Suppression of Enteric Bacteria by Bacteriophages: Importance of Phage Polyvalence in the Presence of Soil Bacteria

Bacteriophages are widely recognized for their importance in microbial ecology and bacterial control. However, little is known about how phage polyvalence (i.e., broad host range) affects bacterial suppression and interspecies competition in environments harboring enteric pathogens and soil bacteria. Here we compare the efficacy of polyvalent phage PEf1 versus coliphage T4 in suppressing a model enteric bacterium (<i>E. coli</i> K-12) in mixtures with soil bacteria (<i>Pseudomonas putida</i> F1 and <i>Bacillus subtilis</i> 168). Although T4 was more effective than PEf1 in infecting <i>E. coli</i> K-12 in pure cultures, PEf1 was 20-fold more effective in suppressing <i>E. coli</i> under simulated multispecies biofilm conditions because polyvalence enhanced PEf1 propagation in <i>P. putida</i>. In contrast, soil bacteria do not propagate coliphages and hindered T4 diffusion through the biofilm. Similar tests were also conducted under planktonic conditions to discern how interspecies competition contributes to <i>E. coli</i> suppression without the confounding effects of restricted phage diffusion. Significant synergistic suppression was observed by the combined effects of phages plus competing bacteria. T4 was slightly more effective in suppressing <i>E. coli</i> in these planktonic mixed cultures, even though PEf1 reached higher concentrations by reproducing also in <i>P. putida</i> (7.2 ± 0.4 vs 6.0 ± 1.0 log₁₀PFU/mL). Apparently, enhanced suppression by higher PEf1 propagation was offset by <i>P. putida</i> lysis, which decreased stress from interspecies competition relative to incubations with T4. In similar planktonic tests with more competing soil bacteria species, <i>P. putida</i> lysis was less critical in mitigating interspecies competition and PEf1 eliminated <i>E. coli</i> faster than T4 (36 vs 42 h). Overall, this study shows that polyvalent phages can propagate in soil bacteria and significantly enhance suppression of co-occurring enteric species

Phage Predation Promotes Filamentous Bacterium <i>Piscinibacter</i> Colonization and Improves Structural and Hydraulic Stability of Microbial Aggregates

Although bacteria–phage interactions have broad environmental applications and ecological implications, the influence of phage predation on bacterial aggregation and structural stability remains largely unexplored. Herein, we demonstrate that inefficient lytic phage predation can promote host filamentous bacterium Piscinibacter colonization onto non-host Thauera aggregates, improving the structural and hydraulic stability of the dual-species aggregates. Specifically, phage predation at 103–104 PFU/mL (i.e., multiplication of infection at 0.01–0.1) promoted initial Piscinibacter colonization by 10–15 folds and resulted in 29–31% higher abundance of Piscinibacter in the stabilized aggregates than that in the control aggregates without phage predation. Transcriptomic analysis revealed upregulated genes related to quorum sensing (by 15–92 folds) and polysaccharide secretion (by 10–90 folds) within the treated aggregates, which was consistent with 120–172% higher content of polysaccharides for the treated dual-species aggregates. Confocal laser scanning microscopic images further confirmed the increase of filamentous bacteria and polysaccharides (both with wider distribution) within the dual-species aggregates. Accordlingly, the aggregates’ structural strength (via atomic force microscopes) and shear resistance (via hydraulic stress tests) increased by 77 and 42%, respectively, relative to the control group. In the long-term experiments, the enhanced hydraulic stability of the treated aggregates could facilitate dwelling bacteria propagation in flow-through conditions. Overall, our study demonstrates that phage predation can promote bacterial aggregation and enhance aggregate structural stability, revealing the beneficial role of lytic phage predation on bacterial symbiosis and environmental adaptivity

Biofilm Control in Flow-Through Systems Using Polyvalent Phages Delivered by Peptide-Modified M13 Coliphages with Enhanced Polysaccharide Affinity

Eradication of biofilms that may harbor pathogens in water distribution systems is an elusive goal due to limited penetration of residual disinfectants. Here, we explore the use of engineered filamentous coliphage M13 for enhanced biofilm affinity and precise delivery of lytic polyvalent phages (i.e., broad-host-range phages lysing multiple host strains after infection). To promote biofilm attachment, we modified the M13 major coat protein (pVIII) by inserting a peptide sequence with high affinity for Pseudomonas aeruginosa (P. aeruginosa) extracellular polysaccharides (commonly present on the surface of biofilms in natural and engineered systems). Additionally, we engineered the M13 tail fiber protein (pIII) to contain a peptide sequence capable of binding a specific polyvalent lytic phage. The modified M13 had 102- and 5-fold higher affinity for P. aeruginosa-dominated mixed-species biofilms than wildtype M13 and unconjugated polyvalent phage, respectively. When applied to a simulated water distribution system, the resulting phage conjugates achieved targeted phage delivery to the biofilm and were more effective than polyvalent phages alone in reducing live bacterial biomass (84 vs 34%) and biofilm surface coverage (81 vs 22%). Biofilm regrowth was also mitigated as high phage concentrations induced residual bacteria to downregulate genes associated with quorum sensing and extracellular polymeric substance secretion. Overall, we demonstrate that engineered M13 can enable more accurate delivery of polyvalent phages to biofilms in flow-through systems for enhanced biofilm control

Control of Antibiotic-Resistant Bacteria in Activated Sludge Using Polyvalent Phages in Conjunction with a Production Host

Bacteriophage-based microbial control could help address a growing need to attenuate the proliferation of antibiotic-resistant bacteria (ARB) in wastewater treatment plants (WWTPs). However, the infectivity of commonly isolated narrow-host-range phages decreases quickly upon addition to activated sludge (i.e., plaque-forming units had a half-life of 0.63 h). Here, we show that polyvalent (broad-host-range) phages proliferate and thrive in activated sludge microcosms, especially when added along with their production hosts. Polyvalent phage cocktails (PER01 and PER02) were significantly more effective than narrow-host-range coliphage cocktails (MER01 and MER02) in suppressing a model ARB [β-lactam-resistant <i>Escherichia coli</i> NDM-1, initially present at 6.2 ± 0.1 log₁₀ colony-forming units (CFU)/mL]. After 5 days, the NDM-1 concentration significantly decreased to 3.8 ± 0.2 log₁₀ CFU/mL in the presence of the polyvalent phage cocktail, compared to 4.7 ± 0.3 log₁₀ CFU/mL for the coliphage cocktail treatment. Because of the presence of alternative hosts, polyvalent phages reached greater densities, which increased the probability of ARB infection. The fraction of surviving <i>E. coli</i> harboring the <i>bla</i>_NDM‑1 resistance gene was also significantly lower for the polyvalent phage cocktail treatment (0.57 ± 0.07) than for the control (0.74 ± 0.08). Therefore, polyvalent phages safely produced with nonpathogenic hosts could offer a novel approach to controlling problematic ARB in WWTPs and mitigating the propagation and discharge of associated resistance genes to the environment

1,4-Dioxane Biodegradation by <i>Mycobacterium dioxanotrophicus</i> PH-06 Is Associated with a Group‑6 Soluble Di-Iron Monooxygenase

1,4-Dioxane (dioxane) is a groundwater contaminant of emerging concern for which bioremediation may be a promising strategy. Several bacterial strains can metabolize dioxane or degrade it cometabolically. However, the molecular basis of dioxane biodegradation is only partially understood, and the gene coding for dioxane/tetrahydrofuran (THF) monooxygenase in Pseudonocardia dioxanivorans CB1190 is the only well-characterized catabolic gene. Here, we identify a novel group-6 propane monooxygenase gene cluster (<i>prmABCD</i>) in Mycobacterium dioxanotrophicus PH-06, which is a bacterium with superior dioxane degradation kinetics compared with CB1190. Whole genome sequencing of PH-06 revealed the existence of a single soluble di-iron monooxygenase (SDIMO). RNA sequencing and reverse transcription quantitative PCR (RT-qPCR) subsequently confirmed that all four components of this gene cluster are upregulated when PH-06 is grown on dioxane compared with growth on acetate or glucose as negative controls. This first characterization of a group-6 SDIMO associated with dioxane biodegradation suggests that dioxane-degrading genes may be more diverse than previously appreciated. A primer/probe set designed to target the large hydroxylase subunit of this gene cluster exhibited high selectivity (no false positives) and high sensitivity (detection limit = 3000–4000 gene copies/mL culture), which may be useful to help assess the presence of dioxane degraders at contaminated sites and minimize false negatives

Elevated Levels of Pathogenic Indicator Bacteria and Antibiotic Resistance Genes after Hurricane Harvey’s Flooding in Houston

Urban flooding can dramatically affect the local microbial landscape and increase the risk of waterborne infection in flooded areas. Hurricane Harvey, the most destructive hurricane since Katrina in 2005, damaged more than 100000 homes in Houston and flooded numerous wastewater treatment plants. Here we surveyed microbial communities in floodwater inside and outside residences, bayou water, and residual bayou sediment collected immediately postflood. Levels of <i>Escherichia coli</i>, a fecal indicator organism, were elevated in bayou water samples as compared to historical levels, as were relative abundances of key indicator genes of anthropogenic sources of antibiotic resistance (<i>sul</i>1/16S rRNA and <i>intI</i>1/16S rRNA) based on 6 month postflood monitoring. Quantitative polymerase chain reaction measurements showed that gene markers corresponding to putative pathogenic bacteria were more abundant in indoor floodwater than in street floodwater and bayou water. Higher abundances of 16S rRNA and <i>sul</i>1 genes were also observed in indoor stagnant waters. Sediments mobilized by floodwater exhibited an increased abundance of putative pathogens postflood in both residential areas and public parks. Overall, this study demonstrates that extreme flooding can increase the level of exposure to pathogens and associated risks