Recovery of Previously Uncultured Bacterial Genera from Three Mediterranean Sponges

Sponges often harbour a dense and diverse microbial community. Presently, a large discrepancy exists between the cultivable bacterial fraction from sponges and the community in its natural environment. Here, we aimed to acquire additional insights into cultivability of (previously uncultured) bacteria from three sponge species, namely Aplysina aerophoba, Corticium candelabrum and Petrosia ficiformis, by studying bacterial growth on five media in the form of 60 communities scraped from plates without antibiotics, as well as in the form of individual isolates that were grown on these media supplemented with antibiotics. We applied (double-)barcoded 16S ribosomal RNA (rRNA) gene amplicon sequencing for species identification. We show that previously uncultured bacteria can be cultivated using conventional plating and that application of antibiotics in the media can serve to capture a greater bacterial diversity. Moreover, we present criteria to address an important caveat of the plate scraping method whereby bacteria may be detected that did not actually grow. Fourteen out of 27 cultivated novel taxa (<95% identity of the 16S rRNA gene amplicon to reported species) belong to Actinobacteria, which indicates the presence of a large untapped reservoir of bioactive compounds. Three Flavobacteriaceae spp. were isolated that potentially constitute two new genera and one new species.</p

Correction to: Recovery of Previously Uncultured Bacterial Genera from Three Mediterranean Sponges

The original version of this article unfortunately contained a mistake. In the “Nucleotide Sequence Accession Numbers” section, the accession number “PRJEB4784” that links to the deposited data is incorrect

High throughput cultivation-based screening on porous aluminum oxide chips allows targeted isolation of antibiotic resistant human gut bacteria

The emergence of bacterial pathogens that are resistant to clinical antibiotics poses an increasing risk to human health. An important reservoir from which bacterial pathogens can acquire resistance is the human gut microbiota. However, thus far, a substantial fraction of the gut microbiota remains uncultivated and has been little-studied with respect to its resistance reservoir-function. Here, we aimed to isolate yet uncultivated resistant gut bacteria by a targeted approach. Therefore, faecal samples from 20 intensive care patients who had received the prophylactic antibiotic treatment selective digestive decontamination (SDD), i.e. tobramycin, polymyxin E, amphotericin B and cefotaxime, were inoculated anaerobically on porous aluminium oxide chips placed on top of poor and rich agar media, including media supplemented with the SDD antibiotics. Biomass growing on the chips was analysed by 16S rRNA gene amplicon sequencing, showing large inter-individual differences in bacterial cultivability, and enrichment of a range of taxonomically diverse operational taxonomic units (OTUs). Furthermore, growth of Ruminococcaceae (2 OTUs), Enterobacteriaceae (6 OTUs) and Lachnospiraceae (4 OTUs) was significantly inhibited by the SDD antibiotics. Strains belonging to 16 OTUs were candidates for cultivation to pure culture as they shared 95% sequence identity with the closest type strain and had a relative abundance of 2%. Six of these OTUs were detected on media containing SDD antibiotics, and as such were prime candidates to be studied regarding antibiotic resistance. One of these six OTUs was obtained in pure culture using targeted isolation. This novel strain was resistant to the antibiotics metrodinazole and imipenem. It was initially classified as member of the Ruminococcaceae, though later it was found to share 99% nucleotide identity with the recently published Sellimonas intestinalis BR72T. In conclusion, we show that high-throughput cultivation-based screening of microbial communities can guide targeted isolation of bacteria that serve as reservoirs of antibiotic resistance.</p

Characterization of Enterococcus isolates colonizing the intestinal tract of intensive care unit patients receiving selective digestive decontamination

Enterococci have emerged as important opportunistic pathogens in intensive care units (ICUs). In this study, enterococcal population size and Enterococcus isolates colonizing the intestinal tract of ICU patients receiving Selective Digestive Decontamination (SDD) were investigated. All nine patients included in the study showed substantial shifts in the enterococcal 16S rRNA gene copy number in the gut microbiota during the hospitalization period. Furthermore, 41 Enterococcus spp. strains were isolated and characterized from these patients at different time points during and after ICU hospitalization, including E. faecalis (n = 13), E. faecium (n = 23), and five isolates that could not unequivocally assigned to a specific species (E. sp. n = 5)Multi locus sequence typing revealed a high prevalence of ST 6 in E. faecalis isolates (46%) and ST 117 in E. faecium (52%). Furthermore, antibiotic resistance phenotypes, including macrolide and vancomycin resistance, as well as virulence factor-encoding genes [asa1, esp-fm, esp-fs, hyl, and cyl (B)] were investigated in all isolates. Resistance to ampicillin and tetracycline was observed in 25 (61%) and 19 (46%) isolates, respectively. Furthermore, 30 out of 41 isolates harbored the erm (B) gene, mainly present in E. faecium isolates (78%). The most prevalent virulence genes were asa1 in E. faecalis (54%) and esp (esp-fm, 74%; esp-fs, 39%). Six out of nine patients developed nosocomial enterococcal infections, however, corresponding clinical isolates were unfortunately not available for further analysis. Our results show that multiple Enterococcus species, carrying several antibiotic resistance and virulence genes, occurred simultaneously in patients receiving SDD therapy, with varying prevalence dynamics over time. Furthermore, simultaneous presence and/or replacement of E. faecium STs was observed-, reinforcing the importance of screening multiple isolates to comprehensively characterize enterococcal diversity in ICU patients.</p

Comparative genomic analysis of Flavobacteriaceae : insights into carbohydrate metabolism, gliding motility and secondary metabolite biosynthesis

BACKGROUND: Members of the bacterial family Flavobacteriaceae are widely distributed in the marine environment and often found associated with algae, fish, detritus or marine invertebrates. Yet, little is known about the characteristics that drive their ubiquity in diverse ecological niches. Here, we provide an overview of functional traits common to taxonomically diverse members of the family Flavobacteriaceae from different environmental sources, with a focus on the Marine clade. We include seven newly sequenced marine sponge-derived strains that were also tested for gliding motility and antimicrobial activity. RESULTS: Comparative genomics revealed that genome similarities appeared to be correlated to 16S rRNA gene- and genome-based phylogeny, while differences were mostly associated with nutrient acquisition, such as carbohydrate metabolism and gliding motility. The high frequency and diversity of genes encoding polymer-degrading enzymes, often arranged in polysaccharide utilization loci (PULs), support the capacity of marine Flavobacteriaceae to utilize diverse carbon sources. Homologs of gliding proteins were widespread among all studied Flavobacteriaceae in contrast to members of other phyla, highlighting the particular presence of this feature within the Bacteroidetes. Notably, not all bacteria predicted to glide formed spreading colonies. Genome mining uncovered a diverse secondary metabolite biosynthesis arsenal of Flavobacteriaceae with high prevalence of gene clusters encoding pathways for the production of antimicrobial, antioxidant and cytotoxic compounds. Antimicrobial activity tests showed, however, that the phenotype differed from the genome-derived predictions for the seven tested strains. CONCLUSIONS: Our study elucidates the functional repertoire of marine Flavobacteriaceae and highlights the need to combine genomic and experimental data while using the appropriate stimuli to unlock their uncharted metabolic potential

Nationwide surveillance reveals frequent detection of carbapenemase-producing Enterobacterales in Dutch municipal wastewater

Carbapenemase-producing Enterobacterales (CPE) pose a threat to public health necessitating restriction of further spread. Tools to efficiently monitor the prevalence of these still relatively rare resistant bacteria and insight in routes of dissemination are pivotal for the development of prevention strategies. By analysis of untreated and treated wastewater from 100 municipal wastewater treatment plants (WWTPs) together serving over 40% of the Dutch population, this study investigated both the distribution of CPE in the Dutch population, and WWTPs as a source of CPE in the aquatic environment. CPE were detected at 89% of the WWTPs, in 87 influents and 53 effluents. Overall, 15 different CPE-types were detected based on species and carbapenemase gene. The most widely distributed were E. coli carrying blaOXA-48-like genes, which were detected at 88 WWTPs including small WWTPs without connected health care institutions (HCI). BlaOXA-48-like-positive-K. pneumoniae, blaNDM- or blaKPC-positive E. coli, and blaNDM- or blaKPC-positive K. pneumoniae were detected at 33, 20, and 14 WWTPs, respectively. Mean influent and effluent CPE concentrations were 7.9×102 cfu/l and 11 cfu/l. The total daily number of CPE discharged by the 100 WWTPs was estimated to be 2.2×1011 cfu. In multivariate analysis, CPE concentrations in untreated wastewater were positively associated with WWTP size and E. coli concentrations, but not with the presence of HCI. Based on the total number of CPE (9.7×1012 cfu) and ESBL-E. coli (2.41015 cfu) in influents, and a prevalence of approximately 5% in the Dutch population for ESBL-E. coli, the prevalence of CPE in the Dutch population was roughly estimated to be 0.02%. Wastewater surveillance is an efficient tool to monitor the distribution of CPE in the population at a national level and may supplement human surveillance data. CPE are emitted to the aquatic environment with treated wastewater and associated public health risks need to be determined

Nationwide surveillance reveals frequent detection of carbapenemase-producing Enterobacterales in Dutch municipal wastewater

Carbapenemase-producing Enterobacterales (CPE) pose a threat to public health necessitating restriction of further spread. Tools to efficiently monitor the prevalence of these still relatively rare resistant bacteria and insight in routes of dissemination are pivotal for the development of prevention strategies. By analysis of untreated and treated wastewater from 100 municipal wastewater treatment plants (WWTPs) together serving over 40% of the Dutch population, this study investigated both the distribution of CPE in the Dutch population, and WWTPs as a source of CPE in the aquatic environment. CPE were detected at 89% of the WWTPs, in 87 influents and 53 effluents. Overall, 15 different CPE-types were detected based on species and carbapenemase gene. The most widely distributed were E. coli carrying blaOXA-48-like genes, which were detected at 88 WWTPs including small WWTPs without connected health care institutions (HCI). BlaOXA-48-like-positive-K. pneumoniae, blaNDM- or blaKPC-positive E. coli, and blaNDM- or blaKPC-positive K. pneumoniae were detected at 33, 20, and 14 WWTPs, respectively. Mean influent and effluent CPE concentrations were 7.9×102 cfu/l and 11 cfu/l. The total daily number of CPE discharged by the 100 WWTPs was estimated to be 2.2×1011 cfu. In multivariate analysis, CPE concentrations in untreated wastewater were positively associated with WWTP size and E. coli concentrations, but not with the presence of HCI. Based on the total number of CPE (9.7×1012 cfu) and ESBL-E. coli (2.41015 cfu) in influents, and a prevalence of approximately 5% in the Dutch population for ESBL-E. coli, the prevalence of CPE in the Dutch population was roughly estimated to be 0.02%. Wastewater surveillance is an efficient tool to monitor the distribution of CPE in the population at a national level and may supplement human surveillance data. CPE are emitted to the aquatic environment with treated wastewater and associated public health risks need to be determined

Nationwide surveillance reveals frequent detection of carbapenemase-producing Enterobacterales in Dutch municipal wastewater

Carbapenemase-producing Enterobacterales (CPE) pose a threat to public health necessitating restriction of further spread. Tools to efficiently monitor the prevalence of these still relatively rare resistant bacteria and insight in routes of dissemination are pivotal for the development of prevention strategies. By analysis of untreated and treated wastewater from 100 municipal wastewater treatment plants (WWTPs) together serving over 40% of the Dutch population, this study investigated both the distribution of CPE in the Dutch population, and WWTPs as a source of CPE in the aquatic environment. CPE were detected at 89% of the WWTPs, in 87 influents and 53 effluents. Overall, 15 different CPE-types were detected based on species and carbapenemase gene. The most widely distributed were E. coli carrying blaOXA-48-like genes, which were detected at 88 WWTPs including small WWTPs without connected health care institutions (HCI). BlaOXA-48-like-positive-K. pneumoniae, blaNDM- or blaKPC-positive E. coli, and blaNDM- or blaKPC-positive K. pneumoniae were detected at 33, 20, and 14 WWTPs, respectively. Mean influent and effluent CPE concentrations were 7.9×102 cfu/l and 11 cfu/l. The total daily number of CPE discharged by the 100 WWTPs was estimated to be 2.2×1011 cfu. In multivariate analysis, CPE concentrations in untreated wastewater were positively associated with WWTP size and E. coli concentrations, but not with the presence of HCI. Based on the total number of CPE (9.7×1012 cfu) and ESBL-E. coli (2.41015 cfu) in influents, and a prevalence of approximately 5% in the Dutch population for ESBL-E. coli, the prevalence of CPE in the Dutch population was roughly estimated to be 0.02%. Wastewater surveillance is an efficient tool to monitor the distribution of CPE in the population at a national level and may supplement human surveillance data. CPE are emitted to the aquatic environment with treated wastewater and associated public health risks need to be determined