Significance This study illustrates the dynamics of the oral microbiome during long-term starvation. After an initial ecological collapse, only three species were recoverable and displayed significant transcriptional activity: Klebsiella pneumoniae, Klebsiella oxytoca, and Providencia alcalifaciens. Klebsiella spp. are significant human pathogens and are frequently resistant to multiple classes of antibiotics. In addition to its status as a clinical scourge in its own right, K. pneumoniae has emerged as a chief facilitator in the transfer of drug resistance genes from the environment to pathogens. Hospital surfaces contaminated with oral fluids are well-documented sources of outbreaks of drug-resistant Enterobacteriaceae; therefore, the ability of Klebsiella to outcompete its neighbors during starvation and survive long-term in saliva is particularly noteworthy.

Abstract It is well-understood that many bacteria have evolved to survive catastrophic events using a variety of mechanisms, which include expression of stress-response genes, quiescence, necrotrophy, and metabolic advantages obtained through mutation. However, the dynamics of individuals leveraging these abilities to gain a competitive advantage in an ecologically complex setting remain unstudied. In this study, we observed the saliva microbiome throughout the ecological perturbation of long-term starvation, allowing only the species best equipped to access and use the limited resources to survive. During the first several days, the community underwent a death phase that resulted in a ∼50–100-fold reduction in the number of viable cells. Interestingly, after this death phase, only three species, Klebsiella pneumoniae, Klebsiella oxytoca, and Providencia alcalifaciens, all members of the family Enterobacteriaceae, appeared to be transcriptionally active and recoverable. Klebsiella are significant human pathogens, frequently resistant to multiple antibiotics, and recently, ectopic colonization of the gut by oral Klebsiella was documented to induce dysbiosis and inflammation. MetaOmics analyses provided several leads for further investigation regarding the ecological success of the Enterobacteriaceae. The isolates accumulated single nucleotide polymorphisms in known growth advantage in stationary phase alleles and produced natural products closely resembling antimicrobial cyclic depsipeptides. The results presented in this study suggest that pathogenic Enterobacteriaceae persist much longer than their more benign neighbors in the salivary microbiome when faced with starvation. This is particularly significant, given that hospital surfaces contaminated with oral fluids, especially sinks and drains, are well-established sources of outbreaks of drug-resistant Enterobacteriaceae.

Many bacteria are well-equipped to deal with exposure to adverse environmental events such as starvation, oxidative stress, and antimicrobials, as well as fluctuations in temperature, pH, and osmolality. Diverse strategies for coping with these stresses have evolved, including expression of stress response genes (1), quiescence (2), necrotrophy (3), and growth advantages gained through mutation (4). Although these systems are increasingly understood, little is known regarding the dynamics of individual species leveraging these abilities to gain a competitive advantage in an ecologically complex setting. In the case of long-term starvation, the bulk of research on bacterial dynamics and survival mechanisms has been performed using monospecies cultures of Escherichia coli (4⇓⇓⇓⇓–9). In E. coli, populations exhibited a death phase at ∼3 d of starvation, resulting in a loss of >99% of the population (4). This death phase was followed by a long-term stationary phase, in which viable cell counts plateau virtually indefinitely (i.e., for years) (4). During the long-term stationary phase, various subpopulations carrying advantageous mutations (growth advantage in stationary phase [GASP] mutants) arose and came to dominate the culture, displacing their less-fit siblings (4). These mutations frequently resulted in increased ability to catabolize one or more amino acids as a source of carbon and energy (4). Therefore, it was likely that in a complex multispecies community, a succession of species and strains would increase and decrease in relative abundance throughout the course of the experiment, based on which species were most fit in the changing environment.

To our knowledge, there have been no previous reports on the interplay of a complex community of human-associated bacteria subjected to long-term starvation. To begin to address this knowledge gap, the present study monitored a saliva-derived complex oral microbiota during starvation in saline solution and saliva for 100 d. The bacterial community residing in the human oral cavity is a particularly unique microbiota that undergoes cyclical expansions and contractions of microbial diversity as a result of both host food ingestion intervals and hygienic practices, such as brushing and use of mouthwash. These perturbations respectively result in cycles of relative feast or famine and regular killing or removal of large swaths of the microbial population, after which ecological succession begins anew (10). Dental caries and periodontitis represent two extremely prevalent and costly diseases that are now recognized as the result of localized ecological catastrophes of the oral microbiome (11⇓⇓–14). One of the major impediments to the study of complex, human-associated microbial communities is the difficulty cultivating such diverse ecologies in a well-controlled laboratory setting. The oral microbiota was chosen as a model system for this pilot study because of the existence of a well-established in vitro culture system using media that allows for the growth of a diversity of species approaching that of an in vivo human mouth (15).

Discussion This study provides an account of a complex, human-associated microbial community experiencing the ecological perturbation of long-term starvation. The finding that Klebsiella and Providencia species were the apparent sole survivors in a community after long-term starvation is significant and highly intriguing. K. pneumoniae, K. oxytoca, and P. alcalifaciens are all members of the Enterobacteriaceae family of Proteobacteria. K. pneumoniae is a significant pathogen and represents the ‘K’ in the ESCKAPE pathogens, a group of organisms frequently resistant to multiple antibiotics (16). K. oxytoca and P. alcalifaciens are also opportunistic pathogens and are frequently drug resistant (17, 18). Oral K. pneumoniae was recently shown to induce inflammation and dysbiosis in the gut after ectopic colonization, and it was hypothesized that the oral cavity provides a reservoir for would-be intestinal pathogens, such as Klebsiella (33). Furthermore, aside from being a substantial pathogen in its own right, evidence is accumulating that K. pneumoniae serves as a key trafficker of drug resistance loci from the environment to human pathogens (34). The mechanisms employed by these Enterobacteriaceae to outlast their neighbors during long-term starvation await investigation. The Enterobacteriaceae encode among the largest genomes in the oral microbiome (35) and, as such, have added metabolic flexibility compared with Streptococci and other common constituents of the oral cavity (36). It is likely that during long-term starvation, species with reduced genomes have less metabolic flexibility and are at a significant disadvantage to Enterobacteriaceae (37). Klebsiella are diazotrophs, and all Enterobacteriaceae are capable of using nitrate, S-oxides, and N-oxides as terminal electron acceptors (36). Thus, the concept that these abilities were advantageous during long-term starvation remains an attractive hypothesis. In addition, the MetaOmics analyses performed in this study provided several additional hypotheses for further investigation. The increased abundance of SNPs in several genes in K. pneumoniae and P. alcalifaciens may represent GASP mutations, which were originally discovered in E. coli, another member of the family Enterobacteriaceae (4). Overall analysis of the Enterobacteriaceae transcriptome indicated that the species may be attempting to conserve energy and use passive transport to locate a novel food source. Meanwhile, several intriguing cyclic depsipeptides may have been employed by the Enterobacteriaceae to kill their neighbors. The results presented here also illustrate the value of RNA-based detection methods. Although a large number of taxa were present at all points, according to sequencing of 16S amplicons, as well as metagenomes, sequencing of mRNA revealed a much more drastic reduction of species during and after the death phase. This loss of diversity was also reflected in the sequencing of the outgrowth communities and the plating assay, from which only the three Enterobacteriaceae species were recoverable at day 20 under the conditions tested, despite the presence of DNA from a multitude of species in the community at that time. Because some bacteria are known to enter a viable but not culturable state during adverse growth conditions, the lack of taxonomic diversity at the transcriptional level during the later points of starvation serves as an important validation that the colony-forming units per milliliter and recoverable species on solid media reported here are not largely underestimated. The ability of the Enterobacteriaceae to survive longer than other members of the saliva microbial community may have a great deal of clinical significance. Although the long-term starvation model used in this study is unlikely to simulate the oral cavity, where periods of starvation are much shorter, it is presumably analogous to the succession that occurs when human saliva is deposited in environmental locations not exposed to rapid desiccation. Contaminated hospital surfaces, particularly sinks, have been the source of outbreaks of multidrug-resistant Klebsiella (17, 38, 39). It is therefore easy to imagine a scenario in which Enterobacteriaceae survive for extended periods in mixtures of saliva and water in sinks and drains, where aerosol formation after subsequent use of the sink leads to spread of the infection. This danger is compounded by the frequent horizontal gene transfer of resistance genes employed by Enterobacteriaceae (34, 39). Elucidation of the mechanisms used by the Enterobacteriaceae to survive long-term starvation in a community setting is highly important, and further research is currently in progress.

Materials and Methods More detailed methods with additional references are available in the SI Appendix. The starting bacterial community (S-mix), derived from the saliva of six healthy subjects, ages 25–35 y, has been described previously (15). After overnight growth in SHI medium in microaerophilic conditions (2%O 2 , 5%CO 2 , 93%N 2 ), 1 mL aliquots of S-mix were starved in either 1× PBS or a 1:1 mixture of 1× PBS and cell-free saliva. Colony-forming units per milliliter was determined by growth for 72 h on SHI media agar under microaerophilic conditions. 16S rDNA taxonomic profiling was performed by Illumina sequencing of V2-V4 amplicons, followed by analysis using QIIME. Metagenomes were assembled de novo using SPAdes and aligned using Mauve. Metatranscriptomic reads were mapped to Enterobacteriaceae genomes using Burrows-Wheeler alignment, and genes that were significantly differentially expressed were identified using DeSeq2. Natural products analysis was performed using reverse-phase high-pressure liquid chromatography followed by tandem mass spectrometry, as detailed in the SI Appendix. Raw sequencing data have been deposited in the Sequence Read Archive (SRA) (40, 41).

Acknowledgments The authors thank Roberta Faustoferri for helpful proofreading of the manuscript. This study was supported by National Institutes of Health/National Institute of Dental and Craniofacial Research Grants F32-DE026947 (to J.L.B.), R00-DE024543 (to A.E.), and R01-DE020102 and R01-DE026186 (to X.H., J.S.M., and W.S.).

Footnotes Author contributions: J.L.B., X.H., J.S.M., and W.S. designed research; J.L.B., E.L.H., X.T., A.E., and J.S.M. performed research; J.L.B., E.L.H., R.L., X.H., A.E., J.S.M., and W.S. analyzed data; and J.L.B. wrote the paper.

Conflict of interest statement: J.L.B. is a part-time consultant for uBiome, Inc. W.S. is a part-time chief science officer of C3J Therapeutics, Inc., which has licensed technologies from the University of California Regents that could be indirectly related to this research project.

This article is a PNAS Direct Submission.

Data deposition: The raw sequencing files used in the genomic, metagenomic, and transcriptomic analyses in this study have been deposited in the Sequence Read Archive, https://www.ncbi.nlm.nih.gov/sra [accession nos. PRJNA525688 (genomes) and PRJNA525517 (metagenomes/transcriptomes)].

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1820594116/-/DCSupplemental.