-major issues\n(final major comment and minor comments) about the structure and tone of the manuscript.\nMajor comments\n1. There are some important technical details regarding the genomes newly sequenced. Please, run\nCheckM on your genomes and report the completeness and contamination values. Also, please add\nto Supplementary Table 4 the sequencing statistics (N50, coverage, number of genes, etc.) of the\nnew genomes. These two aspects are relevant to have an idea of the quality of genomes sequenced\nby the authors. In the results, you want to add one or two sentences about the quality of the\ngenomes sequenced (sequencing statistics, completeness and contamination).\nWe fully agree with the reviewer that information regarding genome quality is of essence\nto evaluate any potential impact on downstream analyses. To provide this, we added a\nsection on genome assembly assessment (lines 672-679) using the tools BBMap and checkM.\nA majority of the assessed genomes was of high quality (high completeness, low\ncontamination) and thus suitable for the presented downstream analyses. A full overview\nof the assessed statistics is listed in new Suppl. Table S9. Completeness and contamination\nvalues have also been introduced into Suppl. Table S4 as requested.\n2. Maybe the most salient issue is with the molecular dating analysis. An essential aspect of any\nmolecular dating analysis is to determine if the data has a temporal signal (the mol dating analysis is\nvalid only when there’s such a signal). Did the authors check if their data has a temporal signal? They\nwant to do that using TemEst or any similar program. Please report the value of the regression of\ndates of sampling against the root-to-tip distances. Secondly, why did the authors choose a strict\nmolecular clock? A strict mol clock is not very realistic, biologically speaking. A relaxed molecular\nclock model is a more sensible option. Third, some information is missing regarding the setting of the\nsubstitution model. Please mention the DNA substitution model used for the analysis and other\nparameters (Proportion of Invariant sites, Gamma category counts, etc.). Finally, but as relevant as\nthe first point, ideally the analysis should be run twice or more to evaluate consistency (similar values\nshould be got for the time to the most recent ancestor (tMRCA) in both runs). Please evaluate the\ntrace plot for the tree likelihood and check if there is good mixing in the tree likelihood. Also, please\nmake sure the effective sampling size of tMRCA is higher than 200 in both runs. The confidence of\nthe mol dating analysis is very important given the statement made by the authors that early\nadaptive radiation in A. baumannii occurred around the Neolithic. Of note, even if there is a weak or\nlousy temporal signal (and thus the Mol dating analysis is not reliable), I think the rest of the data and\nanalyses in this study are very valuable.\nFollowing the suggestion of the reviewer, we performed temporal signal analysis on the\ndata through TempEST. As the reviewer correctly estimated, the detected temporal signal\nof the data set was overall rather weak, potentially impacting the accuracy of the time-\nbased analysis. However, we believe that the novelty of the results outweighs the initially\ndetected weak signal. Thus, to compensate for the lowered temporal signal, we based our\nfurther parameter selection on previously published methods utilized for the research on\ninternational clone 1, including the decision to adapt a strict molecular clock and an GTR-\ngamma DNA substitution model (Holt et al., 2016). To address the reviewer’s question, we\nhave added a section containing details of the temporal signal analysis (lines 718-726). We\nconsequently toned down regarding our claims based on the molecular clock but kept the\nmain conception considering the overall evidence. Moreover, we critically mentioned the\nweaknesses in the “Limitations” section (lines 627-631).\n3. There are a couple of recent phylogenomic studies that have studied non-human A. baumannii\nisolates that are relevant to the subject of this paper. A recent genomic and phenotypic study found\nA. baumannii in agricultural surface water, urban streams, and even milk tanks (ref A below). Another\nrecent study found a significant diversity of novel clones of A. baumannii in grass (ref B). It would be\ninteresting to know if the OXA-51 allelic variants found in ref B are similar to the O.XA-51 variants\nfound by the authors in their soil and plant isolates. This could indicate that certain clades are\npreferentially found in soil/plant habitats\nRef A\nhttps://pubmed.ncbi.nlm.nih.gov/38440986/\nRef B\nhttps://pubmed.ncbi.nlm.nih.gov/37439781/\nWe have compared our dataset with that of the two recent publications. On the basis of\nMLST which is more reliable compared to OXA-51-like variants alone, we identified 3 STs of\nRef. A that were also represented in our dataset (2 stork isolates, 1 earthworm isolate), and\ninsufficient for a discussion on clades preferentially associated with soil/plant habitats even\nif we assume the stork isolates to stem from the same habitats. However, we have briefly\nmentioned and cited the references to further illustrate the One Health relevance (see lines\n617-620).\n4. Given that most molecular epidemiology studies of A. baumannii use MLST as the standard\napproach, please state how many new STs (either new combinations of known alleles or even new\nallelic variants for some loci) were found as per both Pasteur and Oxford MLST schemes. Also,\nconsidering the 125 novel OXA-51 variants (line 306 and listed in Supp Table 3) please state the\nsource of the isolates (soil, bird, etc.) and briefly mention which source has most of the novel\nvariants.\nThe requested information on the number of new sequence types (ST) has now been added\nto the manuscript (see lines 681-683) and the Suppl. Table 3 has been amended to give an\noverview on the sources of isolates with novel OXA-51 variants. Most of them were\nassociated with isolates from white stork nestling as has been now stated in the manuscript\n(see line 305).\n5. Finally, some edits can enhance the manuscript. For one, the abstract could be more focused. Also,\nalthough the discussion is a great debate on many aspects of the ecology (and even evolution) of A.\nbaumannii, it is a bit of a whopper. It feels rather long and some points, although very interesting,\nare not directly connected to the findings of the paper. For instance, the section about the\ncomparison of the population structure of A. baumannii and P. aeruginosa. Regarding the methods,\nthe sections “garden compost” and “Collection of rodents, rats and shrews” can be included in the\nfirst section “Sample collection and processing”.\nThe abstract is now considerably condensed (see lines 84-103). As requested, we have\nremoved the comparison of population structures of A. baumannii and P. aeruginosa and\nfurther streamlined the discussion outlined above. We changed the Methods section\naccording to the reviewer’s advice (see lines 645-656).\nMinor comments\nI think the data linking A. baumannii dispersion and fungi are interesting but not conclusive. Maybe\nthe authors want to tone down this a little bit.\nWe have toned down in these parts on the request of several reviewers. Moreover, we\nhave restructured the corresponding discussion sections to improve the presentation.\nLine 63: instead of “around 400” please give the actual number.\nCorrected: 401 (see line 63)\nLines 66-68: maybe you want to mention how many different types of sources you sampled.\nWe have refrained from following this advice to save space and because of the ambiguity\nto define what “different” sources are.\nLine 99: provide the actual number of genomes sequenced.\nCorrected: 401 (see line 94)\nLine 141-143: This a clear case of reverse Zoonosis, If I were you, I would say explicitly that.\nDone, thank you! (see line 128)\nLines 262-266: how many of those OXA-51 variants were novel?\n122 were novel (see line 303)\nLine 330: “unique genes” can be misleading; “gene families” or “homologous groups” are better\noptions\nChanged to “gene families” (see line 329 and also abstract lines 95-96)\nLine 624: issue is misspelled.\nOmitted in the course of streamlining the discussion!\nReviewer #2:\n\"The manuscript, titled \"On the ecology of Acinetobacter baumannii – jet stream rider and\nopportunist by nature,\" aims to describe the ecology of A. baumannii by analyzing 1,450 isolates, of\nwhich approximately 400 were genome sequenced. It is an interesting and well-written manuscript;\nhowever, several points need to be addressed before it is ready for publication.\nSome general and specific aims are:\n- My main concern is the claim that this work has resolved the ecology of A. baumannii by analysing\naround 1500 strains, of which approximately 400 were sequenced. Given the widespread distribution\nof A. baumannii species in the environment, resolving the ecology of Acinetobacter baumannii\nrequires a geographically evenly distributed sampling approach, as there might be several additional\nhidden niches in the environment, including other animals, etc., that play a pivotal role as reservoirs\nof strains. I believe the conclusions drawn are biased towards the samples available for this study\n(i.e. storks and specific geographical regions). Conclusions are valid for the regions studied NOT at\nthe global level. I am concerned\nbecause even including the publicly available GenBank genomes also does not normalise the sample biases as only a tiny fraction of the genomes available in GenBank are from environmental samples. I think to address this, the tone and working around this needs to be modified throughout the text.\nThank you! These comments helped us a lot to clarify statements and claims. However, we\nfelt that our previous statements already made clear, that our study has not completely\n“resolved the ecology of A. baumannii”. However, now we more explicitly pointed out\nlimitations and biases and toned down on claims and conclusions, also in line with concerns\nof other reviewers (see below). But please also acknowledge our previous statements, e.g.\nin the “Limitations” section where we already had written “Although we could demonstrate\nworldwide spread of lineages isolated in Poland and Germany, not all lineages necessarily\nspread worldwide. Ecology of A. baumannii in other climates and geographic regions may\ndiffer considerably.” Please see also another previous statement “Although our sample\nsetting, essentially restricted to samples from Poland and Germany, represents a significant portion\nof the worldwide known diversity, evidently, our picture remains incomplete.” (now in lines 446-\n448”).\n- Lines 112-115: Not sure about this section of the conclusion, \"Taken together, the intrinsic\nresistance endowment and potential to acquire antibiotic resistance can be explained by coevolution\nwith antibiotic-producing fungi and other microorganisms within the soil,\" as I think it includes a lot\nof generalisations. For example, parts of the widespread antimicrobial genomic islands found in A.\nbaumannii international clones have been shown to have been acquired from other clinically\nrelevant bacteria such as Salmonella and Enterobacterales. Overall, I believe this study does not\ninclude substantial evidence to support this claim.\nThank you! We have rewritten parts of the Summary and toned down on claims. Clearly,\nacquired resistance genes play a major role in the clinical context. The natural endowment\nof the species, however, already includes lots of resistance genes and efflux pumps\nconferring resistance to chloramphenicol and various beta-lactams amongst others.\nMoreover, the permeability of the outer membrane is extremely low compared to that of\nEnterobacterales for instance. This is what was meant by “intrinsic resistance endowment”\nwhich clearly evolved in natural environments, and most likely, particularly in soil habitats.\n- Lines 138-140: Please include the link in the reference list.\"\nDone! (see line 126)\n- Line 158: Please clarify why the smaller scale? Was the sample size determined through statistical\nmethods?\nDue to the large effort, the sample size was kept small given that we simply wanted to prove\nfor Germany the same tendency as observed for Poland. The determination of the scale was\nwithout statistical means. We have limited the statement on differences in the ecology of\nbacteria and/or storks to a comparison of Poland and Spain to account for the reviewer’s\nconcern of a small sample size of storks from Germany (see line 158).\nLines 169-172: Please include some numbers (e.g. colonisation, mortality rate etc.).\nWe have omitted the statement on mortality because of low statistical power of the\nobservations. Years with high colonization rates with A. baumannii among nestlings\napparently did not lower the breeding success.\n- Line 171: Did you identify increased white blood cell counts in all colonised individuals? Please\nclarify.\nNo, the increase of white blood cell count was not identified in the complete set of colonized\nindividuals, but the difference to non-colonised individuals was highly significant (see Suppl.\nTable S2).\n- Line 176: In the section \"We collected pellets from selected nests\", how did you ensure that the\nsamples are genuinely from pellets and nothing else (e.g. egg shells OR feathers)? Please clarify.\nThank you, this is a very important point. The pellet samples for enrichment cultures were\ntaken from the center of the pellet with sterile tweezers to exclude enrichment of\ncontaminations from the outer surface of the pellet. We have now also specified this in the\nmanuscript (see lines 643-644). To illustrate that A. baumannii is really found inside of the\npellets, we had also provided Suppl. Fig. S4. When breaking open a pellet, the interior can\nbe directly stamped on a CHROMagar Acinetobacter selective agar thus demonstrating the\npresence of A. baumannii inside the pellet.\n- Line 178: This is an interesting observation, the fact that no pellets was positive earlier than May-\nApril, could this be a correlation with the rain season in the area and that pellets might have washed\naway? Could you please elaborate on this?\nThis observation is perfectly matched by our later analyses on soil samples. There is a clear\nseasonality and it appears to be a temperature dependence in the first place. In compost,\nwere temperatures are higher, the season for A. baumannii is longer. As mentioned in the\ndiscussion, seasonality has been also described in cattle. More important, seasonality is also\nobserved in the clinical setting, but only for non-MDR isolates which apparently enter the\nhospital at higher rates during the summer season (see lines 453-463).\n- Line 186: Could this be due to the seasonal conditions?\nYes. Apparently, adult storks are not colonized by A. baumannii when they arrive from their\nwintering grounds, otherwise we should have detected them in pellet material in February-\nMarch. Only after the season for A. baumannii has started in the soil, we also see increasing\nnumbers of positive pellets. This is why your concern regarding regional and global\nstatements is of course highly relevant. We cannot deduce on what happens in tropical and\nsubtropical climates for example (but please see our “Limitations” section). All the more\nremarkable it is, that our data collection covers such a large part of the worldwide known\ndiversity. As far as we can see, only a very special dispersal mechanism can explain this.\n- Line 248: Please specify which regions?\nWe now added “Germany” in the manuscript (see line 244), but the specific place\n“Neuendorf, Schleswig-Holstein” is available through Suppl. Table S4 for the interested\nreader.\n- Line 317-318: Please include the sequence type number (ST#) representing each of the international\nclones. (e.g. OXA-66, ST2 etc.).\nDone! (see lines 316-318)\n- the blaOXA-51-like and blaADC are considered part of the core genome in A. baumannii, so can you\nelaborate on the sequence type(s) of those that lack these genes? And is there a correlation between\nthe lack of both genes (i.e. when blaADC is absent blaOXA-51-like is also absent)?\nThe sequence types scatter for strains lacking either bla or bla . We found not a\nOXA-51-like ADC\nsingle isolate lacking both genes. A detailed exploration is possible via the microreact tool.\n- Lines 381-389: Please include the sequence type(s) of those with AMR genes. This is important\ngiven that all the significance of A. baumannii is in the context of AMR.\nOnly 12 isolates of our collection exhibit AMR gene counts above 3. Six of these are human\nclinical isolates representing STPas 2, 106, 1357, 2402, and 2409, four are animal isolates of\nveterinary and livestock origin representing STPas 23, 241 and 866. Only two isolates of\nenvironmental and wildlife origin reach a count of 4 AMR genes (STPas 23 and 309). This is\navailable via Suppl. Table S4 and now mentioned in lines 380-384.\n- Line 402: Please be specific here, how many isolates were recovered? Was a sufficient number of\nstrains recovered to support the claim (i.e. airborne spread)?\nWe describe here the first piece of evidence which arose from a few preliminary\nexperiments with tubes mounted above the compost. Of these five samples, four were\npositive for A. baumannii. The positive finding stimulated more systematic experiments\nwith sterile plant material deposited in the garden. These deposition experiments were\nsuccessful in four out of four cases. These numbers have now been provided in the\nmanuscript (see lines 411-415).\n- Line 412: in all samples tested? Please clarify!\nThis kind of experiment has been performed multiple times by three students\nindependently over the years. They all consistently demonstrated the coating of different\nspores from Aspergillus and Penicillium species with different strains of A. baumannii. The\nlevel of coating illustrated in Fig. 7 is representative of the level of coating visualized by\nbright field and fluorescence microscopy. We shall be happy to provide additional pictures\nif this is requested.\n- Line 436: This is not accurate, several publications including genome sequence data have\ndocumented the isolation of IC1 (ST1) from natural environments. Please fix this.\nFirst of all, we noted that IC1, IC2 and IC3 are missing in our own collection of environmental\nisolates. This is accurate. Secondly, the environmental isolates described by others\nbelonging to IC1-3 typically exhibit signs of escape from hospital or livestock production, e.g.\ntypical resistance profiles, or at least are from environments of ambient human activity.\nThey therefore do not represent original environmental isolates. Unfortunately, we could\nnot identify the genomes mentioned by the reviewer.\n- Lines 441-461: I can see a lot of merits in comparing the population structures of A. baumannii and\nPseudomonas aeruginosa, however, it seems very out of place in the context of this study given the\ncompletely different evolutionary stories and global/clonal expansions that these two genera have\nexperienced. Please remove this section.\nAs suggested, we have removed this section!\n- The discussion is very long, the manuscript will benefit more from a more concise discussion.\nWe followed this suggestion and have reduced and streamlined the discussion!\nReviewer #3:\nDear Authors,\nI enjoyed and spent some time reviewing your manuscript ‘On the ecology of Acinetobacter\nbaumannii – jet stream rider and opportunist by nature’\nYour manuscript is conspicuous, well clearly presented and participated and it contributes to greater\nunderstanding the ecology and evolution of A. baumannii in natural habitats, that to date is really\npoorly understood outside the hospital.\nMy compliments for the supplementary datasets that are rich in content and very useful for data\nvisualization and interpretations.\nThank you!\nAll the comments and suggestions I have made are of a minor nature and I would be happy for the\nmanuscript to be accepted for publication in Nature Communication once these have been\nappropriately addressed point by point as follows:\n- Why authors identified white storks as a model system to study the ecology of A. baumannii? Why\nnot other wild or synanthropic birds?\nAs outlined earlier (Wilharm et al., 2017), we started with the hypothesis that the ability of\nA. baumannii to efficiently grow at temperatures above 40°C could point to an adaptation\nto hosts with core body temperatures around 40°C such as birds. White storks are\nintensively studied and nestlings regularly ringed on a broad scale. This means that there is\nno extra sampling campaign necessary. Moreover, the sampling is very easy due to the large\nsize of the nestlings and it is well established. The nestlings show a so-called akinesia that\nprevents them from falling out of the nest. Further, we considered migratory birds to be\nparticularly interesting due to the possibility of intercontinental transfer between Africa and\nEurope. All in all, storks were simply the most easily accessible starting point. By chance, it\nturned out to be a fascinating model system.\n- Considering the huge epidemiological study, I wonder if the authors also found other pathogens of\ninterest from the samples analyzed.\nFor most of the time we used selective media and enrichment methods as outlined in the\nMaterials and Methods section so that we only rarely isolated microorganisms other than\nAcinetobacter. Only in the beginning of our studies we also systematically used non-\nselective media that indeed revealed a plethora of potential pathogens including E. coli,\nKlebsiella and Pseudomonas. The results are unpublished except for a few novel species\ndiscovered. Having this wealth in mind, we kept a considerable part of our sampling material\nin the freezer that can be used for future studies.\n- Regarding the Diversity in terms of OXA-51 variants collected from Poland and Germany and the\nPhylogenomics-based representation of the diversity, I agree with the choice of at least one strain\nrepresenting each OXA variant for whole genome sequencing.\nThank you!\n- Very useful and well done the ‘Phylogenetic distribution of strains carrying selected variants similar\nto OXA-51’ illustrated in Suppl. Fig. S14 and deepened in the microreaction project\nThank you!\nRegarding ‘Compost, soil, rhizosphere and the forest paradox’ (line 257), really interesting the\nsampling of the soil originating from a single compost across a period of seven months that yielded\n86 isolates with 20 different variants of the OXA-51 family, and earthworms within the same compost\nthat yielded 10 different variants of OXA-51.\nInstead, (line 298-301) although very interesting as data, considering the low number of samples\nexamined, I would go with more moderation in stating that A. baumannii can thrive along streams\nand rivers outside of forests.\nThank you! We have attenuated our statements accordingly (see lines 295-297).\nThe decomposition of plant material by fungi would seem to lay the foundation for the proliferation\nof A. baumannii. You could clearly assume that decomposition of plant material by fungi would seem\nto appear to set the stage for proliferation of A. baumannii.\n- Line 398: Regarding the Colonization of compost material and linkages to the fungal world, the\nexperiment is not so clear to me. How many tubes did you mounted? How many trials have you\nconducted? Please specify\nRegarding colonization of compost material, these deposition experiments with sterile\nmaterial yielded A. baumannii in 4 out of 4 cases. Before, we had conducted preliminary\nexperiments in which we mounted 5 tubes above the compost. Of these 5 samples, 4 were\npositive for A. baumannii. These numbers have now been provided in the manuscript (see\nlines 411-415). The interaction between fungi and A. baumannii in vitro have been studied\nmultiple times over the years by different students with consistent reproducibility. We have\nrewritten parts of the discussion section and also toned down on this topic.\n- Line 845: Collection of rodents, rats and shrews. How many cat-captured rodents have you\ncollected? Particularly, can you describe in detail the capture procedure and whether there are laws\nand permits (especially for rats) in the country of collection (in addition to those already mentioned)\nabout the associated biological risks.\nThe numbers of cat-captured rodents (n=154) and shrews (n=64) have been detailed in\nSupplementary Table S1. Regarding cat-captured animals, only dead animals deposited by\ncats at their respective homes were included. Wild rats were captured by professional pest\ncontrol operators, and hence no capture permit was required (see referenced work by\nRaafat et al., 2020). Other small mammals were captured as described in referenced work\nby Jeske et al., 2021.\nFIGURES\n- Figures 3, 4, 5 are obviously simply representative and at first glance difficult to understand\nespecially because of the amount of isolates represented graphically. The link https://microreact.org\nclearly describes and represents this. But to make the graphic part of the figures, especially 4 and 5,\nmore immediate and understandable, please divide each of the figures into A and B and briefly\ndescribe the individual parts before directing the reader to the link.\nDone!\n- Please enlarge the central part of Fig. S1 ‘Spatial correlogram’ to make it more readable\nDone!\n- I suggest putting letters A, B, C... for each Supplementary Figure consisting of two or more figures\nand where the legend is not prorply specified, to make them even clearer (E.g. Suppl. Fig. S18)\nDone!\nHope my review could help you to improve your work.\nRegards\nThank you again!\nReviewer #4:\nIn this study, Wilharm et al. investigated multiple aspects of ecology and distribution of\nAcinetobacter baumannii. Authors have dedicated a quite a bit of resources for this study and\nprovide some novel insights into the ecology and evolution of A. baumannii complex. However, I find\nthe current form of the manuscript, presentation of the results and the analyses do not justify the\neffort put into this study and data generated by the authors. Also, there are few concerns related to\nmethodology of this study. Below I highlight my major concerns and comments.\n1. Structure and the organization of the manuscript: The current written style of this manuscript\nincludes a collage of mini experiments, at times seems very fragmented.\na. The introduction itself is not giving a proper background to justify why it is important to conduct\nthe study that authors have done, and I don’t think “we intended to unravel the ecology of these\nbacteria in their natural habitats” is a good argument. I would suggest re-writing a proper\nintroduction highlighting the thought process and major gaps that authors are anticipating on filling\nwith their work. Also, it is a bit weird to see the first citation in the introduction is the 90th citation in\nthe reference list.\nWe felt that “…to unravel the ecology of these bacteria in their natural habitats” is a good\nargument, because this piece was missing and we expected it to contribute to our\nunderstanding of its tenacity and success in the hospital setting. We have tried to improve\nthe introduction (see lines 133-144). Further, we switched to the reference style of the\njournal now.\nb. Detailed results related to isolation information of the A. baumannii can go into the\nsupplementary and just provide a summary of what was isolated.\nWe have reduced the section as much as possible but also received contrary advice from\nother reviewers to provide even more information.\nc. Authors need to analyze the genomes bit more thoroughly. This is the most interesting dataset of\nthe study and there are many analyses that authors can do to identify the ecology and evolution of\nthis species complex (see my major point 3).\nThank you! We have extended our genome-based analyses. See below our response to\nmajor point 3!\nd. Supplementary: There are two supplementary files and one includes the figures and the other\nincludes a mix of things which also have some interesting results on GIS based analyses. I think these\ntwo documents need to be merged (one of this document even have a figure 1). I also think the GIS\nanalyses need to be highlighted in the main text results and to show whether the number of isolates\nhave an association with the proximity to human activity. This is what I saw with your transect\nstudy, where you only isolate A. baumannii after the village (Fig S12).\nDue to the conflicting advice from other reviewers we kept the GIS analyses in the\nSupplements but tried to better organize the supplementary material as advised.\ne. Discussion is extremely long and highly speculative, while making huge statements without\nadequate support. I think authors need to first define their objectives and make the discussion more\nstreamlined and avoid jargon.\nWe have considerably streamlined the discussion and as detailed below our hypothesis on\nthe airborne global spread of A. baumannii received significant support from a recent study.\nWe further removed the highly speculative statements mentioned by the reviewer and use\nof jargon.\n2. Uneven sampling:\na. Authors have done an amazing job with sampling White storks in Germany and Poland. Then they\nhave attempted to sample other bird species (grey-herons, kestrels and lack storks), which have a\nvery small number of individuals compared to the white stork samples and only sampled the cloaca\nof the birds. This raises the question whether this uneven sampling led to the observed lack of\nisolation of A. baumannii from other bird species. Second, in White storks it seems like A. baumannii\nis isolated more from choanae and for other species authors took cloacal samples. Again, this makes\nme wonder if authors sampled choanae in other birds whether they would have gotten different set\nof results.\nWe concur in the assessment of uneven sampling both in terms of numbers and in terms of\ndifferences of the sampling materials. We have acknowledged this, now stating\n“…apparently differ from white stork regarding carriage of A. baumannii although this might\nbe influenced by differences in sampling depth and sampling types.” (see lines 250-252).\nHowever, even if we compare the same kind of sample material, none of the other three\norganisms reaches the prevalence levels of the white stork.\nb. Autoclaved compost test: This was an interesting approach but why did you only do this in a\ngarden. I think it would make more sense to conduct this experiment both in the garden setting,\nsemi-natural area and in the forest environment. This way you will have the opportunity to test your\nhypotheses related to airborne nature of dispersion of A. baumannii away from human settlements.\nDefinitely, it will be interesting to learn in which environment we see the highest airborne\nload but this was out of the scope of this study, also because of the enormous effort to\nobtain permissions. At least, in global support of our hypothesis of airborne dispersal of A.\nbaumannii, a very recent paper by Rodó et al. (PMID: 39250672) explicitly reports on the\npossibility of long-distance transport of pathogens including A. baumannii based on\ntropospheric sampling.\n3. Genome analyses: I feel like this is the most important dataset of the study and authors just looked\nat the surface of this dataset.\na. It would be good to show genes/gene families/SNP differences between environmental isolates\nand clinical isolates in a figure (it should be a main figure).\nb. Given that authors have identify clades of environmental, animal and clinical isolates I think\nauthors should conduct positive selection analyses to investigate if there are certain genes under\npositive selection when A. baumannii become a hospital strain. Moreover, this will allow you to\nidentify if there are particular genes that show converged positive selection when strains become\nclinical isolates across the clades.\nc. Molecular clock analysis: What Acinetobacter species did the authors used as the outgroup when\ndoing this analysis? Also given the diversity and the geographic distribution of the genomes you have,\nyou should conduct ancestral state reconstruction to see if you can point our a potential origin of the\nradiation of this species clade.\nWe thank the reviewer for these important comments regarding the data we analyzed. We\nagree that throughout this study we only scratched the surface of the rich data sets but feel\nlike the presented results will have a major contribution on future research within this field\nand that it is time to involve the scientific community to make full benefit out of these\nunique data. To comply with your suggestion (a), we performed gene family enrichment\nanalyses (see novel Fig. 6 and novel Suppl. Figure 21). We are especially thankful for this\nsuggestion given that it revealed a number of significant differences between\nenvironmental and clinical isolates’ genomes which we are now presenting in Fig. 6 and\ncommenting in lines 392-398.\n(b) Positive selection analyses are beyond the scope of this study but will be surely\nperformed by members of the scientific community soon and we would happily support\nsuch studies.\n(c) See comments on the molecular clock analysis above in response to reviewer 1. We need\nto expand our dataset until we reach a better temporal signal. We followed the work by\nHolt et al. (2016).\n4. Tracing the food sources: I think the proper way to do this is first to conduct metabarcoding of the\npallet material. Then identify food items common in pallets with A. baumannii isolates and then\ncollect the wild individuals (like what you did with earthworms) and then see if those wild diet items\ncarry A. baumannii.\nDefinitely, this would have been an elegant way that will however be applied in the future\nto complete the tracing studies.\n5. I also wonder why authors did not conduct whole microbiome sequencing of their samples to\nconfirm that isolation actually represent the presence of A. baumannii. This is because the isolation\nalone might not give the full picture of the presence of A. baumannii in a given sample. Have you\ndone any preliminary work to make sure you always identify A. baumannii from samples when the\nbacteria are present?\nWe agree, metagenomics studies are an important next step, however beyond the scope of\nthis work. As already mentioned in the “Limitations” section of the Discussion, we have\npublished evidence that we are not isolating A. baumannii from every sample that contains\nit. This has to be addressed in the future to complete our picture.\nMinor comments\nL127: Citations are weird. Please double check.\nReferences were updated and included in the reference list.\nL154: Is “average rate of 25%” mean they were isolated from 25% of samples. In that case isn’t it the\nprevalence?\nCorrect! We rephrased accordingly.\nL164: Terminology for birds is “Cloacal” not “Rectal”.\nCorrected throughout!\nL233: Can you split the isolate numbers of the bacteria according to different earthworm genera?\nWe have now provided this information in Supplementary Table S1.\nL254-256: I think this is an issue of sampling depths and sampling types.\nWe have acknowledged this, now stating “…apparently differ from white stork regarding\ncarriage of A. baumannii although this might be influenced by differences in sampling depth\nand sampling types.” (see lines 250-252).\nOxa-51 variants: What is your cutoff value to call a strain as a different variant? Is it 1bp difference?\nAlso, what is the length of this segment?\nVariants are only indexed if differing on the protein level, synonymous nucleotide\nsubstitutions justify no variant designation (see lines 191-193). The protein has a length of\n274 amino acids.\nL330-331: Can you summaries the geographic distribution of your genomes? This will help readers to\neasily see if there are geographic biases in the isolates you used in this study.\nThe geographical distribution of all genomes included (those selected from NCBI and ours)\nis available in Suppl. Table S4. The geographical distribution of the genomes generated\nwithin this study is highly biased (mainly from Poland and Germany) and accessible via the\nMicroreact tool and illustrated in Fig. 4. However, the NCBI dataset is also highly biased:\nmost genomes deposited have been contributed by a few countries worldwide.\nL413: Is the incubation temperature you used natural (e.g., environmental temperature)? Also, if the\nadherence of A. baumannii inhibit the germination of fungal spores, can that also impact other\nproperties of the spores (like the dispersal ability)?\nStandard incubation condition was at 37°C, both for adherence and germination assays (we\nhave now included this information in the Materials and Methods section (see also below),\nrelevant at least for the compost setting. However, adherence and germination assays also\nwork at 27°C. We don’t know how adherence and dispersal interfere. We have refrained\nfrom such experiments because of biosafety concerns.\n441-461: Very weird section to start your discussion with, given that the study was not a comparative\nstudy between P. aeruginosa and A. baumannii.\nWe have omitted this section on the comparison between P. aeruginosa and A. baumannii,\nalso in agreement with other reviewers’ recommendation.\n462-501: This part is also very speculative, and I struggle to find how this relate to authors findings,\nexcept for that authors show the ability of the bacteria to adhere to fungi. I think if authors want to\ndiscuss more “Evidence for interrelatedness of A. baumannii life cycle with the fungal world” they\nneed to conduct more experiment to properly show the claims they make here.\nTo extend the work on the interrelatedness of A. baumannii with fungi is beyond the scope\nof this study. Therefore, we have toned down on the evidence that we provide to comply\nwith the reviewers’ doubts. Moreover, we rewrote parts of the discussion and we updated\nliterature in Suppl. Table S8. More and more evidence is accumulating in the literature but\nthe topic is still clearly understudied. Given the high impact this has on the clinical outcome,\nwe felt unable to completely omit this part. In particular, we believe awareness and research\nneed to be stimulated.\nL509: Basidiomycota is a very broad and diverse division of fungi and don’t think using this is a good\nargument here.\nWe have specified “such as Fistulina hepatica” (see line 511).\nL541-585: I am not sure how your findings support A. baumannii uses air currents to travel 1000s of\nkms between continents. Maybe I missed something here?\nThank you! This question helped us to optimize the presentation of our line of argument:\nWe identified several isolates from comparably pristine habitats in Poland that are closely\nrelated to clinical isolates described in countries all over the world (e.g. Canada, Thailand,\nJapan). These pairs of environmental & clinical isolates each share most recent common\nancestors that existed only a few decades ago. In comparison to bacteria that are known to\nspread in association with humans and which exhibit the highest dispersion rates (Louca,\n2022), our findings suggest significantly higher dispersion rates for A. baumannii. How can\nwe explain multiple independent long-distance spreading events within decades? Humans\nare not regularly colonized with A. baumannii. The genomes of our isolates show no signs\nof escape from hospitals or livestock production (e.g. in terms of acquisition of resistance\ndeterminants and mobile genetic elements). Accordingly, there is no evidence of spread due\nto human activity. A. baumannii is known to have the capability of airborne spread in\nhospitals and we have demonstrated repeatedly airborne colonization of sterile plant\nmaterial in the garden. Aerosolization of Acinetobacter is in full agreement with the\nliterature both in terms of metagenomics studies and in terms of resistance to desiccation\nand radiation. Especially striking, A. baumannii has been detected in glacier microbiomes all\nover the world including Antarctica. Moreover, a very recent paper by Rodó et al. (PMID:\n39250672) explicitly reports on the possibility of long-distance transport of pathogens\nincluding A. baumannii based on tropospheric sampling. Arguably, we have no direct\nevidence that A. baumannii reaches jet streams, although not unlikely, and therefore\nremoved allusions to this picture (see modification of title).\nL559-560: This I don’t underatand. There seem to be an association with your isolates and presence\nof humans and then here you say “All in all, there is no evidence that the observed global pattern of\ndistribution of environmental A. baumannii is caused by human activity”. You have data to test this!\nThank you again! To clarify, human activity since the Neolithic generated numerous novel\nhabitats including pastures and nutrient-rich waters while reducing non-conducive forest\nareas. This fueled early-on radiation within the species leading to a high diversity of ancient\nlineages. Environmental A. baumannii isolated in our study exhibit little to no signs of\nselection pressure as exerted by nowadays human activity, use of antibiotics in human\nhealth care and livestock production in particular. This has already been tested and\npresented in Suppl. Table S4 and Suppl. Figures S18 & S19 (see also modifications in the text\nin lines 380-384). Thus, there is no contradiction, ancient and modern human activity have\ndifferent impact on the evolution of A. baumannii.\nL660-668: This section seems like it is repeating things. Please add this section to other\ncorresponding sections.\nDone!\nL809: Did the incubation happened at room temperature?\nIncubation was at 37°C, which has now been stated in the manuscript (see lines 753-760).\nL822-828: Results for this methods section is not in the “Results” section. I think it is very interesting\nand should be in the main paper.\nThank you! We agree that these results are very interesting, however due to the request to\nstreamline and reduce the manuscript, we had to decide to keep it as it is; at least several\naspects are provided in the Supplements (see Suppl. Fig. 20 and Suppl. Material S1).\nFigures: Please provide better legends for the figures. The interactive link is cool but you need to\nprovide enough information these figure legends to guide reads (figures and legends should be able\nto stand alone). Also please provide information on the color codes associated with phylogenies.\nWe provide developed figure legends of the figures to improve accessibility.\nPoint-by-point response:\nReviewer #4 (Remarks to the Author):\nThis study by Wilharm et al., provides an interesting dataset on A. baumannii, but I do not think\nauthors are doing enough justification for this amazing dataset they have (i.e, genomes)\nproduced here. This version of the manuscript still provides a simple story on where and how to\nisolate these bacteria (which is very interesting) and present some hypothesis on how this\nbacteria species might spread around the world. However, I still think the major value of this study are the new genomes,\nthus, I still think more comparative analyses of these genomes are necessary to properly understand\nthe underlying genomic features that might drive A. baumannii to become clinically relevant strains\n(which should also play the center role).\nThank you for acknowledging our data collection. We agree in the need for more detailed\ncomparative genomic analyses to better understand the particular success of a few lineages\nin hospitals as compared to the huge diversity found in the environment. However, such\nanalyses require the isolation of IC 1-, IC 2- and IC 3-related strains from pristine\nenvironments which we could not achieve here (we already pronounced this in the\nmanuscript in lines 415-417). This is especially important regarding IC 2, which is by far the\nmost successful lineage in hospitals worldwide. Among the reasons, why we failed to isolate\nIC 2, the most plausible explanations are that IC 2 isolates (i) do not propagate in soil and\nwhite storks where most of our collection comes from, and/or (ii) their natural habitats are\ngeographically restricted (see below), and/or (iii) they don’t spread efficiently outside of\nhospitals (especially no efficient airborne spread). Therefore we believe this manuscript\nneeds to be published: to stimulate further environmental studies all over the world to\ncomplete our picture!\nGiven that sampling is bias toward Germany and Poland, I don’t think (even with the great number of\nisolates produce by this study), authors have enough evidence/data to give a global overview of this\nspecies complex. What you have are amazing set of genomes that you can answer many more\ninteresting questions related to how A. baumannii become virulent in hospital settings. Such a story\nwill fit well with the aims and interest the broad readership of this journal.\nWe agree that our collection does not give a global overview of the species complex.\nHowever, we would like to emphasize that we did not claim to have provided a global\noverview. On the contrary, we stated in the “Limitations” section (lines 588 ff) that there is\nevidence of understudying because of the use of a specific enrichment method and that\nthe ecology of A. baumannii may differ in other climates and geographic regions. What is\nmore, as outlined above, we missed to isolate some very important lineages (IC 1 -3) and\nhave also clearly stated this. Nevertheless, the fact that our local sample set was able to\nrepresent more than half of the hitherto known diversity in terms of OXA-51 variants and\nto more than double the pangenome of the species is clear evidence of the global\nrelevance of our study, let alone the close relationship of globally distributed isolates that\nwe could demonstrate.\nSecondly, I still don’t agree with authors proposed mechanism of the spread of this species complex,\nmainly because the proposed mechanism is based on many non-tested assumptions or over\nextrapolating some findings. Also, authors tend to ignore potential steppingstone colonization\nmethods, spread by other migrant species (not just storks), and potential combination of many\nmethods including the airborne spread as authors suggested. I think, to receive a better outcome\nfrom this amazing work, the scope of the study should change and generate more specific research\nquestions (related to the evolution, and genetic underpinnings of virulence) instead of just saying you\nare trying to “improve the understanding of the ecology of A. baumannii in natural habitats…”.\nWe do not exclude that a combination of different mechanisms contributes to spreading of\nthe species. Importantly, we do not even claim that storks contribute significantly to global\nspread of A. baumannii. However, our claim of long-distance airborne spread of\nA. baumannii is in full agreement with recent work by others as discussed in our\nmanuscript, describing the presence of A. baumannii in glaciers worldwide and in\ntropospheric samples. Our trials to “improve the understanding of the ecology of\nA. baumannii in natural habitats…” turned A. baumannii from a nosocomial pathogen with\nunclear origin into one of the few bacterial pathogens really accessible for studies in its\nnatural environments."}, "author_response": ""}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have addressed my comments. Congratulations to Gottfried and colleagues for all their hard work.", "reviewer_3": "Dear Authors,\nI noted all my suggestions and those of the other reviewers have been well accepted and incorporated into the text.\nThe manuscript is now certainly more robust and deserves to be published without further revisions, in my opinion.\nGood luck and congratulations\nKind regards", "reviewer_4": "This study by Wilharm et al., provides an interesting dataset on A. baumannii, but I do not think authors are doing enough justification for this amazing dataset they have (i.e, genomes) produced here. This version of the manuscript still provides a simple story on where and how to isolate these bacteria (which is very interesting) and present some hypothesis on how this bacteria species might spread around the world. However, I still think the major value of this study are the new genomes, thus, I still think more comparative analyses of these genomes are necessary to properly understand the underlying genomic features that might drive A. baumannii to become clinically relevant strains (which should also play the center role).\nGiven that sampling is bias toward Germany and Poland, I don’t think (even with the great number of isolates produce by this study), authors have enough evidence/data to give a global overview of this species complex. What you have are amazing set of genomes that you can answer many more interesting questions related to how A. baumannii become virulent in hospital settings. Such a story will fit well with the aims and interest the broad readership of this journal.\nSecondly, I still don’t agree with authors proposed mechanism of the spread of this species complex, mainly because the proposed mechanism is based on many non-tested assumptions or over extrapolating some findings. Also, authors tend to ignore potential steppingstone colonization methods, spread by other migrant species (not just storks), and potential combination of many methods including the airborne spread as authors suggested. I think, to receive a better outcome from this amazing work, the scope of the study should change and generate more specific research questions (related to the evolution, and genetic underpinnings of virulence) instead of just saying you are trying to “improve the understanding of the ecology of A. baumannii in natural habitats…”."}, "author_response": ""}]

Decision letter