Genetic Variation and Preliminary Indications of Divergent Niche Adaptation in Cryptic Clade II of Escherichia.

The evolution, habitat, and lifestyle of the cryptic clade II of Escherichia, which were first recovered at low frequency from non-human hosts and later from external environments, were poorly understood. Here, the genomes of selected strains were analyzed for preliminary indications of ecological differentiation within their population. We adopted the delta bitscore metrics to detect functional divergence of their orthologous genes and trained a random forest classifier to differentiate the genomes according to habitats (gastrointestinal vs external environment). Model was built with inclusion of other Escherichia genomes previously demonstrated to have exhibited genomic traits of adaptation to one of the habitats. Overall, gene degradation was more prominent in the gastrointestinal strains. The trained model correctly classified the genomes, identifying a set of predictor genes that were informative of habitat association. Functional divergence in many of these genes were reflective of ecological divergence. Accuracy of the trained model was confirmed by its correct prediction of the habitats of an independent set of strains with known habitat association. In summary, the cryptic clade II of Escherichia displayed genomic signatures that are consistent with divergent adaptation to gastrointestinal and external environments.

Correction: Regev, Y., et al. Molecular Identification and Characterization of Vibrio Species and Mycobacterium Species in Wild and Cultured Marine Fish from the Eastern Mediterranean Sea. Microorganisms 2020, 8, 863.

We would like to change the authors' affiliation of paper [...].

Molecular Identification and Characterization of Vibrio Species and Mycobacterium Species in Wild and Cultured Marine Fish from the Eastern Mediterranean Sea.

In contrast to numerous documented pathogens and infectious diseases of aquaculture, there is a lack of baseline data and information regarding pathogenic agents' prevalence in wild marine fish populations. This study focused on two common fish pathogenic microorganisms, namely Mycobacterium species and Vibrio species, both of which are known to be major causes of fish loss, occasionally to the extent of being a limiting factor in fish production. Both microorganisms are known as zoonotic agents. In total, 210 wild marine indigenous and Lessepsian fish from four different species from the eastern Mediterranean Sea were sampled and tested for Vibrio species and Mycobacterium species during a two-year period (2016-2017). Using PCR with 16S rRNA primers, we detected different strain variations of Mycobacterium species and Vibrio species and, based on the sequencing results, the overall prevalence for Vibrio species in wild fish in 2016 was significantly higher compared to 2017. No significant difference was detected for Mycobacterium species prevalence in wild fish between 2016 and 2017. In addition, 72 gilthead seabream (Sparus aurata) from an Israeli offshore marine farm were also examined during the two-year period (2017-2018). The results suggest that Mycobacterium species prevalence was significantly higher in 2018, while in 2017 there was no positive results for Mycobacterium species. In addition, there was no significant difference between both years in regard to the prevalence of Vibrio species for maricultured fish. These results highlight the necessity of continuous molecular monitoring in order to evaluate the prevalence of pathogenic microorganisms in both wild and cultured fish populations.

Draft Genome Sequences of 16 Strains of Escherichia Cryptic Clade II Isolated from Intertidal Sediment in Hong Kong.

The genus Escherichia includes several cryptic clades. Among them, the members of cryptic clade II have rarely been found, and their genome sequences remain largely uninvestigated. Here, we report the draft genome sequences of 16 strains of Escherichia cryptic clade II that were isolated from intertidal sediment in Hong Kong.

Tracking the relative concentration between Bacteroidales DNA markers and culturable Escherichia coli in fecally polluted subtropical seawater: potential use in differentiating fresh and aged pollution.

Routine water quality monitoring practices based on the enumeration of culturable Escherichia coli provides no information about the source or age of fecal pollution. An emerging strategy is to use culturable E. coli and the DNA markers of Bacteroidales complementarily for microbial source tracking. In this study, we consistently observed in seawater microcosms of 3 different conditions that culturable E. coli decayed faster (T99 = 1.14 - 4.29 days) than Bacteroidales DNA markers did (T99 = 1.81 - 200.23 days). Concomitantly, the relative concentration between Bacteroidales DNA markers and culturable E. coli increased over time in all treatments. Particularly, the increase during the early stage of the experiments (before T99 of E. coli was reached) was faster than during the later stage (after T99 of E. coli was attained). We propose that the tracking of the relative concentration between Bacteroidales DNA markers and culturable E. coli provides an opportunity to differentiate a pollution that is relatively fresh from one that has aged. This method, upon further investigation and validation, could be useful in episodic pollution events where the surge of E. coli concentration causes noncompliance to the single sample maximum criterion that mandates high frequency follow-up monitoring.

A peptide identification-free, genome sequence-independent shotgun proteomics workflow for strain-level bacterial differentiation.

Shotgun proteomics is an emerging tool for bacterial identification and differentiation. However, the identification of the mass spectra of peptides to genome-derived peptide sequences remains a key issue that limits the use of shotgun proteomics to bacteria with genome sequences available. In this proof-of-concept study, we report a novel bacterial fingerprinting method that enjoys the resolving power and accuracy of mass spectrometry without the burden of peptide identification (i.e. genome sequence-independent). This method uses a similarity-clustering algorithm to search for mass spectra that are derived from the same peptide and merge them into a unique consensus spectrum as the basis to generate proteomic fingerprints of bacterial isolates. In comparison to a traditional peptide identification-based shotgun proteomics workflow and a PCR-based DNA fingerprinting method targeting the repetitive extragenic palindromes elements in bacterial genomes, the novel method generated fingerprints that were richer in information and more discriminative in differentiating E. coli isolates by their animal sources. The novel method is readily deployable to any cultivable bacteria, and may be used for several fields of study such as environmental microbiology, applied microbiology, and clinical microbiology.

Use of food waste as fish feeds: effects of prebiotic fibers (inulin and mannanoligosaccharide) on growth and non-specific immunity of grass carp (Ctenopharyngodon idella).

The effects of inulin and mannanoligosaccharide (MOS) on the growth performance and non-specific immunity of grass carp were studied. Two doses of prebiotic fiber with 0.2 or 2% of the fibers are being mixed into fish feed pellets. Fish growth as well as selected non-specific immune parameters of grass carp were tested in a feeding trial, which lasted for 8 weeks. Fish was fed at 2.5% body mass per day. INU02, INU2, and MOS2 significantly improved relative weight gain, specific growth rate, protein efficiency ratio, and food conversion ratio of grass carp fed with food waste-based diet. In terms of non-specific immune response, grass carp showed significant improvement in all three tested parameters (total serum immunoglobin, bactericidal activity, and anti-protease activity). Adding 2% of inulin (INU2) into food waste diets seemed to be more preferable than other supplemented experimental diets (INU02, MOS02, MOS2), as it could promote growth of grass carp as well as improving the non-specific immune systems of grass carp.

Sperm proteome of Mytilus galloprovincialis: Insights into the evolution of fertilization proteins in marine mussels.

Cataloging the sperm proteome of an animal can improve our understanding of its sperm-egg interaction and speciation, but such data are available for only a few free-spawning invertebrates. This study aimed to identify the sperm proteome of Mytilus galloprovincialis, a free-spawning marine mussel. We integrated public transcriptome datasets by de novo assembly, and applied SDS-PAGE coupled LC-MS/MS analysis to profile the sperm proteome, resulting in the identification of 550 proteins. Comparing the homologous sperm protein coding genes between M. galloprovincialis and its closely related species M. edulis revealed that fertilization proteins have the highest mean nonsynonymous substitution rate (Ka/Ks = 0.62) among 11 functional groups, consistent with previous reports of positive selection of several fertilization proteins in Mytilus. Moreover, 78 sperm proteins in different functional groups have Ka/Ks values > 0.5, indicating the presence of many candidate sperm proteins for further analysis of rapid interspecific divergence. The MS data are available in ProteomeXchange with the identifier PXD001665.

Differential utility of the Bacteroidales DNA and RNA markers in the tiered approach for microbial source tracking in subtropical seawater.

Source tracking of fecal pollution is an emerging component in water quality monitoring. It may be implemented in a tiered approach involving Escherichia coli and/or Enterococcus spp. as the standard fecal indicator bacteria (FIB) and the 16S rRNA gene markers of Bacteroidales as source identifiers. The relative population dynamics of the source identifiers and the FIB may strongly influence the implementation of such approach. Currently, the relative performance of DNA and RNA as detection targets of Bacteroidales markers in the tiered approach is not known. We compared the decay of the DNA and RNA of the total (AllBac) and ruminant specific (CF128) Bacteroidales markers with those of the FIB in seawater spiked with cattle feces. Four treatments of light and oxygen availability simulating the subtropical seawater of Hong Kong were tested. All Bacteroidales markers decayed significantly slower than the FIB in all treatments. Nonetheless, the concentrations of the DNA and RNA markers and E. coli correlated significantly in normoxic seawater independent of light availability, and in hypoxic seawater only under light. In hypoxic seawater without light, the concentrations of RNA but not DNA markers correlated with that of E. coli. Generally, the correlations between Enterococcus spp. and Bacteroidales were insignificant. These results suggest that either DNA or RNA markers may complement E. coli in the tiered approach for normoxic or hypoxic seawater under light. When light is absent, either DNA or RNA markers may serve for normoxic seawater, but only the RNA markers are suitable for hypoxic seawater.

Draft Genome Sequences of Three Escherichia coli Strains Investigated for the Effects of Lysogeny on Niche Diversification.

During the course of investigating the effects of lysogeny on niche diversification of Escherichia coli, we used the temperate phages induced from one E. coli strain to infect another and created an isogenic lysogen of the latter. The draft genome sequences of the three E. coli strains are reported herein.

Draft Genome Sequence of Escherichia coli E1728 Isolated from Marine Sediment in Hong Kong.

Recent findings of Escherichia coli persisting autochthonously in environmental matrices outside animal bodies have revealed largely unknown facets of the lifestyle and ecophysiology of the species that have yet to be explored. Here, we report the draft genome sequence of E. coli E1728 isolated from marine sediment.

Biogeography of bacterioplankton in the tropical seawaters of Singapore.

Knowledge about the biogeography of marine bacterioplankton on the global scale in general and in Southeast Asia in particular has been scarce. This study investigated the biogeography of bacterioplankton community in Singapore seawaters. Twelve stations around Singapore island were sampled on different schedules over 1 year. Using PCR-DNA fingerprinting, DNA cloning and sequencing, and microarray hybridization of the 16S rRNA genes, we observed clear spatial variations of bacterioplankton diversity within the small area of the Singapore seas. Water samples collected from the Singapore Strait (south) throughout the year were dominated by DNA sequences affiliated with Cyanobacteria and Alphaproteobacteria that were believed to be associated with the influx of water from the open seas in Southeast Asia. On the contrary, water in the relatively polluted Johor Strait (north) were dominated by Betaproteobacteria, Gammaproteobacteria, and Bacteroidetes and that were presumably associated with river discharge and the relatively eutrophic conditions of the waterway. Bacterioplankton diversity was temporally stable, except for the episodic surge of Pseudoalteromonas, associated with algal blooms. Overall, these results provide valuable insights into the diversity of bacterioplankton communities in Singapore seas and the possible influences of hydrological conditions and anthropogenic activities on the dynamics of the communities.

Improving the performance of an end-point PCR assay commonly used for the detection of Bacteroidales pertaining to cow feces.

Bacteroidales are normal gut flora of warm-blooded animals. Since each host species carries a different diversity of Bacteroidales, the detection of host-associated gene markers of Bacteroidales has emerged as a promising tool for the tracking of the source of fecal pollution in aquatic ecosystems. To detect cow-associated Bacteroidales, a commonly used method has been an end-point PCR assay with the 16S rRNA genes primers CF128F (cow-associated) and Bac708R (all Bacteroidales). The PCR assay has demonstrated high rates of true-positive detection (i.e., high sensitivity) in all previous studies. However, the assay also had high rates of false-positive detection to the samples of non-target hosts in some cases (i.e., low specificity). In opposite to the reason many investigators have proposed, our results suggested that false detection was not necessarily due to the presence of the target sequence of CF128F in the feces of non-target hosts. Instead, we found sequences of non-target hosts having single internal mismatches with CF128F. Those mismatches were well tolerated in PCR, partly due to the universality of Bac708R. To improve the detection performance, we designed a novel primer CF592R (targeting the same clade of sequences as CF128F) to substitute Bac708R. The use of CF529R alleviated false detection and also led to a tenfold reduction in detection limit in the samples tested, compared to the use of Bac708R. Many other end-point PCR assays that detect the 16S rRNA genes in Bacteroidales also use a host-associated primer to couple with Bac708R, and low specificity or sensitivity has been reported. Based on our findings for CF128F, we suggest that the suitability of Bac708R in those PCR assays needs to be revisited.

Hydrothermally generated aromatic compounds are consumed by bacteria colonizing in Atlantis II Deep of the Red Sea.

Hydrothermal ecosystems have a wide distribution on Earth and many can be found in the basin of the Red Sea. Production of aromatic compounds occurs in a temperature window of ∼60-150 °C by utilizing organic debris. In the past 50 years, the temperature of the Atlantis II Deep brine pool in the Red Sea has increased from 56 to 68 °C, whereas the temperature at the nearby Discovery Deep brine pool has remained relatively stable at about 44 °C. In this report, we confirmed the presence of aromatic compounds in the Atlantis II brine pool as expected. The presence of the aromatic compounds might have disturbed the microbes in the Atlantis II. To show shifted microbial communities and their metabolisms, we sequenced the metagenomes of the microbes from both brine pools. Classification based on metareads and the 16S rRNA gene sequences from clones showed a strong divergence of dominant bacterial species between the pools. Bacteria capable of aromatic degradation were present in the Atlantis II brine pool. A comparison of the metabolic pathways showed that several aromatic degradation pathways were significantly enriched in the Atlantis II brine pool, suggesting the presence of aromatic compounds. Pathways utilizing metabolites derived from aromatic degradation were also significantly affected. In the Discovery brine pool, the most abundant genes from the microbes were related to sugar metabolism pathways and DNA synthesis and repair, suggesting a different strategy for the utilization of carbon and energy sources between the Discovery brine pool and the Atlantis II brine pool.

Vertical stratification of microbial communities in the Red Sea revealed by 16S rDNA pyrosequencing.

The ecosystems of the Red Sea are among the least-explored microbial habitats in the marine environment. In this study, we investigated the microbial communities in the water column overlying the Atlantis II Deep and Discovery Deep in the Red Sea. Taxonomic classification of pyrosequencing reads of the 16S rRNA gene amplicons showed vertical stratification of microbial diversity from the surface water to 1500 m below the surface. Significant differences in both bacterial and archaeal diversity were observed in the upper (20 [corrected] and 50 m) and deeper layers (200 and 1500 m). There were no obvious differences in community structure at the same depth for the two sampling stations. The bacterial community in the upper layer was dominated by Cyanobacteria whereas the deeper layer harbored a large proportion of Proteobacteria. Among Archaea, Euryarchaeota, especially Halobacteriales, were dominant in the upper layer but diminished drastically in the deeper layer where Desulfurococcales belonging to Crenarchaeota became the dominant group. The results of our study indicate that the microbial communities sampled in this study are different from those identified in water column in other parts of the world. The depth-wise compositional variation in the microbial communities is attributable to their adaptations to the various environments in the Red Sea.

Host-specific 16S rRNA gene markers of Bacteroidales for source tracking of fecal pollution in the subtropical coastal seawater of Hong Kong.

This study investigated the diversity of Bacteroidales communities in the feces of eight host species in Hong Kong (subtropical Asia), including human (in the form of sewage), cow, pig, horse, cat, dog, rabbit and rat. The analysis of terminal restriction fragment length polymorphism (TRFLP) in the 16S rRNA genes revealed significant differences in Bacteroidales communities among all host species, with the exception of dog and cat. Manual examination of TRFLP profiles resulted in six terminal restriction fragments (TRFs) that were potentially specific to the sewage (one TRF), cow (three TRFs) or pig (two TRFs) samples. All six TRFs were (1) present in 100% of the samples of the respective target host, (2) absent in other hosts or present only in low frequency and low intensity, and (3) verified for sizes using in silico digestion of DNA sequences in clone libraries. The six TRFs could reliably indicate the source of fecal contaminations in natural seawater amended with sewage, cow or pig fecal samples. In field tests conducted for two polluted and one unpolluted coastal site, the sewage-specific TRF was detected in all seawater samples of the sites known to be impacted by raw and treated sewage. However, only two of three cow-specific TRFs were detected for the two polluted sites, which also received fecal input from feral cows. No pig-specific TRF was detected, although one of the coastal sites was chronically polluted by pig farm run-offs. Nevertheless, the total absence of the six potentially host-specific TRFs in the seawater of an unpolluted site demonstrated the specificity of the TRFs as gene markers in indicating actual pollution.

Response of bacterioplankton community structures to hydrological conditions and anthropogenic pollution in contrasting subtropical environments.

Bacterioplankton community structures under contrasting subtropical marine environments (Hong Kong waters) were analyzed using 16S rRNA gene denaturing gradient gel electrophoresis (DGGE) and subsequent sequencing of predominant bands for samples collected bimonthly from 2004 to 2006 at five stations. Generally, bacterial abundance was significantly higher in the summer than in the winter. The general seasonal variations of the bacterial community structure, as indicated by cluster analysis of the DGGE pattern, were best correlated with temperature at most stations, except for the station close to a sewage discharge outfall, which was best explained by pollution-indicating parameters (e.g. biochemical oxygen demand). Anthropogenic pollutions appear to have affected the presence and the intensity of DGGE bands at the stations receiving discharge of primarily treated sewage. The relative abundance of major bacterial species, calculated by the relative intensity of DGGE bands after PCR amplification, also indicated the effects of hydrological or seasonal variations and sewage discharges. For the first time, a systematic molecular fingerprinting analysis of the bacterioplankton community composition was carried out along the environmental and pollution gradient in a subtropical marine environment, and it suggests that hydrological conditions and anthropogenic pollutions altered the total bacterial community as well as the dominant bacterial groups.

Particle-attached and free-living bacterial communities in a contrasting marine environment: Victoria Harbor, Hong Kong.

Diversity of particle-attached and free-living marine bacteria in Victoria Harbor, Hong Kong, and its adjacent coastal and estuarial environments was investigated using DNA fingerprinting and clone library analysis. Denaturing gradient gel electrophoresis (DGGE) analysis of 16S rRNA genes showed that bacterial communities in three stations of Victoria Harbor were similar, but differed from those in adjacent coastal and estuarine stations. Particle-attached and free-living bacterial community composition differed in the Victoria Harbor area. DNA sequencing of 28 bands from DGGE gel showed Alphaproteobacteria was the most abundant group, followed by the Bacteroidetes, and other Proteobacteria. Bacterial species richness (number of DGGE bands) differed among stations and populations (particle-attached and free-living; bottom and surface). BIOENV analysis indicated that the concentrations of suspended solids were the major contributing parameter for the spatial variation of total bacterial community structure. Samples from representative stations were selected for clone library (548 clones) construction and their phylogenetic distributions were similar to those of sequences from DGGE. Approximately 80% of clones were affiliated to Proteobacteria, Bacteroidetes and Cyanobacteria. The possible influences of dynamic pollution and hydrological conditions in the Victoria Harbor area on the particle-attached and free-living bacterial community structures were discussed.

Recent advances in molecular techniques for the detection of phylogenetic markers and functional genes in microbial communities.

The detection and analysis of nucleic acids extracted from microbial communities are the ultimate ways to determine the diversity and functional capability of microbial communities in the environments. However, it remains a challenge to use molecular techniques for unequivocal determination and quantification of microbial species composition and functional activities. Considerable efforts have been made to enhance the capability of molecular techniques. Here an update of the recent developments in molecular techniques for environmental microbiology is provided.

Shewanella irciniae sp. nov., a novel member of the family Shewanellaceae, isolated from the marine sponge Ircinia dendroides in the Bay of Villefranche, Mediterranean Sea.

Strain UST040317-058(T), comprising non-pigmented, rod-shaped, facultatively anaerobic, Gram-negative cells that are motile by means of single polar flagella, was isolated from the surface of a marine sponge (Ircinia dendroides) collected from the Mediterranean Sea. Comparative 16S rRNA gene sequence-based phylogenetic analysis placed the strain in a separate cluster with the recognized bacterium Shewanella algae IAM 14159(T), with which it showed a sequence similarity of 95.0 %. The sequence similarity between strain UST040317-058(T) and its other (six) closest relatives ranged from 91.6 to 93.8 %. Strain UST040317-058(T) showed oxidase, catalase and gelatinase activities. The typical respiratory quinones for shewanellas, menaquinone MK-7 and ubiquinones Q-7 and Q-8, were also detected. The predominant fatty acids in strain UST040317-058(T) were i15 : 0, 16 : 0, 17 : 1omega8c and summed feature 3 (comprising i15 : 0 2-OH and/or 16 : 1omega7c), altogether representing 56.9 % of the total. The DNA G+C content was 39.9 mol%. The strain could be differentiated from other Shewanella species by its inability to reduce nitrate or produce H(2)S and by 10-22 additional phenotypic characteristics. On the basis of the phylogenetic and phenotypic data presented in this study, strain UST040317-058(T) represents a novel species in the genus Shewanella, for which the name Shewanella irciniae sp. nov. is proposed. The type strain is UST040317-058(T) (=JCM 13528(T)=NRRL B-41466(T)).