Comparative Genomics of Pseudomonas stutzeri Complex: Taxonomic Assignments and Genetic Diversity

Pseudomonas stutzeri is a species complex with extremely broad phenotypic and genotypic diversity. However, very little is known about its diversity, taxonomy and phylogeny at the genomic scale. To address these issues, we systematically and comprehensively defined the taxonomy and nomenclature for this species complex and explored its genetic diversity using hundreds of sequenced genomes. By combining average nucleotide identity (ANI) evaluation and phylogenetic inference approaches, we identified 123 P. stutzeri complex genomes covering at least six well-defined species among all sequenced Pseudomonas genomes; of these, 25 genomes represented novel members of this species complex. ANI values of ≥∼95% and digital DNA-DNA hybridization (dDDH) values of ≥∼60% in combination with phylogenomic analysis consistently and robustly supported the division of these strains into 27 genomovars (most likely species to some extent), comprising 16 known and 11 unknown genomovars. We revealed that 12 strains had mistaken taxonomic assignments, while 16 strains without species names can be assigned to the species level within the species complex. We observed an open pan-genome of the P. stutzeri complex comprising 13,261 gene families, among which approximately 45% gene families do not match any sequence present in the COG database, and a large proportion of accessory genes. The genome contents experienced extensive genetic gain and loss events, which may be one of the major mechanisms driving diversification within this species complex. Surprisingly, we found that the ectoine biosynthesis gene cluster (ect) was present in all genomes of P. stutzeri species complex strains but distributed at very low frequency (43 out of 9548) in other Pseudomonas genomes, suggesting a possible origin of the ancestors of P. stutzeri species complex in high-osmolarity environments. Collectively, our study highlights the potential of using whole-genome sequences to re-evaluate the current definition of the P. stutzeri complex, shedding new light on its genomic diversity and evolutionary history.

Genetic Variability among Swine Influenza Viruses in Italy: Data Analysis of the Period 2017–2020

Swine play an important role in the ecology of influenza A viruses (IAVs), acting as mixing vessels. Swine (sw) IAVs of H1N1 (including H1N1pdm09), H3N2, and H1N2 subtypes are enzootic in pigs globally, with different geographic distributions. This study investigated the genetic diversity of swIAVs detected during passive surveillance of pig farms in Northern Italy between 2017 and 2020. A total of 672 samples, IAV-positive according to RT-PCR, were subtyped by multiplex RT-PCR. A selection of strains was fully sequenced. High genotypic diversity was detected among the H1N1 and H1N2 strains, while the H3N2 strains showed a stable genetic pattern. The hemagglutinin of the H1Nx swIAVs belonged to HA-1A, HA-1B, and HA-1C lineages. Increasing variability was found in HA-1C strains with the circulation of HA-1C.2, HA-1C.2.1 and HA-1C.2.2 sublineages. Amino acid deletions in the HA-1C receptor binding site were observed and antigenic drift was confirmed. HA-1B strains were mostly represented by the Δ146-147 Italian lineage HA-1B.1.2.2, in combination with the 1990s human-derived NA gene. One antigenic variant cluster in HA-1A strains was identified in 2020. SwIAV circulation in pigs must be monitored continuously since the IAVs’ evolution could generate strains with zoonotic potential.

Genome Size, Cytotype Diversity and Reproductive Mode Variation of Cotoneaster integerrimus (Rosaceae) from the Balkans

Cotoneaster integerrimus represents a multiploid and facultative apomictic system of widely distributed mountain populations. We used flow cytometry to determine genome size, ploidy level, and reproduction mode variation of the Balkan populations, supplemented by analysis of nuclear microsatellites in order to address: (i) geographic distribution and variation of cytotypes among the populations; (ii) variation of reproduction mode and the frequency of sexuality; (iii) pathways of endosperm formation among the sampled polyploids and their endosperm balance requirements; (iv) genotypic diversity and geographic distribution of clonal lineages of polyploids. The prevalence of apomictic tetraploid cytotype followed by sexual diploids and extremely rare triploids was demonstrated. This prevalence of tetraploids affected the populations’ structure composed from clonal genotypes with varying proportions. The co-occurrence of diploids and tetraploids generated higher cytotype, reproductive mode, and genotypic diversity, but mixed-ploidy sites were extremely rare. The endosperm imbalance facilitates the development and the occurrence of intermediate triploids in mixed-ploidy populations, but also different tetraploid lineages elsewhere with unbalanced endosperm. All these results showed that the South European populations of C. integerrimus have higher levels of cytotype and reproductive diversity compared to the Central European ones. Therefore, the South European populations can be considered as a potential reservoir of regional and global diversity for this species.

Multicenter, Prospective Validation of a Phenotypic Algorithm to Guide Carbapenemase Testing in Carbapenem-Resistant Pseudomonas aeruginosa using the ERACE-PA Global Surveillance Program

Background Carbapenemase-producing, carbapenem-resistant Pseudomonas aeruginosa (CP-CRPA) are a global challenge. However, detection efforts can be laborious because numerous mechanisms produce carbapenem resistance. An MIC based algorithm (imipenem or meropenem-resistant plus ceftazidime-non-susceptible plus cefepime-non-susceptible) was proposed to identify isolates most likely to harbor a carbapenemase; however, prospective validation in geographies displaying genotypic diversity and varied carbapenemase prevalence is warranted. Methods CRPA were collected during the ERACE-PA global surveillance program from 17 sites in 12 countries. Isolates underwent susceptibility testing following local standards to ceftazidime, cefepime, and ceftolozane/tazobactam. Isolates underwent initial phenotypic carbapenemase screening followed by molecular testing if positive. The primary algorithm criteria were applied and results compared to phenotypic carbapenemase results to assess the performance of the algorithm. A secondary criteria of (the algorithm criteria or imipenem or meropenem-resistant plus ceftolozane/tazobactam-non-susceptible) was assessed. Results 807 CRPA were assessed and 464 isolates met the algorithm criteria described above. Overall, testing was reduced by 43% compared with testing all CRPA. Carbapenemase-positive isolates missed by the algorithm were largely driven by GES. Addition of the criteria of imipenem or meropenem-resistant plus ceftolozane/tazobactam-non-susceptible decreased the number of CP-CRPA missed by the algorithm (21 versus 40 isolates, respectively) still reducing number of isolates tested by 39%. Conclusions Application of the initial algorithm (imipenem or meropenem-resistant plus ceftazidime-non-susceptible plus cefepime-non-susceptible) performed well in a global cohort with 33% phenotypically carbapenemase-positive isolates. Addition of imipenem or meropenem-resistant plus ceftolozane/tazobactam-non-susceptible reduced the number of phenotypically carbapenemase-positive isolates missed and may be useful in areas with a prominence of GES.

Comparison of Diplodia Tip Blight Pathogens in Spanish and North American Pine Ecosystems

Diplodia tip blight is the most ubiquitous and abundant disease in Spanish Pinus radiata plantations. The economic losses in forest stands can be very severe because of its abundance in cones and seeds together with the low genetic diversity of the host. Pinus resinosa is not genetically diverse in North America either, and Diplodia shoot blight is a common disease. Disease control may require management designs to be adapted for each region. The genetic diversity of the pathogen could be an indicator of its virulence and spreading capacity. Our objective was to understand the diversity of Diplodia spp. in Spanish plantations and to compare it with the structure of American populations to collaborate in future management guidelines. Genotypic diversity was investigated using microsatellite markers. Eight loci (SS9–SS16) were polymorphic for the 322 isolates genotyped. The results indicate that Diplodia sapinea is the most frequent Diplodia species present in plantations of the north of Spain and has high genetic diversity. The higher genetic diversity recorded in Spain in comparison to previous studies could be influenced by the intensity of the sampling and the evidence about the remarkable influence of the sample type.

Phylogeography and phylogeny of Rhinoviruses collected from Severe Acute Respiratory Infection (SARI) cases over successive epidemic periods in Tunisia

Rhinoviruses (RV) are a major cause of Severe Acute Respiratory Infection (SARI) in children, with high genotypic diversity in different regions. However, RV type diversity remains unknown in several regions of the world. In this study, the genetic variability of the frequently circulating RV types in Northern Tunisia was investigated, using phylogenetic and phylogeographic analyses with a specific focus on the most frequent RV types: RV-A101 and RV-C45. This study concerned 13 RV types frequently circulating in Northern Tunisia. They were obtained from respiratory samples collected in 271 pediatric SARI cases, between September 2015 and November 2017. A total of 37 RV VP4-VP2 sequences, selected among a total of 49 generated sequences, was compared to 359 sequences from different regions of the world. Evolutionary analysis of RV-A101 and RV-C45 showed high genetic relationship between different Tunisian strains and Malaysian strains. RV-A101 and C45 progenitor viruses’ dates were estimated in 1981 and 1995, respectively. Since the early 2000s, the two types had a wide spread throughout the world. Phylogenetic analyses of other frequently circulating strains showed significant homology of Tunisian strains from the same epidemic period, in contrast with earlier strains. The genetic relatedness of RV-A101 and RV-C45 might result from an introduction of viruses from different clades followed by local dissemination rather than a local persistence of an endemic clades along seasons. International traffic may play a key role in the spread of RV-A101, RV-C45, and other RVs.

Genetic Factors Associated with Variation in Abundance of the Invasive Yellow Crazy Ant (Anoplolepis gracilipes)

<p>A key component of successful invasion is the ability of an introduced population to reach sufficient abundance to persist, spread, and alter or dominate the recipient biological community. Genetic diversity is one of many factors that may contribute to population dynamics, but has important ramifications for biological fitness, and thus invasion success in the long term. I explored genetic factors associated with variation in abundance (i.e., differential invasion success) of the yellow crazy ant Anoplolepis gracilipes in the Indo-Pacific region, primarily focussing on Arnhem Land in Australia's Northern Territory. I explored five aspects that I hypothesised could contribute to variation in the abundance of this ant: 1) I investigated the unusual reproductive mode of A. gracilipes, and tested whether it involved dependent-lineage genetic caste determination (DL GCD) in Arnhem Land. In DL GCD systems populations require hybridisation between genetically distinct groups to produce both reproductive and worker castes. Asymmetry in the ratio of different lineages may result in low abundance and population collapse. I found no evidence for a DL GCD system in A. gracilipes, and thus its abundance in Arnhem Land does not appear to be constrained by any lineage ratio asymmetry. Worker reproduction (either of males or asexual production of other workers) also appeared unlikely. The reproductive mode of the species remains fascinating but enigmatic; 2) I explored whether multiple source populations were responsible for the observed variation in abundance in Arnhem Land (i.e., is abundance associated with propagule pressure, or populations from different sources), and if the population has diverged since introduction. The A. gracilipes population in Arnhem Land stemmed from a single source, and thus propagule pressure was apparently not responsible for variation in abundance. In contrast to many invasive ants, population divergence has occurred since introduction; 3) I tested the hypotheses that genetic variation was associated with variation in abundance in Arnhem Land, and that ecological success was density-dependent. While the population divergence found in Chapter 3 was not related to variation in abundance, genotypic diversity was higher in more abundant nest clusters. These more abundant nest clusters were in turn associated with lower native ant species diversity, and a difference in composition of the invaded ant community (i.e., greater ecological success); 4) I revisited the invasion of the yellow crazy ant in Tokelau to determine whether a haplotype that was linked to greater abundance and dominance of the ant community has increased in distribution. Although ants of the inferred dominant haplotype were implicated in most new invasions, their abundance was substantially lower than previously observed in Tokelau; 5) I conducted a preliminary analysis of the metagenomic diversity of A. gracilipes endogenous parasites and symbionts among populations from Christmas Island, Okinawa, Samoa and Arnhem Land. Bacterial community composition and diversity differed between the study populations. Variation in abundance among A. gracilipes populations in Arnhem Land was not due to parasite load on populations with low abundance. However, low abundance of A. gracilipes was correlated with lower microbial diversity overall, and higher prevalence of some groups, notably two that confer antibiotic properties. Together, my findings suggest that propagule pressure, reproductive mode and haplotype-specific effects do not appear to be associated with variation in A. gracilipes abundance. Other genetic factors I investigated do appear to be associated with the variation in A. gracilipes abundance and effects on the invaded ant communities. Genotypic diversity was positively related to the abundance of A. gracilipes in Arnhem Land, and this relationship may be affected by population divergence through population bottlenecks. In addition, differences in bacterial diversity among populations highlighted several candidate bacteria that could be associated with variation in abundance, which would be a topic of future work. Although genetic factors are often implicated in the successful establishment of invasive species, my thesis demonstrates that genetic factors may also be associated with post-establishment population dynamics.</p>

Genetic Factors Associated with Variation in Abundance of the Invasive Yellow Crazy Ant (Anoplolepis gracilipes)

<p>A key component of successful invasion is the ability of an introduced population to reach sufficient abundance to persist, spread, and alter or dominate the recipient biological community. Genetic diversity is one of many factors that may contribute to population dynamics, but has important ramifications for biological fitness, and thus invasion success in the long term. I explored genetic factors associated with variation in abundance (i.e., differential invasion success) of the yellow crazy ant Anoplolepis gracilipes in the Indo-Pacific region, primarily focussing on Arnhem Land in Australia's Northern Territory. I explored five aspects that I hypothesised could contribute to variation in the abundance of this ant: 1) I investigated the unusual reproductive mode of A. gracilipes, and tested whether it involved dependent-lineage genetic caste determination (DL GCD) in Arnhem Land. In DL GCD systems populations require hybridisation between genetically distinct groups to produce both reproductive and worker castes. Asymmetry in the ratio of different lineages may result in low abundance and population collapse. I found no evidence for a DL GCD system in A. gracilipes, and thus its abundance in Arnhem Land does not appear to be constrained by any lineage ratio asymmetry. Worker reproduction (either of males or asexual production of other workers) also appeared unlikely. The reproductive mode of the species remains fascinating but enigmatic; 2) I explored whether multiple source populations were responsible for the observed variation in abundance in Arnhem Land (i.e., is abundance associated with propagule pressure, or populations from different sources), and if the population has diverged since introduction. The A. gracilipes population in Arnhem Land stemmed from a single source, and thus propagule pressure was apparently not responsible for variation in abundance. In contrast to many invasive ants, population divergence has occurred since introduction; 3) I tested the hypotheses that genetic variation was associated with variation in abundance in Arnhem Land, and that ecological success was density-dependent. While the population divergence found in Chapter 3 was not related to variation in abundance, genotypic diversity was higher in more abundant nest clusters. These more abundant nest clusters were in turn associated with lower native ant species diversity, and a difference in composition of the invaded ant community (i.e., greater ecological success); 4) I revisited the invasion of the yellow crazy ant in Tokelau to determine whether a haplotype that was linked to greater abundance and dominance of the ant community has increased in distribution. Although ants of the inferred dominant haplotype were implicated in most new invasions, their abundance was substantially lower than previously observed in Tokelau; 5) I conducted a preliminary analysis of the metagenomic diversity of A. gracilipes endogenous parasites and symbionts among populations from Christmas Island, Okinawa, Samoa and Arnhem Land. Bacterial community composition and diversity differed between the study populations. Variation in abundance among A. gracilipes populations in Arnhem Land was not due to parasite load on populations with low abundance. However, low abundance of A. gracilipes was correlated with lower microbial diversity overall, and higher prevalence of some groups, notably two that confer antibiotic properties. Together, my findings suggest that propagule pressure, reproductive mode and haplotype-specific effects do not appear to be associated with variation in A. gracilipes abundance. Other genetic factors I investigated do appear to be associated with the variation in A. gracilipes abundance and effects on the invaded ant communities. Genotypic diversity was positively related to the abundance of A. gracilipes in Arnhem Land, and this relationship may be affected by population divergence through population bottlenecks. In addition, differences in bacterial diversity among populations highlighted several candidate bacteria that could be associated with variation in abundance, which would be a topic of future work. Although genetic factors are often implicated in the successful establishment of invasive species, my thesis demonstrates that genetic factors may also be associated with post-establishment population dynamics.</p>

Genotypic Diversity of Ciprofloxacin Nonsusceptibility and Its Relationship with Minimum Inhibitory Concentrations in Nontyphoidal Salmonella Clinical Isolates in Taiwan

This study analyzed the genetic diversity of ciprofloxacin (CIP) nonsusceptibility and the relationship between two major mechanisms and minimum inhibitory concentrations (MICs) of CIP in nontyphoidal Salmonella (NTS). Chromosomal mutations in quinolone resistance-determining regions (QRDRs) and plasmid-mediated quinolone resistance (PMQR) genes were searched from ResFinder, ARG-ANNOT, and PubMed for designing the sequencing regions in gyrA, gyrB, parC, and parE, and the 13 polymerase chain reactions for PMQR genes. We found that QRDR mutations were detected in gyrA (82.1%), parC (59.0%), and parE (20.5%) but not in gyrB among the 39 isolates. Five of the 13 PMQR genes were identified, including oqxA (28.2%), oqxB (28.2%), qnrS (18.0%), aac(6′)-Ib-cr (10.3%), and qnrB (5.1%), which correlated with the MICs of CIP within 0.25–2 μg/mL, and it was found that oxqAB contributed more than qnr genes to increase the MICs. All the isolates contained either QRDR mutations (53.8%), PMQR genes (15.4%), or both (30.8%). QRDR mutations (84.6%) were more commonly detected than PMQR genes (46.2%). QRDR mutation numbers were significantly associated with MICs (p < 0.001). Double mutations in gyrA and parC determined high CIP resistance (MICs ≥ 4 μg/mL). PMQR genes contributed to intermediate to low CIP resistance (MICs 0.25–2 μg/mL), thus providing insights into mechanisms underlying CIP resistance.