Increasing throughput, decreasing cost, and rapid development of informatics and sequencing pipelines has reshaped the field of microbial ecology, allowing researchers to survey a breadth of new environments [34, 61–63]. Recently, the first ICU survey to utilize next generation sequencing technology was published  and showed a surprising amount of bacterial diversity for an environment under constant attack via aggressive sanitation and antibiotic treatment efforts. The consortia were generally diverse, but some consortia contained a high representation of members of the family Enterobacteriaceae, typically considered to be gut microbes. Shortly after this publication, a study characterizing a snapshot of surfaces and sinks in two NICU rooms corroborated high proportions of fecal coliform bacteria on surface samples . Certainly the NICU has the capacity to retain enteric microbes, but their propensity to migrate to the gut remains unclear.
Next-generation sequencing surveys in the ICU have reported high levels of community diversity. Poza et al. found 1,145 distinct OTUs in an ICU in Spain  and subsequent studies reported 1,621 and 3,925 OTUs in a NICU in the US and in an Austrian ICU, respectively [9, 10]. While comparing these studies is difficult due to differences in sample size and protocols, we can begin to appreciate the need to better understand why so many types of bacteria can be found in a regularly cleaned environment. Our study, the first time series survey of an ICU using next-generation sequencing technologies, unveiled over 20,000 OTUs across 2 NICU rooms occupied by different infants with partial time overlap. Our study is distinct from prior NICU surveys in that it used amplicon-EMIRGE, a 16S rRNA gene assembly software which can be more sensitive in OTU detection  and provide increased confidence when making lower taxonomic level classifications . The increase in OTUs from study to study might be attributed to increases in sequencing read lengths and, in this study, increased information from reassembled, full-length genes, but the biological relevance of this increase is unclear. Notably, of the over 20,000 OTUs characterized here, only 984 were found in 2 or more samples. Further surveys are needed, integrating time-series sampling and samples from multiple surface types from different hospitals, to better characterize the expected number of OTUs in an ICU and the implications of this number for ICU occupants.
The increased sensitivity provided by EMIRGE was helpful when evaluating temporal patterns, especially pertaining to source-sink characterization. Similarly, our source-sink analyses benefited from the increased number of samples and timepoints relative to prior studies [8–10], which did not attempt to identify source-sink relationships. The SourceTracker results suggest the most probable room reservoir for gut colonists is tubing followed by surfaces, incubators, and hands (Figure 6). The tubing area sampled, the hub of the silastic nasogastric feeding tube, is the closest in proximity to the infant and, since SourceTracker is not bidirectional, it is difficult to tease out the directionality in this exchange . Incubators from both infants also appear to mirror successional patterns in the infant’s GIT, but without finer scale temporal sampling it is difficult to determine the true source and sink. The observation that hands tend to show a variable amount of potential fecal colonist is likely due to the variability in sampling and hand hygiene, as hand samples were taken both before and after infants received care from healthcare providers. A good example of this is infant 1’s DOL 27 hand sample in which the large spike in Escherichia likely came from a swab collected directly after contact with the infant (Figure 5).
Given the large inventory of sequences and the time-series dataset, it was possible to identify likely reservoirs of microorganisms in the room environment, prior to their appearance in the GIT (for example, the asterisked OTUs in Figure 4). Many of these sequences had perfect or near perfect identity between room and GIT 16S rRNA genes. Two notable examples include the Klebsiella pneumoniae in infant 1 and Finegoldia magna in infant 2, whose fecal to room sequence best hits averaged 99.4% and 99.6% identity respectively. Infant 1’s K. pneumoniae is first detectable in the gut on DOL 9, but NICU samples first detect the organism on electronic and sink samples starting at DOL 3, our earliest sampling point. Interestingly, the K. pneumoniae is outcompeted in the gut, yet is reintroduced on two separate occasions. This could be a byproduct of our detection limits, but the relatively high abundance of Klebsiella in many NICU samples and its availability at all timepoints, suggests the opportunity for reinoculation from multiple room reservoirs. The F. magna in infant 2’s samples exhibit similar patterns in that it is initially a high-ranking taxa that is out competed by other Firmicutes, but is reintroduced later in the time series.
If the environment is a reservoir for gut colonizing microbes in our cohort, then it is likely infants housed in close proximity will share the same strain. The 16S rRNA gene survey shows the availability of reservoirs of colonizing populations (likely with multiple strain variants) in the infant’s immediate environment. However, it cannot discriminate at the strain level, so the mere existence of a phylotype in the room prior to gut colonization is not a direct measure of BE to infant transfer. The current work resolves this, by using extensive genome sequence comparison of E. faecalis from the gut of two infants housed in the same ward to establish that environment to room occupant transfer occurs in the NICU. The mode of acquisition of infant 2’s abundant strain by infant 1 is unclear, but nosocomial infection by enterococci is not uncommon.
Enterococci are particularly difficult to classify due the plasticity of their genomes. Upwards of 25% of E. faecalis genomes may be comprised of mobile or acquired elements . Recent experiments attribute this genome flexibility partially to the ability to produce transconjugant hybrid strains in which several 100 kb fragments can be transferred between donor and recipient strain . Transfer of these genome fragments is dependent on pheromone-responsive plasmids, which were found in all strains studied here. The ability to form hybrids not only confounds the ability to confirm identical strains, unless the entire genome has been recovered, it also provides a competitive advantage in the hospital BE where enterococci have been problematic for decades [65, 67]. Enterococci are notoriously hardy and are able to persist on medical equipment and hospital surfaces for long periods of time [65, 68]. They are able to withstand chlorine, heat, some alcohol treatments, and possibly most concerning, several types of antibiotics . Their genome plasticity and ability to easily acquire new genes from other strains make them particularly well suited to thrive in the hospital environment.
Gut colonists must withstand selective pressures both inside and out of the gut. Two obvious forms of selection in the NICU come from hospital cleaning and the broad use of antibiotics. All rooms were cleaned daily using wet solutions containing QACs and all infants were administered multiple types of antibiotics. Incorrect administration of biocides, through misuse or unintended mixing with existing fluids (that is, water from sink samples or removing sanitizing agents via water rinsing), could enrich for resistance genes . Even if used to factory standards, if surface-dried cells or biofilms remain, biocide activity could be ineffective and contribute to cross resistance to biocides and antibiotics . Biofilm forming communities can be upwards of 1,000 times more resistant to QACs than their planktonic forms  and biofilm formation can be triggered by the types of antibiotics administered in this study . This may be a contributing factor as to why a recent study found enteric microbial communities to be relatively unaltered before and after routine NICU surface cleaning . Certain types of biofilms in many Enterobacteriaceae, including those studied here, contain amyloid fibers, called curli. Curli have been implicated in adhesion to abiotic surfaces, such as polystyrene, Teflon, and stainless steel, and contribute to adhesion to host epithelial cells and invasion by Escherichia coli in the gut . This type of dual-purpose adaptation may allow enteric organisms to persist on NICU surfaces until transmission to a more favorable environment such as the gut. Efflux pumps are another multipurpose adaptation conferring competitive advantages inside and out of the gut. Numerous pumps from every major class of efflux pump were identified here and, collectively, can function to pump out QACs and administered antibiotics. Previous studies have positively correlated high QAC minimum inhibitory concentrations (MICs) with increased antibiotic resistance markers in enteric microbes , indicating biocide efflux may be an important function for microbes in the ICU. Efflux and biofilm formation are two of many possible explanations as to how colonizers combat both biocides administered during NICU cleaning and host-administered antibiotics.