Antibiotic resistance, virulence, and phylogenetic analysis of Escherichia coli strains isolated from free-living birds in human habitats

Wild birds can be colonized by bacteria, which are often resistant to antibiotics and have various virulence profiles. The aim of this study was to analyze antibiotic resistance mechanisms and virulence profiles in relation to the phylogenetic group of E. coli strains that were isolated from the GI tract of wildfowl. Out of 241 faecal samples, presence of E. coli resistant to a cephalosporin (ESBL/AmpC) was estimated for 33 isolates (13,7%). Based on the analysis of the coexistence of 4 genes encoding ESBLs/AmpC (blaCTX-M, blaTEM, blaSHV, blaAmpC) and class 1 and 2 integrons genes (intI1, intI2) a subset of two resistance profiles was observed among the investigated E. coli isolates carrying blaAmpC, blaSHV, and blaCTX-M, blaTEM, class 1 and 2 integrons, respectively. The E. coli isolates were categorized into 4 phylogenetic groups A (39.4%), B2 (24.25%), D (24.25%) and B1 (12.1%). The pathogenic B2 and D groups were mainly typical for the Laridae family. Among the 28 virulence factors (Vfs) detected in pathogenic phylogenetic groups B2 and D, 7 were exclusively found in those groups (sfa, vat, tosA, tosB, hly, usp, cnf), while 4 VFs (fecA, fyuA, irp2, kspMTII) showed a statistically significant association (P≤0.05) with phylogroups A and B1. Our results indicated that strains belonging to commensal phylogroups A/B1 possess extensive iron acquisition systems (93,9%) and autotransporters (60,6%), typical for pathogens, hence we suggest that these strains evolve towards higher levels of virulence. This study, which is a point assessment of the virulence and drug resistance potential of wild birds, confirms the importance of taking wild birds as a reservoir of strains that pose a growing threat to humans. The E. coli analyzed in our study derive from different phylogenetic groups and possess an arsenal of antibiotic resistance genes and virulence factors that contribute to their ability to cause diseases.

Genome-wide identification of the Liriodendron chinense WRKY gene family and its diverse roles in response to multiple abiotic stress

Background Liriodendron chinense (Lchi) is a tree species within the Magnoliaceae family and is considered a basal angiosperm. The too low or high temperature or soil drought will restrict its growth as the adverse environmental conditions, thus improving L. chinense abiotic tolerance was the key issues to study. WRKYs are a major family of plant transcription factors known to often be involved in biotic and abiotic stress responses. So far, it is still largely unknown if and how the LchiWRKY gene family is tied to regulating L. chinense stress responses. Therefore, studying the involvement of the WRKY gene family in abiotic stress regulation in L. chinense could be very informative in showing how this tree deals with such stressful conditions. Results In this research, we performed a genome-wide analysis of the Liriodendron chinense (Lchi) WRKY gene family, studying their classification relationships, gene structure, chromosomal locations, gene duplication, cis-element, and response to abiotic stress. The 44 members of the LchiWRKY gene family contain a significant amount of sequence diversity, with their lengths ranging from 525 bp to 40,981 bp. Using classification analysis, we divided the 44 LchiWRKY genes into three phylogenetic groups (I, II, II), with group II then being further divided into five subgroups (IIa, IIb, IIc, IId, IIe). Comparative phylogenetic analysis including the WRKY families from 17 plant species suggested that LchiWRKYs are closely related to the Magnolia Cinnamomum kanehirae WRKY family, and has fewer family members than higher plants. We found the LchiWRKYs to be evenly distributed across 15 chromosomes, with their duplication events suggesting that tandem duplication may have played a major role in LchiWRKY gene expansion model. A Ka/Ks analysis indicated that they mainly underwent purifying selection and distributed in the group IId. Motif analysis showed that LchiWRKYs contained 20 motifs, and different phylogenetic groups contained conserved motif. Gene ontology (GO) analysis showed that LchiWRKYs were mainly enriched in two categories, i.e., biological process and molecular function. Two group IIc members (LchiWRKY10 and LchiWRKY37) contain unique WRKY element sequence variants (WRKYGKK and WRKYGKS). Gene structure analysis showed that most LchiWRKYs possess 3 exons and two different types of introns: the R- and V-type which are both contained within the WRKY domain (WD). Additional promoter cis-element analysis indicated that 12 cis-elements that play different functions in environmental adaptability occur across all LchiWRKY groups. Heat, cold, and drought stress mainly induced the expression of group II and I LchiWRKYs, some of which had undergone gene duplication during evolution, and more than half of which had three exons. LchiWRKY33 mainly responded to cold stress and LchiWRKY25 mainly responded to heat stress, and LchiWRKY18 mainly responded to drought stress, which was almost 4-fold highly expressed, while 5 LchiWRKYs (LchiWRKY5, LchiWRKY23, LchiWRKY14, LchiWRKY27, and LchiWRKY36) responded equally three stresses with more than 6-fold expression. Subcellular localization analysis showed that all LchiWRKYs were localized in the nucleus, and subcellular localization experiments of LchiWRKY18 and 36 also showed that these two transcription factors were expressed in the nucleus. Conclusions This study shows that in Liriodendron chinense, several WRKY genes like LchiWRKY33, LchiWRKY25, and LchiWRKY18, respond to cold or heat or drought stress, suggesting that they may indeed play a role in regulating the tree’s response to such conditions. This information will prove a pivotal role in directing further studies on the function of the LchiWRKY gene family in abiotic stress response and provides a theoretical basis for popularizing afforestation in different regions of China.

The evolution of insect visual opsin genes with specific consideration of the influence of ocelli and life history traits

Background Visual opsins are expressed in the compound eyes and ocelli of insects and enable light detection. Three distinct phylogenetic groups of visual opsins are found in insects, named long (LW), short (SW) and ultraviolet (UV) wavelength sensitive opsins. Recently, the LW group was found to be duplicated into the LW2b and the LW2a opsins. The expression of LW2b opsins is ocelli specific in some insects (e.g., bees, cricket, scorpion flies), but the gene was not found in other orders possessing three or less ocelli (e.g., dragonflies, beetles, moths, bugs). In flies, two LW2b homologs have been characterised, with one expressed in the ocelli and the other in the compound eyes. To date, it remains unclear which evolutionary forces have driven gains and losses of LW opsins in insects. Here we take advantage of the recent rapid increase in available sequence data (i.e., from insect genomes, targeted PCR amplification, RNAseq) to characterize the phylogenetic relationships of 1000 opsin sequences in 18 orders of Insects. The resulting phylogeny discriminates between four main groups of opsins, and onto this phylogeny we mapped relevant morphological and life history traits. Results Our results demonstrate a conserved LW2b opsin only present in insects with three ocelli. Only two groups (Brachycera and Odonata) possess more than one LW2b opsin, likely linked to their life history. In flies, we hypothesize that the duplication of the LW2b opsin occurred after the transition from aquatic to terrestrial larvae. During this transition, higher flies (Brachycera) lost a copy of the LW2a opsin, still expressed and duplicated in the compound eyes of lower flies (Nematocera). In higher flies, the LW2b opsin has been duplicated and expressed in the compound eyes while the ocelli and the LW2b opsin were lost in lower flies. In dragonflies, specialisation of flight capabilities likely drove the diversification of the LW2b visual opsins. Conclusion The presence of the LW2b opsin in insects possessing three ocelli suggests a role in specific flight capabilities (e.g., stationary flight). This study provides the most complete view of the evolution of visual opsin genes in insects yet, and provides new insight into the influence of ocelli and life history traits on opsin evolution in insects.

A Genomic Perspective Across Earth’s Microbiomes Reveals That Genome Size in Archaea and Bacteria Is Linked to Ecosystem Type and Trophic Strategy

Our view of genome size in Archaea and Bacteria has remained skewed as the data has been dominated by genomes of microorganisms that have been cultivated under laboratory settings. However, the continuous effort to catalog Earth’s microbiomes, specifically propelled by recent extensive work on uncultivated microorganisms, provides an opportunity to revise our perspective on genome size distribution. We present a meta-analysis that includes 26,101 representative genomes from 3 published genomic databases; metagenomic assembled genomes (MAGs) from GEMs and stratfreshDB, and isolates from GTDB. Aquatic and host-associated microbial genomes present on average the smallest estimated genome sizes (3.1 and 3.0 Mbp, respectively). These are followed by terrestrial microbial genomes (average 3.7 Mbp), and genomes from isolated microorganisms (average 4.3 Mbp). On the one hand, aquatic and host-associated ecosystems present smaller genomes sizes in genera of phyla with genome sizes above 3 Mbp. On the other hand, estimated genome size in phyla with genomes under 3 Mbp showed no difference between ecosystems. Moreover, we observed that when using 95% average nucleotide identity (ANI) as an estimator for genetic units, only 3% of MAGs cluster together with genomes from isolated microorganisms. Although there are potential methodological limitations when assembling and binning MAGs, we found that in genome clusters containing both environmental MAGs and isolate genomes, MAGs were estimated only an average 3.7% smaller than isolate genomes. Even when assembly and binning methods introduce biases, estimated genome size of MAGs and isolates are very similar. Finally, to better understand the ecological drivers of genome size, we discuss on the known and the overlooked factors that influence genome size in different ecosystems, phylogenetic groups, and trophic strategies.

Prevalence, Risk Factors, and Genetic Characterization of Extended-Spectrum Beta-Lactamase Escherichia coli Isolated From Healthy Pregnant Women in Madagascar

Antimicrobial resistance is a major public health concern worldwide affecting humans, animals and the environment. However, data is lacking especially in developing countries. Thus, the World Health Organization developed a One-Health surveillance project called Tricycle focusing on the prevalence of ESBL-producing Escherichia coli in humans, animals, and the environment. Here we present the first results of the human community component of Tricycle in Madagascar. From July 2018 to April 2019, rectal swabs from 492 pregnant women from Antananarivo, Mahajanga, Ambatondrazaka, and Toamasina were tested for ESBL-E. coli carriage. Demographic, sociological and environmental risk factors were investigated, and E. coli isolates were characterized (antibiotic susceptibility, resistance and virulence genes, plasmids, and genomic diversity). ESBL-E. coli prevalence carriage in pregnant women was 34% varying from 12% (Toamasina) to 65% (Ambatondrazaka). The main risk factor associated with ESBL-E. coli carriage was the rainy season (OR = 2.9, 95% CI 1.3–5.6, p = 0.009). Whole genome sequencing was performed on 168 isolates from 144 participants. blaCTX–M–15 was the most frequent ESBL gene (86%). One isolate was resistant to carbapenems and carried the blaNDM–5 gene. Most isolates belonged to commensalism associated phylogenetic groups A, B1, and C (90%) and marginally to extra-intestinal virulence associated phylogenetic groups B2, D and F (10%). Multi locus sequence typing showed 67 different sequence types gathered in 17 clonal complexes (STc), the most frequent being STc10/phylogroup A (35%), followed distantly by the emerging STc155/phylogroup B1 (7%), STc38/phylogroup D (4%) and STc131/phylogroup B2 (3%). While a wide diversity of clones has been observed, SNP analysis revealed several genetically close isolates (n = 34/168) which suggests human-to-human transmissions. IncY plasmids were found with an unusual prevalence (23%), all carrying a blaCTX–M–15. Most of them (85%) showed substantial homology (≥85%) suggesting a dissemination of IncY ESBL plasmids in Madagascar. This large-scale study reveals a high prevalence of ESBL-E. coli among pregnant women in four cities in Madagascar associated with warmth and rainfall. It shows the great diversity of E. coli disseminating throughout the country but also transmission of specific clones and spread of plasmids. This highlights the urgent need of public-health interventions to control antibiotic resistance in the country.

Phylogenetic Groups and Antimicrobial Resistance Genes in Escherichia coli from Different Meat Species

Escherichia coli isolated from meat of different animal species may harbour antimicrobial resistance genes and may thus be a threat to human health. The objectives of this study were to define antimicrobial resistance genes in E. coli isolates from pork, beef, chicken- and turkey meat and analyse whether their resistance genotypes associated with phylogenetic groups or meat species. A total number of 313 E. coli samples were isolated using standard cultural techniques. In 98% of resistant isolates, a dedicated resistance gene could be identified by PCR. Resistance genes detected were tet(A) and tet(B) for tetracycline resistance, strA and aadA1 for streptomycin resistance, sulI and sulII for resistance against sulphonamides, dfr and aphA for kanamycin resistance and blaTEM for ampicillin resistance. One stx1 harbouring E. coli isolated from pork harboured the tet(A) gene and belonged to phylogenetic group B2, whilst another stx1 positive isolate from beef was multi-resistant and tested positive for blaTEM,aphA, strA–B, sulII, and tet(A) and belonged to phylogenetic group A. In conclusion, the distribution of resistance elements was almost identical and statistically indifferent in isolates of different meat species. Phylogenetic groups did not associate with the distribution of resistance genes and a rather low number of diverse resistance genes were detected. Most E. coli populations with different resistance genes against one drug often revealed statistically significant different MIC values.

The evolutionary history of Shigella flexneri serotype 6 in Asia

Shigella flexneri serotype 6 is an understudied cause of diarrhoeal diseases in developing countries, and has been proposed as one of the major targets for vaccine development against shigellosis. Despite being named as S. flexneri , Shigella flexneri serotype 6 is phylogenetically distinct from other S. flexneri serotypes and more closely related to S. boydii . This unique phylogenetic relationship and its low sampling frequency have hampered genomic research on this pathogen. Herein, by utilizing whole genome sequencing (WGS) and analyses of Shigella flexneri serotype 6 collected from epidemiological studies (1987–2013) in four Asian countries, we revealed its population structure and evolutionary history in the region. Phylogenetic analyses supported the delineation of Asian Shigella flexneri serotype 6 into two phylogenetic groups (PG-1 and −2). Notably, temporal phylogenetic approaches showed that extant Asian S. flexneri serotype 6 could be traced back to an inferred common ancestor arising in the 18th century. The dominant lineage PG-1 likely emerged in the 1970s, which coincided with the times to most recent common ancestors (tMRCAs) inferred from other major Southeast Asian S. flexneri serotypes. Similar to other S. flexneri serotypes in the same period in Asia, genomic analyses showed that resistance to first-generation antimicrobials was widespread, while resistance to more recent first-line antimicrobials was rare. These data also showed a number of gene inactivation and gene loss events, particularly on genes related to metabolism and synthesis of cellular appendages, emphasizing the continuing role of reductive evolution in the adaptation of the pathogen to an intracellular lifestyle. Together, our findings reveal insights into the genomic evolution of the understudied Shigella flexneri serotype 6, providing a new piece in the puzzle of Shigella epidemiology and evolution.

Systematic Analysis and Biochemical Characterization of the Caffeoyl Shikimate Esterase Gene Family in Poplar

Caffeoyl shikimate esterase (CSE) hydrolyzes caffeoyl shikimate into caffeate and shikimate in the phenylpropanoid pathway. In this study, we performed a systematic analysis of the CSE gene family and investigated the possible roles of CSE and CSE-like genes in Populus. We conducted a genome-wide analysis of the CSE gene family, including functional and phylogenetic analyses of CSE and CSE-like genes, using the poplar (Populus trichocarpa) genome. Eighteen CSE and CSE-like genes were identified in the Populus genome, and five phylogenetic groups were identified from phylogenetic analysis. CSEs in Group Ia, which were proposed as bona fide CSEs, have probably been lost in most monocots except Oryza sativa. Primary functional classification showed that PoptrCSE1 and PoptrCSE2 had putative function in lignin biosynthesis. In addition, PoptrCSE2, along with PoptrCSE12, might also respond to stress with a function in cell wall biosynthesis. Enzymatic assay of PoptoCSE1 (Populus tomentosa), -2 and -12 showed that PoptoCSE1 and -2 maintained CSE activity. PoptoCSE1 and 2 had similar biochemical properties, tissue expression patterns and subcellular localization. Most of the PoptrCSE-like genes are homologs of AtMAGL (monoacylglycerol lipase) genes in Arabidopsis and may function as MAG lipase in poplar. Our study provides a systematic understanding of this novel gene family and suggests the function of CSE in monolignol biosynthesis in Populus.

Multiple Novel Clades of Anopheline Mosquitoes Caught Outdoors in Northern Zambia

Residual vector populations that do not come in contact with the most frequently utilized indoor-directed interventions present major challenges to global malaria eradication. Many of these residual populations are mosquito species about which little is known. As part of a study to assess the threat of outdoor exposure to malaria mosquitoes within the Southern and Central Africa International Centers of Excellence for Malaria Research, foraging female anophelines were collected outside households in Nchelenge District, northern Zambia. These anophelines proved to be more diverse than had previously been reported in the area. In order to further characterize the anopheline species, sequencing and phylogenetic approaches were utilized. Anopheline mosquitoes were collected from outdoor light traps, morphologically identified, and sent to Johns Hopkins Bloomberg School of Public Health for sequencing. Sanger sequencing from 115 field-derived samples yielded mitochondrial COI sequences, which were aligned with a homologous 488 bp gene segment from known anophelines (n = 140) retrieved from NCBI. Nuclear ITS2 sequences (n = 57) for at least one individual from each unique COI clade were generated and compared against NCBI’s nucleotide BLAST database to provide additional evidence for taxonomical identity and structure. Molecular and morphological data were combined for assignment of species or higher taxonomy. Twelve phylogenetic groups were characterized from the COI and ITS2 sequence data, including the primary vector species Anopheles funestus s.s. and An. gambiae s.s. An unexpectedly large proportion of the field collections were identified as An. coustani and An. sp. 6. Six phylogenetic groups remain unidentified to species-level. Outdoor collections of anopheline mosquitoes in areas frequented by people in Nchelenge, northern Zambia, proved to be extremely diverse. Morphological misidentification and underrepresentation of some anopheline species in sequence databases confound efforts to confirm identity of potential malaria vector species. The large number of unidentified anophelines could compromise the malaria vector surveillance and malaria control efforts not only in northern Zambia but other places where surveillance and control are focused on indoor-foraging and resting anophelines. Therefore, it is critical to continue development of methodologies that allow better identification of these populations and revisiting and cleaning current genomic databases.

panModule: detecting conserved modules in the variable regions of a pangenome graph

The recent years have seen the rise of pangenomes as comparative genomic tools to better understand the evolution of gene content among microbial genomes in close phylogenetic groups such as species. While the core or persistent genome is often well-known as it includes essential or ubiquitous genes, the variable genome is usually less characterized and includes many genes with unknown functions even among the most studied organisms. It gathers important genes for strain adaptation that are acquired by horizontal gene transfer. Here, we introduce panModule, an original method to identify conserved modules in pangenome graphs built from thousands of microbial genomes. These modules correspond to synteny blocks composed of consecutive genes that are conserved in a subset of the compared strains. Identifying conserved modules can provide insights on genes involved in the same functional processes, and as such is a very helpful tool to facilitate the understanding of genomic regions with complex evolutionary histories. The panModule method was benchmarked on a curated dataset of conserved modules in Escherichia coli genomes. Its use was illustrated through a study of a high pathogenicity island in Klebsiella pneumoniae that allowed a better understanding of this region. panModule is freely available and accessible through the PPanGGOLiN software suite (https://github.com/labgem/PPanGGOLiN).