Multi-drug resistant Enterobacter bugandensis species isolated from the International Space Station and comparative genomic analyses with human pathogenic strains

Enterobacter species are facultative anaerobic, Gram-stain-negative, and saprophytic microorganisms found in soil, sewage, and as a commensal enteric flora of the human gastrointestinal tract [1]. They have been associated with nosocomial infection in humans, causing bacteremia, endocarditis, septic arthritis, osteomyelitis, skin and soft tissue infections, lower respiratory tract, urinary tract, and intra-abdominal infections [2, 3]. Some Enterobacter have also been reported plant pathogens [4]. Antibiotic resistance and its clinical implications have been extensively studied in genus Enterobacter, especially Enterobacter cloacae, which is resistant to cephalosporins, ampicillin, amoxicillin, and cefoxitin [5, 6].

In an ongoing effort of the International Space Station (ISS) Microbial Observatory investigation, the National Aeronautics and Space Administration (NASA) is cataloging the total and viable microbial communities of crew-associated environments using cultivation and molecular techniques of microbial detection [7, 8]. As a result, five isolates belonging to the Enterobacter bugandensis group of bacteria from two different locations of the ISS were isolated [9]. Since the initial molecular screening identified these strains as Enterobacter but the identification was not able to resolve their taxonomy to species level, detailed genomic characterizations were warranted in addition to the traditional microbiological characterization. Due to its unstable taxonomic structure, methods utilized for the speciation of Enterobacter varied widely. Commercial biochemical typing systems such as API® 20E [10] or Vitek® 2, and matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) [11] methods have been used, but with limited success. On the basis of 16S rRNA analysis, Enterobacter was structured as a polyphyletic genus and most of the species could not be resolved [1]. Therefore, multilocus sequence typing (MLST) analysis was found to be more appropriate for phylogenetic classification of Enterobacter species [12].

To resolve this question further, whole genome sequencing (WGS) and de novo assembly was performed on all five ISS E. bugandensis strains, creating MLST and genome variation profiles of the ISS strains [13]. Furthermore, comparative genome alignment of the ISS strains with all publicly available 1291 Enterobacter genomes revealed that genomes of these five ISS strains were highly similar to only three clinical E. bugandensis with very high genome similarities and formed a unique ecotype. They are (a) EB-247 strain [13], isolated from neonatal blood of a patient from Tanzania, (b) 153_ECLO strain [14], isolated from the urine of a neonatal patient strain admitted to the University of Washington Medical Center, Seattle, WA and (c) MBRL 1077 strain, a carbapenemase-producing strain [15] isolated from the wound of a 72-year-old woman with a history of cutaneous scleroderma, medically complicated obesity, and venous insufficiency. In this study, comparative genomic analyses of five ISS strains and three clinical isolates were carried out to elucidate antimicrobial resistance (AMR) phenotypic properties, MDR gene profiles, and genes related to potential virulence and pathogenic potential of the ISS Enterobacter strains.

Sample collection from ISS environmental surfaces, processing, cultivation of bacteria were already reported [9]. When 105 bacterial strains isolated from various ISS locations were analyzed for their phylogenetic affiliations, five isolates were identified as Enterobacter bugandensis. The five Enterobacter isolates characterized during this study were isolated from two different locations of the ISS flight in March 2015. Four isolates were isolated from the waste and hygiene compartment (WHC), and one strain from the Advanced Resistive Exercise Device (ARED) foot platform of ISS.

In summary, a comparative phenotypic and genotypic analyses of ISS isolates identified as E. bugandensis were carried out. Additional genomic analyses revealed a close genetic relatedness between ISS isolates and nosocomial earth isolates. MLST and whole genome SNP tree placed ISS and nosocomial isolates to a separate clade when phylogenetically aligned with other member of genus Enterobacter. A detailed functional and antimicrobial resistance analysis reveals that the ISS isolates have a 79% probability of being a human pathogen and share similar antimicrobial resistance pattern with E. bugandensis EB-247, MBRL-1077 and 153_ECLO strains, making them relevant for future missions and crew health considerations.

A total of 112 identified genes of the ISS strains were involved in virulence, disease, and defense. Genes associated with resistance to antibiotics and toxic compounds, including the multidrug resistance tripartite system (also known as 3-protein systems) as shown in a polychlorinated biphenyl-degrader, Burkholderia xenovorans LB400 [39], was noticed in the ISS strain. This protein forms the basic structure and plays a crucial role in, the functioning of an efflux pump rendering a microbe drug resistant [40, 41]. A multiple antibiotic resistance (MAR) locus or MAR operon was observed in ISS strains, which codes for protein MarA, MarB, MarC, and MarR, and regulate more than 60 genes, including upregulation of drug efflux systems that have been reported in Escherichia coli K12 [42–44]. Aminoglycoside adenylyltransferases, whose role is spectinomycin 9-O-adenylyltransferases, which confers microbial resistance to the aminoglycosides in Salmonella enterica, was also seen in ISS strains [45]. Similarly, resistance to fluoroquinolones due to a mutation in gyrA gene in S. enterica [46], and fosfomycin resistance due to the presence of FosA protein-coding gene, which catalyzes the addition of glutathione to C1 of the oxirane in Serratia marcescens [47], were observed in ISS strains. Multiple copies of multi-drug resistance (MDR) genes highly homologous to S. marcescens, a pathogen, were identified in the ISS Enterobacter genomes, which gives an indication that these strains may be a potential human pathogen. When tested with PathogenFinder [48] algorithm, strain IF2SW-P2^T had > 77% probability to be a human pathogen. When compared with E. cloacae ATCC 13047, which is a well-described human pathogen [49], all five ISS strains showed a > 79% probability score.

Astronauts have been taking beta-lactam based medical drugs for approximately two decades, and ß-lactamase (superfamily I [metal dependent hydrolases] and E.C.3.5.2.6) was present in all strains under study, while penicillin-binding proteins (PPB4B) were only present in MBRL-1077. Fluoroquinolone resistance due to gyrase and topoisomerase mutation was present in all the strains. Metal-dependent hydrolases, cation efflux system protein CusA, cobalt-zinc-cadmium resistance protein, cobalt-zinc-cadmium resistance protein CzcA, DNA-binding heavy metal response regulator, Co/Zn/Cd efflux system membrane fusion protein, zinc transporter ZitB were found in both ISS isolate and nosocomial organism understudy. These genes principally help in detoxification of periplasm by exporting toxic metal cation outside the cell. Determinants of the metal resistance are usually located on the plasmid and readily acquired from the environment and also complement antibiotic resistance [50, 51]. The plasmid encoded putative transcriptional regulators containing the CopG/Arc/MetJ DNA-binding domain and a metal-binding domain were present in the ISS strains (Additional file 2: Table S2). Further studies are required for phenotypic characterization to confirm this trait. Presence of active beta lactamase gene, efflux pump, and RND (resistance, nodulation and cell division protein family) protein family renders broad-spectrum resistance to ISS isolates from drugs and natural inhibitors.

We have recently observed that competency of bacteria to acquire foreign genetic material increases in microgravity (in preparation) and similar mechanism for metal resistance of ISS strain was also predicted. Antimicrobial and metal resistance is also conferred by RND genes [52], which were present in all the strains under study. Genomic analysis reveals the presence of genes associated with MDR efflux pump, belonging to RND, which are reported to be the major contributors of resistance to antibiotic and other toxic compounds to the bacteria [41]. RND efflux system, inner membrane transporter CmeB, membrane fusion protein CmeA, outer membrane lipoprotein CmeC, outer membrane lipoprotein NodT family were found in all strains. These become important for the future space studies, as MDR has been reported to play role in the physiological function and confer resistance to the substances like bile, hormone and host defense molecule [53], which can make bacteria a dominant persistor and lead to pathogenicity in humans.

The genomic characterizations showed that the ISS Enterobacter strains might potentially exhibit pathogenicity to human. However, the pathogenicity of the ISS strains compared to clinical strains isolated from patients should be explored in vivo experiments before making any assumption about whether these potential AMR gene markers are due to spaceflight changes or not. Moreover, the transit time and route for the organisms from the ISS may have some small impact on the response or physiological traits of the bacteria. WGS is still an important tool to monitor transmission routes of opportunistic pathogen bacteria [25, 26]. To avoid this, future missions could utilize Nanopore sequencing directly in microgravity as well as additional function and taxonomic classification methods [26, 54], and then leverage the above detailed analytic steps to gauge relevance for crew health and safety.