Reservoirs of Non-baumannii Acinetobacter Species

Acinetobacter spp. are ubiquitous gram negative and non-fermenting coccobacilli that have the ability to occupy several ecological niches including environment, animals and human. Among the different species, Acinetobacter baumannii has evolved as global pathogen causing wide range of infection. Since the implementation of molecular techniques, the habitat and the role of non-baumannii Acinetobacter in human infection have been elucidated. In addition, several new species have been described. In the present review, we summarize the recent data about the natural reservoir of non-baumannii Acinetobacter including the novel species that have been described for the first time from environmental sources and reported during the last years.


INTRODUCTION
Implementation of molecular techniques in research laboratories has greatly improved the identification of Acinetobacter species. Among these techniques, 16S-rRNA, RNA polymerase subunit B (rpoB), and DNA gyrase subunit B (gyrB) gene sequencing, as well as DNA-DNA hybridization and whole genome sequencing provide good informative data for Acinetobacter taxonomic studies (Rafei et al., 2014;Jung and Park, 2015). Based on these methods, novel species have been reported and the genus now contains 51 species with valid published names (http://apps. szu.cz/anemec/Classification.pdf. (Accessed October, 2015).
Acinetobacter species are ubiquitous in nature and can be found in different environmental sources such as hydrocarbon contaminated areas, activated sludge, sewage, dump sites, but also on vegetables, animals, and humans (Doughari et al., 2011). The ability to dominate in so many ecological niches led thus some authors to consider these bacteria as microbial weeds (Cray et al., 2013).
Among the different species, Acinetobacter baumannii is the leading one. It has emerged in recent decades as a clinically relevant pathogen causing a wide range of nosocomial infections, community-acquired infections or war and natural disaster-related infections (Peleg et al., 2008). Nevertheless, the role of non-baumannii Acinetobacter in human infections is increasingly reported thanks to technological advances such as molecular biology that allow correct identification of the bacteria at the species level. Thus, for example, several cases concerning multidrug resistant Acinetobacter pittii and Acinetobacter nosocomialis strains that caused infections in health-care facilities have been reported around the world (Karah et al., 2011;Kouyama et al., 2012;Yang et al., 2012;Schleicher et al., 2013;Fitzpatrick et al., 2015). Acinetobacter calcoaceticus which is mainly an environmental species has been described in several cases of pneumonia and bacteraemia (Malhotra et al., 2012). Acinetobacter kookii was a novel species isolated from beet fields in Germany, from soil in the Netherlands and in Korea, and from sediments of fish farms in Malaysia and Thailand (Choi et al., 2013). Acinetobacter venetianus was a novel species isolated from seawater in Israel, oil in Italy, aquaculture ponds in Denmark and from the sea in Japan (Vaneechoutte et al., 2009). Finally, Acinetobacter bohemicus and Acinetobacter albensis were two novel species described for the first time in Czech Republic and recovered from natural ecosystems such as soil, mud and water (Krizova et al., , 2015a. Noteworthy, development of new high throughput sequencing techniques allowed metagenomics studies that could improve our understanding of bacterial microbiota surviving in different environmental sites. For example, Acinetobacter spp. were found in soil samples contaminated with petroleum hydrocarbons (Sarma et al., 2004;Bordenave et al., 2007;Obuekwe et al., 2009) and in sediments and water samples in Asian countries collected either from fish pond contaminated with organic waste or from fish and shrimp farms (Huys et al., 2007;Xiong et al., 2015). However, even if metagenomics can provide information on bacterial diversity, in these studies isolates were not characterized at the species level.
In the recent years, several new Acinetobacter species have been described in Korea and China. Acinetobacter antiviralis and Acinetobacter oleivorans were two novel species isolated from tobacco plant roots and rice paddy in Korea (Lee et al., 2009;Kang et al., 2011). In China, Acinetobacter refrigeratoris, Acinetobacter puyangensis, Acinetobacter qingfengensis, Acinetobacter populi, Acinetobacter guangdongensis, and Acinetobacter harbinensis were six novel species that have been isolated from a refrigerator, popular bark, abandoned lead-zinc ore mine site and surface water of a river respectively (Li et al., 2013(Li et al., , 2014a(Li et al., ,b, 2015bFeng et al., 2014a,b).
Further microbiome studies have been conducted to investigate the bacterial population in the floral nectar of some plants. Interestingly, it has been shown that Acinetobacter was the main bacterial taxa founded (Fridman et al., 2012;. Besides, Acinetobacter boissieri and Acinetobacter nectaris were two novel species that were isolated from nectar samples of plants in Spain (Álvarez-Pérez et al., 2013).
Recently, it has been shown that the environment could constitute a potential reservoir for Acinetobacter spp. resistant isolates. Indeed, carbapenemase and extended-spectrum betalactamase producing strains have been isolated from hospital sewage, soil samples around animal farms, but also in polluted rivers (Zong and Zhang, 2013;Maravić et al., 2015;Wang and Sun, 2015; Table 1), highlighting the potential role of these bacteria in the dissemination of antibiotic resistance genes through the environment.

Food
Presence of Acinetobacter spp. in the food chain has also been studied. From 1999, Berlau et al. isolated A. guillouiae, A. calcoaceticus, A. pittii, A. lwoffii, and A. bereziniae on vegetables purchased from markets in the United Kingdom or harvested from gardens during the summer (Berlau et al., 1999a).   Seifert et al., 1997;Berlau et al., 1999b;Chu et al., 1999;Patil and Chopade, 2001 (Continued) Frontiers in Microbiology | www.frontiersin.org  Seifert et al., 1997;Berlau et al., 1999b;Chu et al., 1999 (Continued) Frontiers in Microbiology | www.frontiersin.org  In a subsequent study conducted in Hong Kong on vegetables, A. pittii and Acinetobacter genomic species 10 and 16 have been found (Houang et al., 2001). Different Acinetobacter species have also been isolated from fish, meat, cheese and milk samples. In Lebanon, Rafei et al. reported the isolation of non-baumannii Acinetobacter including A. pittii, A. calcoaceticus, A. bereziniae, and A. soli from raw cow meat, raw cheese, raw cow milk and vegetable samples (Rafei et al., 2015), and more recently, they isolated a carbepenem resistant A. calcoaceticus from vegetables (Al Atrouni et al., 2016). Acinetobacter spp. have been reported in previous studies from milk samples collected from dairy herds In the United States (Jayarao and Wang, 1999) and Kenya (Ndegwa et al., 2001). The isolation rate was 1.3 and 5% respectively. Acinetobacter spp. have been reported also from mastitic milk and raw bulk tank milk samples in Korea (Nam et al., 2009;Gurung et al., 2013).

Animals
While several published studies reported the isolation of A. baumannii from animals such as ducks, pigeons, chicken, donkey, rabbits, pets (cats, dogs), mules, livestock (goats,pigs, cattle, caws), horses, lice and arthropods (Gouveia et al., 2008;Hamouda et al., 2008Hamouda et al., , 2011Bouvresse et al., 2011;Endimiani et al., 2011;Kempf et al., 2012a,b;Belmonte et al., 2014;Rafei et al., 2015), few studies reported the isolation of non-baumannii Acinetobacter from animals.  Scola et al., 2001), and recently A. radioresistens and A. schindleri were detected from head lice collected from primary school pupils in Thailand (Sunantaraporn et al., 2015). Acinetobacter spp. were also detected from aquatic animals (Huys et al., 2007;Geiger et al., 2009) but also in the gut of some arthropods like tsetse fly in Angola, Africa (Guardabassi et al., 1999). Besides, Acinetobacter apis was a novel species isolated from the intestinal tract of a honey bee in Korea . Furthermore, other studies have been performed to investigate the intestinal ecosystem of fish using metagenomic approaches. As results, Acinetobacter was remarquably one of the most abundant genera detected. Indeed, the ability to produce antibacterial compounds against several other species as well as environmental factors and nutrition conditions may affect the bacterial community in the fish intestine and explain the dominance of this group (Hovda et al., 2007;Etyemez and Balcázar, 2015).
Finally, recently, Sun et al. reported the isolation of NDM-1 producing A. lwoffii from rectal sample of a cat in China , suggesting that these companion animals may play a crucial role in the dissemination of multidrug resistant bacteria.

Human Carriage
Acinetobacter spp. can be part of the human flora. In a large University Hospital in Cologne, Germany, Seifert et al. performed an epidemiological study to investigate the colonization with Acinetobacter spp. of the skin and mucous membranes of hospitalized patients and healthy controls. They showed that the colonization rate was higher in patients than in controls (75 vs. 42.5%) (Seifert et al., 1997). The hands, the groin, toe webs, the forehead and the ears were the most frequently colonized body sites. Almost all the species isolated were non-baumannii Acinetobacter including A. lwoffii (47%), A. johnsonii (21%), A. radioresistens (12%), A. pittii (11%), and A. junii (5%). In contrast, A. baumannii and A. bereziniae were rarely detected and the authors did not find A. calcoaceticus or A. haemolyticus on the skin or the mucous membranes (Seifert et al., 1997).
Berlau et al. performed a similar study to investigate the presence of Acinetobacter spp. on the skin (forearm, forehead, toe web) of 192 healthy volunteers in the United Kingdom. As in the previous study, they found that the colonization rate was around 40% with A. lwoffii being the most frequently isolated species and the forearm being the most frequently colonized area. However, the distribution of the other species was different, Acinetobacter genomic species 15BJ (12%), A. radioresistens (8%) and only one individual carried the Acinetobacter baumanniicalcoaceticus complex (Berlau et al., 1999b). In another study conducted in Hong Kong, Chu et al. showed that the skin carriage rate of student nurses and new nurses from the community was 32 and 66% respectively with A. pittii being the most common species (Chu et al., 1999). The authors reported also a potential seasonal variability in skin colonization (Chu et al., 1999). Patil et al. studied skin carriage on six body sites (antecubital fossa, axilla, forehead with hairline, neck, outer surface of nose and toe webs) from volunteers in India. It was found that non-baumannii Acinetobacter were the most frequently isolated species including A. lwoffii, A. junii, A. haemolyticus, A. calcoaceticus, and A. pittii. In this study the antecubital fossa had the highest colonization frequency (48.5%) and the men volunteers were more colonized than the women (Patil and Chopade, 2001).
Likewise, Acinetobacter spp. have been also isolated from fecal samples. A study performed by Dijkshoorn et al. in United Kingdom and the Netherlands to investigate the intestinal carriage of Acinetobacter spp. showed that from 226 fecal samples collected randomly from the community 38 were positive. The species commonly isolated were: A. johnsonii, A. guillouiae, and A. junii (Dijkshoorn et al., 2005).
Genomic approaches have also been used to study the bacterial community of some human samples. Thus, Zakharkina et al. reported Acinetobacter spp. from airway microbiota of healthy individuals (Zakharkina et al., 2013), while Urbaniak et al. reported the detection of these microorganism from human milk samples (Urbaniak et al., 2014). Recently, in another work conducted to study the microbial diversity of intestinal microbiota of healthy volunteers, Li et al. showed that Acinetobacter was present mainly in the duodenum (Li et al., 2015c). According to these findings, we can see the ability of Acinetobacter to survive in commensal samples, suggesting that human could constitute a potential reservoir for this opportunistic bacterium. However, the origin and the factors that can influence this colonization remained unclear.

GLOBAL REMARKS
Referring to these results, we showed here that the environment is the main reservoir of Acinetobacter spp. and interestingly the bacteria have been mainly isolated from sites in contact with human, animal or in areas polluted with hydrocarbon. Therefore, it has been suggested that Acinetobacter spp. belong to the small minority of species that are able to dominate within an open habitat (Cray et al., 2013). Indeed, the microorganisms are exposed in the environment to multiple factors that affect their growth and act as stress parameters such as desiccation conditions, temperature, air humidity and other parameters that are subjected to dynamic changes. Unlike some other Gram negative bacteria, Acinetobacter spp. are able to survive in a dry environment for long periods of time and support desiccation conditions (Wendt et al., 1997;Wagenvoort and Joosten, 2002). This tolerance may be due to different mechanisms such as over expression of proteins involved in the antimicrobial resistance, efflux pumps, down regulation of proteins involved in the cell cycle, transcription and translation in order to enter in a dormant state (Gayoso et al., 2014). Furthermore, hydrocarbons and polysaccharides are macromolecules available in the environment and may constitute a primary substrate for these microorganisms. Acinetobacter species can catabolize the polysaccharides via the production of xylanase which is a key enzyme to degrade complex extracellular substances such as hemicelluloses. It has also been shown that pollution of environmental sites either with fuel oil or metals can affect the microbial diversity and only few types of bacteria such as Acinetobacter spp. were able to resist and dominate such polluted areas (Bordenave et al., 2007;Zhao et al., 2014). Moreover, these bacteria are able to degrade various pollutants and organic compounds and have an important role in environmental bioremediation (Adegoke et al., 2012;Cray et al., 2013). Finally, Acinetobacter spp. have developed strategies to inhibit the growth of competing species either by acidification of the environment (secretion of organic acids) or by production inhibitory biosurfactants (Cray et al., 2013).
In this review, we showed also that the use of DNA based methods contribute to the progress in the field of the diversity of the genus Acinetobacter. As a result, a large number of well characterized species were available and Acinetobacter remains an interesting model for taxonomist to study the natural diversity as well as the evolutionary history of this bacterium. In fact, recent studies suggested that climatic changes and pollution have the potential to alter the species distribution in the environment (Coelho et al., 2013). Other theories consider that evolution of species may be the direct response to climatic modifications (Hoffmann and Sgrò, 2011). These findings raise many questions whether description of new Acinetobacter species was the result of those ecological changes. On the other hand, there is an important question that remains unclearly answered: could these newly described Acinetobacter species have a potential role in human infection? In fact, several studies showed that uncommon and newly described Acinetobacter species such as A. septicus and A. bereziniae were involved in human infection and some of them were resistant to carbapenems (Kilic et al., 2008;Kuo et al., 2010;Sung et al., 2014). Moreover, other studies conducted in France, Croatia, Japan and China reported the detection of multidrug resistant strains of A. schindleri, A. guillouiae, A. soli, A. ursingii and A. beijerinckii isolated from clinical samples (Dortet et al., 2006;Bošnjak et al., 2014;Endo et al., 2014;Fu et al., 2015;Quiñones et al., 2015). Based on these results, one can presume that other species of Acinetobacter will be discovered soon in human infections thanks to more efficient molecular techniques used for bacterial identification.
In conclusion, even if the present data derived from only few studies, it seems that almost all of the Acinetobacter species are widely distributed in nature and that the contaminated environment may enhance the growth of these microorganisms. Further studies are nevertheless required to understand the behavior of Acinetobacter spp. and to elucidate the mode of transmission of those bacteria from these different habitats to humans.

AUTHOR CONTRIBUTIONS
AA, MJ, MH, and MK contributed to the conception and design of the work, and to the acquisition and interpretation of the data. All authors contributed to the drafting of the manuscript and approved the final version to be published.