Host-specific Differences Research Articles

Host range differences between two species of freshwater horsehair worm (Nematomorpha: Chordiidae) Chordodes japonensis and C. formosanus in Japan

Horsehair worms (Nematomorpha: Gordioidea) are endoparasites of terrestrial arthropods, of which two species, Chordodes japonensis and C. formosanus, may have been historically confused due to their morphological similarity. In this study, we conducted field surveys and laboratory parasite inoculation experiments to clarify the characteristics and differences in host range and specificity between the two species. The field surveys revealed that the host ranges of the two species are markedly different. C. formosanus parasitized diverse mantids, while C. japonensis infected only species in the genus Tenodera, as predicted in previous studies. However, the two species had one mantid host, T. angustipennis in common. Parasite inoculation experiments using three species of mantids, revealed parasitism in each of the host-parasite combinations same as in the field study. These results suggest that the observed differences in host patterns are due to physiological factors between the host and parasite. Furthermore, cross-testing conducted on the shared host T. angustipennis showed that the two species coexisted within the same host in some cases, suggesting that the competitive exclusivity of the two species is low. This study also presents a tentative list of host ranges for the two species, comparing and integrating information from this and previous studies. However, the possibility remains that the host ranges of both species may be more diverse.

Cryptosporidium mortiferum n. sp. (Apicomplexa: Cryptosporidiidae), the species causing lethal cryptosporidiosis in Eurasian red squirrels (Sciurus vulgaris)

BackgroundCryptosporidium spp. are globally distributed parasites that infect epithelial cells in the microvillus border of the gastrointestinal tract of all classes of vertebrates. Cryptosporidium chipmunk genotype I is a common parasite in North American tree squirrels. It was introduced into Europe with eastern gray squirrels and poses an infection risk to native European squirrel species, for which infection is fatal. In this study, the biology and genetic variability of different isolates of chipmunk genotype I were investigated.MethodsThe genetic diversity of Cryptosporidium chipmunk genotype I was analyzed by PCR/sequencing of the SSU rRNA, actin, HSP70, COWP, TRAP-C1 and gp60 genes. The biology of chipmunk genotype I, including oocyst size, localization of the life cycle stages and pathology, was examined by light and electron microscopy and histology. Infectivity to Eurasian red squirrels and eastern gray squirrels was verified experimentally.ResultsPhylogenic analyses at studied genes revealed that chipmunk genotype I is genetically distinct from other Cryptosporidium spp. No detectable infection occurred in chickens and guinea pigs experimentally inoculated with chipmunk genotype I, while in laboratory mice, ferrets, gerbils, Eurasian red squirrels and eastern gray squirrels, oocyst shedding began between 4 and 11 days post infection. While infection in mice, gerbils, ferrets and eastern gray squirrels was asymptomatic or had mild clinical signs, Eurasian red squirrels developed severe cryptosporidiosis that resulted in host death. The rapid onset of clinical signs characterized by severe diarrhea, apathy, loss of appetite and subsequent death of the individual may explain the sporadic occurrence of this Cryptosporidium in field studies and its concurrent spread in the population of native European squirrels. Oocysts obtained from a naturally infected human, the original inoculum, were 5.64 × 5.37 μm and did not differ in size from oocysts obtained from experimentally infected hosts. Cryptosporidium chipmunk genotype I infection was localized exclusively in the cecum and anterior part of the colon.ConclusionsBased on these differences in genetics, host specificity and pathogenicity, we propose the name Cryptosporidium mortiferum n. sp. for this parasite previously known as Cryptosporidium chipmunk genotype I.Graphical

Multiple Ehrlichia chaffeensis genes critical for persistent infection in a vertebrate host are identified as nonessential for its growth in the tick vector; Amblyomma americanum.

Ehrlichia chaffeensis is a tick-transmitted monocytic ehrlichiosis agent primarily causing the disease in people and dogs. We recently described the development and characterization of 55 random mutations in E. chaffeensis, which aided in defining the critical nature of many bacterial genes for its growth in a physiologically relevant canine infection model. In the current study, we tested 45 of the mutants for their infectivity ability to the pathogen's tick vector; Amblyomma americanum. Four mutations resulted in the pathogen's replication deficiency in the tick, similar to the vertebrate host. Mutations causing growth defects in both vertebrate and tick hosts included in genes coding for a predicted alpha/beta hydrolase, a putative dicarboxylate amino acid:cation symporter, a T4SS protein, and predicted membrane-bound proteins. Three mutations caused the bacterial defective growth only in the tick vector, which represented putative membrane proteins. Ten mutations causing no growth defect in the canine host similarly grew well in the tick vector. Mutations in 28 genes/genomic locations causing E. chaffeensis growth attenuation in the canine host were recognized as non-essential for its growth in the tick vector. The tick non-essential genes included genes coding for many metabolic pathway- and outer membrane-associated proteins. This study documents novel vector- and host-specific differences in E. chaffeensis for its functional gene requirements.

Attributes of host-specificity better explain the diversified wood-boring longhorn beetles in tropical SW China than plant species diversity

A long-debated question in ecology is whether the hyper-diversity of tropical plant-feeding insects is a direct consequence of high tropical plant diversity or should be attributed to increases in host plant specialization. In this study, we used Cerambycidae (the wood-boring longhorn beetles whose larval stages feed on the xylems of trees and lianas) and plants as study materials to explore which hypothesis is more favoured. Multiple analyses were used to show the differences in host specificity of Cerambycidae in tropical and subtropical forests. From these analyses, we found that the alpha diversity of beetles in tropical forests was significantly higher than that in subtropical forests but not in plants. The relationship between plants and beetles was also closer in tropical areas than in subtropical areas. Our results imply that the wood-boring longhorn beetles show higher degrees of niche conservatism and host-specificity in tropical forests than in subtropical forests. The high diversity of wood-boring longhorn beetles in tropical forests might be explained to a large extent by their more finely partitioned diet breadth.

Intracellular Diversity of WNV within Circulating Avian Peripheral Blood Mononuclear Cells Reveals Host-Dependent Patterns of Polyinfection.

Arthropod-borne virus (arbovirus) populations exist as mutant swarms that are maintained between arthropods and vertebrates. West Nile virus (WNV) population dynamics are host-dependent. In American crows, purifying selection is weak and population diversity is high compared to American robins, which have 100- to 1000-fold lower viremia. WNV passed in robins leads to fitness gains, whereas that passed in crows does not. Therefore, we tested the hypothesis that high crow viremia allows for higher genetic diversity within individual avian peripheral blood mononuclear cells (PBMCs), reasoning that this could have produced the previously observed host-specific differences in genetic diversity and fitness. Specifically, we infected cells and birds with a molecularly barcoded WNV and sequenced viral RNA from single cells to quantify the number of WNV barcodes in each. Our results demonstrate that the richness of WNV populations within crows far exceeds that in robins. Similarly, rare WNV variants were maintained by crows more frequently than by robins. Our results suggest that increased viremia in crows relative to robins leads to the maintenance of defective genomes and less prevalent variants, presumably through complementation. Our findings further suggest that weaker purifying selection in highly susceptible crows is attributable to this higher viremia, polyinfections and complementation.

Host-specific differences in top-expanded TCR clonotypes correlate with divergent outcomes of anti-PD-L1 treatment in responders versus non-responders.

Immune checkpoint inhibitors (ICIs) have revolutionized cancer treatment; however, the responses to ICI treatment are highly variable in different individuals and the underlying mechanisms remain poorly understood. Here, we employed a mouse squamous cell carcinoma (SCC) model where tumor-bearing recipients diverged into responders (R) versus non-responders (NR) upon anti-PD-L1 treatment. We performed in-depth TCRβ sequencing with immunoSEQ platform to delineate the differences in CD8 tumor-infiltrating lymphocytes (TILs). We found that R and NR CD8 TILs both exhibited evidence of clonal expansion, suggesting activation regardless of response status. We detected no differences in clonal expansion or clonal diversity indexes between R vs. NR. However, the top expanded (>1%) TCRβ clonotypes appeared to be mutually exclusive between R and NR CD8 TILs, showing a preferential expansion of distinct TCRβ clonotypes in response to the same SCC tumor in R vs. NR. Notably, the mutual exclusivity of TCR clonotypes in R vs. NR was only observed when top TCRβ clonotypes were counted, because such top-expanded clonotypes are present in the opposite outcome group at a much lower frequency. Many TCRβ sequences were detected in only one recipient at a high frequency, implicating highly individualized anti-tumor immune responses. We conclude that differences in the clonal frequency of top TCR clonotypes between R and NR CD8 TILs may be one of the factors underlying differential anti-PD-L1 responses. This notion may offer a novel explanation for variable ICI responses in different individuals, which may substantially impact the development of new strategies for personalized cancer immunotherapy.

Minimum Core Genome Sequence Typing of Brucella From China

Abstract The Gram-negative bacterial genus Brucella includes six classic species based on host specificity, pathogenicity and phenotypic differences. Four more Brucella species were identified in 2007. Although many Brucella genomes have been sequenced, genome sequences and analysis of Brucella strains isolated in China are still scarce. An efficient genome-based Brucella typing method is also needed. In this study, we used the minimum core genome (MCG) typing method to identify and type Brucella strains. Twenty Brucella isolates from China were newly sequenced. The genome sequences of 55 representative Brucella strains were downloaded. Among the 75 genomes, 1089 genes and 52,030 single nucleotide polymorphisms (SNPs) shared by all isolates were considered as the MCG genes and MCG SNPs. Using these 52,030 MCG SNPs, Brucella was divided into six MCG groups. In addition, average nucleotide identity (ANI) values and the distributions of 184 virulence genes were all computed. The proportions of virulence genes were 90.96%, 93.56%, 95.89%, 86.04%, 85.78% and 91.87% for MCG groups 1 to 6, respectively. The intragroup ANI values were higher than the intergroup values, further confirming the validity of the MCG taxonomy classification. Brucella melitensis and Brucella abortus, the two main Brucella species pathogenic to humans, were well separated from other species. With the development and cost reduction of next-generation sequencing, the MCG typing method can be used for rapid identification of Brucella, which can contribute to the rapid diagnosis of brucellosis and ensure timely and effective treatment.

Diverse susceptibilities and responses of human and rodent cells to orthohantavirus infection reveal different levels of cellular restriction.

Orthohantaviruses are rodent-borne emerging viruses that may cause severe diseases in humans but no apparent pathology in their small mammal reservoirs. However, the mechanisms leading to tolerance or pathogenicity in humans and persistence in rodent reservoirs are poorly understood, as is the manner in which they spread within and between organisms. Here, we used a range of cellular and molecular approaches to investigate the interactions of three different orthohantaviruses–Puumala virus (PUUV), responsible for a mild to moderate form of hemorrhagic fever with renal syndrome in humans, Tula virus (TULV) with low pathogenicity, and non-pathogenic Prospect Hill virus (PHV)–with human and rodent host cell lines. Besides the fact that cell susceptibility to virus infection was shown to depend on the cell type and virus strain, the three orthohantaviruses were able to infect Vero E6 and HuH7 human cells, but only the former secreted infectious particles. In cells derived from PUUV reservoir, the bank vole (Myodes glareolus), PUUV achieved a complete viral cycle, while TULV did not enter the cells and PHV infected them but did not produce infectious particles, reflecting differences in host specificity. A search for mature virions by electron microscopy (EM) revealed that TULV assembly occurred in part at the plasma membrane, whereas PHV particles were trapped in autophagic vacuoles in cells of the heterologous rodent host. We described differential interactions of orthohantaviruses with cellular factors, as supported by the cellular distribution of viral nucleocapsid protein with cell compartments, and proteomics identification of cellular partners. Our results also showed that interferon (IFN) dependent gene expression was regulated in a cell and virus species dependent manner. Overall, our study highlighted the complexity of the host-virus relationship and demonstrated that orthohantaviruses are restricted at different levels of the viral cycle. In addition, the study opens new avenues to further investigate how these viruses differ in their interactions with cells to evade innate immunity and how it depends on tissue type and host species.

Unravelling the Phylogeny of a Common Intestinal Protist: Intrageneric Diversity of Endolimax

Endolimax nana is a common endobiont of the human intestine, but members of the genus have also been reported in non-human hosts and in non-intestinal organs. Limited information is available regarding the genetic diversity of Endolimax, which is necessary to delineate species, host specificity and potential differences in clinical impact on the host. Here, we used cloning of PCR products followed by Sanger sequencing and next-generation PacBio Sequencing to obtain Endolimax-related nuclear ribosomal gene sequences and undertook a phylogenetic analysis to gain additional insight into the taxonomy of Endolimax and related organisms. The new sequences confirmed that E. nana forms a discrete clade within the Archamoebae and is related to Endolimax piscium and Iodamoeba. However, we identified substantial sequence divergence within E. nana and evidence for two distinct clades, which we propose to name E. nana ribosomal lineage 1 and E. nana ribosomal lineage 2. Both of the sequencing approaches applied in the study helped us to improve our understanding of genetic diversity across Endolimax, and it is likely that wider application of next-generation sequencing technologies will facilitate the generation of Endolimax-related DNA sequence data and help complete our understanding of its phylogenetic position and intrageneric diversity.

Tospoviruses Induce Small Interfering RNAs Targeting Viral Sequences and Endogenous Transcripts in Solanaceous Plants.

Tospoviruses infect numerous crop species worldwide, causing significant losses throughout the supply chain. As a defence mechanism, plants use RNA interference (RNAi) to generate virus-derived small-interfering RNAs (vsiRNAs), which target viral transcripts for degradation. Small RNA sequencing and in silico analysis of capsicum and N. benthamiana infected by tomato spotted wilt virus (TSWV) or capsicum chlorosis virus (CaCV) demonstrated the presence of abundant vsiRNAs, with host-specific differences evident for each pathosystem. Despite the biogenesis of vsiRNAs in capsicum and N. benthamiana, TSWV and CaCV viral loads were readily detectable. In response to tospovirus infection, the solanaceous host species also generated highly abundant virus-activated small interfering RNAs (vasiRNAs) against many endogenous transcripts, except for an N. benthamiana accession lacking a functional RDR1 gene. Strong enrichment for ribosomal protein-encoding genes and for many genes involved in protein processing in the endoplasmic reticulum suggested co-localisation of viral and endogenous transcripts as a basis for initiating vasiRNA biogenesis. RNA-seq and RT-qPCR-based analyses of target transcript expression revealed an inconsistent role for vasiRNAs in modulating gene expression in N. benthamiana, which may be characteristic of this tospovirus-host pathosystem.

Variation in haplotypes in single cysts of assemblages C and D, but not of assemblage E of Giardia duodenalis

BackgroundGiardia duodenalis, a single-celled intestinal parasite, is divided into eight assemblages (A-H), with differences in host specificity. Giardia duodenalis reproduces asexually and cycles between the binucleated trophozoite (4 N) and the infectious cyst with four nuclei (16 N). Interaction between the nuclei is limited. Therefore, genetic drift causes differences in genetic make-up between the non-daughter nuclei; the allelic sequence heterozygosity (ASH). The ASH is low (0.01%—0.0023%) for the related assemblages A and E, higher (0.43–0.53) for assemblage B and much higher (0.74% -0.89%) for the assemblage C and D at the root of the phylogenetic tree. The heterozygosity in assemblage F, in the same clade as assemblage A and E, was unknown. The heterozygosity in the sequences of the gdh and dis3 genes was used as proxy for the ASH and whole genome amplification of single cysts followed by cloning and Sanger sequencing of dis3 fragment could reveal the genetic variation within the cyst. The aim of the study was to determine the level of heterozygosity within pooled and single cysts of different assemblages.ResultsThe heterozygosity in gdh and dis3 was determined in pooled cysts of the assemblages A to F. Heterozygosity in the isolates of the assemblages C (n = 2) and D (n = 1) ranged from 0.41% to 0.82% for gdh and dis3 and no heterozygosity was found in the isolates of the assemblages A (n = 4), E (n = 3) and F (n = 3). The heterozygosity in assemblage B (n = 7) was intermediate (0% to 0.62%). Next, the number of haplotypes of dis3 was determined for single cysts of assemblages C, D and E. In the assemblages C and D, two to four haplotypes were found per cyst, while in assemblage E only one haplotype was identified.ConclusionsHaving high heterozygosity is characteristic for the assemblages C and D, while having a low heterozygosity is characteristic for the clade with the assemblages A, E and F.Presence of more than 1 haplotype per cyst in assemblage C and D suggests differences between the non-daughter nuclei, in contrast to the one haplotype in assemblage E.

High prevalence of Ichthyophonus sp. infections in Northeast Atlantic mackerel (Scomber scombrus).

High prevalence of Ichthyophonus sp. infections in Northeast Atlantic mackerel (Scomber scombrus).

Generalist parasites persist in degraded environments: a lesson learned from microsporidian diversity in amphipods.

The present study provides new insight into suitable microsporidian–host associations. It relates regional and continental-wide host specialization in microsporidians infecting amphipods to degraded and recovering habitats across 2 German river catchments. It provides a unique opportunity to infer the persistence of parasites following anthropogenic disturbance and their establishment in restored rivers. Amphipods were collected in 31 sampling sites with differing degradation and restoration gradients. Specimens were morphologically (hosts) and molecularly identified (host and parasites). Amphipod diversity and abundance, microsporidian diversity, host phylogenetic specificity and continental-wide β-specificity were investigated and related to each other and/or environmental variables. Fourteen microsporidian molecular operational taxonomic units (MOTUs), mainly generalist parasites, infecting 6 amphipod MOTUs were detected, expanding the current knowledge on the host range by 17 interactions. There was no difference in microsporidian diversity and host specificity among restored and near-natural streams (Boye) or between those located in urban and rural areas (Kinzig). Similarly, microsporidian diversity was generally not influenced by water parameters. In the Boye catchment, host densities did not influence microsporidian MOTU richness across restored and near-natural sites. High host turnover across the geographical range suggests that neither environmental conditions nor host diversity plays a significant role in the establishment into restored areas. Host diversity and environmental parameters do not indicate the persistence and dispersal of phylogenetic host generalist microsporidians in environments that experienced anthropogenic disturbance. Instead, these might depend on more complex mechanisms such as the production of resistant spores, host switching and host dispersal acting individually or conjointly.

Revisiting the intrageneric structure of the genus Pseudomonas with complete whole genome sequence information: Insights into diversity and pathogen-related genetic determinants.

Pseudomonas spp. exhibit considerable differences in host specificity and virulence. Most Pseudomonas species were isolated exclusively from environmental sources, ranging from soil to plants, but some Pseudomonas species have been detected from versatile sources, including both human host and environmental sources. Understanding genome variations that generate the tremendous diversity in Pseudomonas biology is important in controlling the incidence of infections. With a data set of 704 Pseudomonas complete whole genome sequences representing 186 species, Pseudomonas intrageneric structure was investigated by hierarchical clustering based on average nucleotide identity, and by phylogeny analysis based on concatenated core-gene alignment. Further comparative functional analyses indicated that Pseudomonas species only living in natural habitats lack multiple functions that are important in the regulation of bacterial pathogenesis, indicating the possession of these functions might be characteristic of Pseudomonas human pathogens. Moreover, we have performed pan-genome based homogeneity analyses, and detected genes with conserved structures but diversified functions across the Pseudomonas genomes, suggesting these genes play a role in driving diversity. In summary, this study provided insights into the dynamics of genome diversity and pathogen-related genetic determinants in Pseudomonas, which might help the development of more targeted antibiotics for the treatment of Pseudomonas infections.

Inter-species lateral gene transfer focused on the Chlamydia plasticity zone identifies loci associated with immediate cytotoxicity and inclusion stability.

Chlamydia muridarum actively grows in murine mucosae and is a representative model of human chlamydial genital tract disease. In contrast, C. trachomatis infections in mice are limited and rarely cause disease. The factors that contribute to these differences in host adaptation and specificity remain elusive. Overall genomic similarity leads to challenges in the understanding of these significant differences in tropism. A region of major genetic divergence termed the plasticity zone (PZ) has been hypothesized to contribute to the host specificity. To evaluate this hypothesis, lateral gene transfer was used to generate multiple hetero-genomic strains that are predominately C. trachomatis but have replaced regions of the PZ with those from C. muridarum. In vitro analysis of these chimeras revealed C. trachomatis-like growth as well as poor mouse infection capabilities. Growth-independent cytotoxicity phenotypes have been ascribed to three large putative cytotoxins (LCT) encoded in the C. muridarum PZ. However, analysis of PZ chimeras supported that gene products other than the LCTs are responsible for cytopathic and cytotoxic phenotypes. Growth analysis of associated chimeras also led to the discovery of an inclusion protein, CTL0402 (CT147), and homolog TC0424, which was critical for the integrity of the inclusion and preventing apoptosis.

Identification of Diverse Toxin Complex Clusters and an eCIS Variant in Serratia proteamaculans Pathovars of the New Zealand Grass Grub (Costelytra Giveni) and Manuka Beetle (Pyronota Spp.) Larvae.

ABSTRACTThe grass grub endemic to New Zealand, Costelytra giveni (Coleoptera: Scarabaeidae), and the manuka beetle, Pyronota festiva and P. setosa (Coleoptera: Scarabaeidae), are prevalent pest species. Through assessment of bacterial strains isolated from diseased cadavers of these insect species, 19 insect-active Serratia proteamaculans variants and a single Serratia entomophila strain were isolated. When independently bioassayed, these isolates differed in host range, the rate of disease progression, and 12-day mortality rates, which ranged from 60 to 100% of the challenged larvae. A Pyronota spp.-derived S. proteamaculans isolate caused a transient disease phenotype in challenged C. giveni larvae, whereby larvae appeared diseased before recovering to a healthy state. Genome sequence analysis revealed that all but two of the sequenced isolates contained a variant of the S. entomophila amber-disease-associated plasmid, pADAP. Each isolate also encoded one of seven distinct members of the toxin complex (Tc) family of insect-active toxins, five of which are newly described, or a member of the extracellular contractile injection (eCIS) machine family, with a new AfpX variant designated SpF. Targeted mutagenesis of each of the predicted Tc- or eCIS-encoding regions abolished or attenuated pathogenicity. Host-range testing showed that several of the S. proteamaculans Tc-encoding isolates affected both Pyronota and C. giveni species, with other isolates specific for either Pyronota spp. or C. giveni. The isolation of several distinct host-specific pathotypes of Serratia spp. may reflect pathogen-host speciation.IMPORTANCE New pathotypes of the insect pathogen Serratia, each with differing virulence attributes and host specificity toward larvae of the New Zealand manuka beetle and grass grub, have been identified. All of the Serratia proteamaculans isolates contained one of seven different insect-active toxin clusters or one of three eCIS variants. The diversity of these Serratia-encoded virulence clusters, resulting in differences in larval disease progression and host specificity in endemic scarab larvae, suggests speciation of these pathogens with their insect hosts. The differing virulence properties of these Serratia species may affect their potential infectivity and distribution among the insect populations. Based on their differing geographic isolation and pathotypes, several of these Serratia isolates, including the manuka beetle-active isolates, are likely to be more effective biopesticides in specific environments or could be used in combination for greater effect.

Diversity, host specificity and biogeography in the Cladocorynidae (Hydrozoa, Capitata), with description of a new genus.

The hydrozoan family Cladocorynidae inhabits tropical to temperate waters and comprises the two genera Pteroclava and Cladocoryne. Pteroclava lives in association with some octocorals and hydrozoans, whereas Cladocoryne is more generalist in terms of substrate choice. This work provides a thorough morpho-molecular reassessment of the Cladocorynidae by presenting the first well-supported phylogeny of the family based on the analyses of three mitochondrial and four nuclear markers. Notably, the two nominal genera were confirmed to be monophyletic and both morphological and genetic data led to the formal description of a new genus exclusively associated with octocorals, Pseudozanclea gen. nov. Maggioni & Montano. Accordingly, the diagnosis of the family was updated. The ancestral state reconstruction of selected characters revealed that the symbiosis with octocorals likely appeared in the most recent common ancestor of Pteroclava and Pseudozanclea. Additionally, the presence of euryteles aggregation in the polyp stage and the exumbrellar nematocyst pouches with euryteles represent synapomorphies of all cladocorynid taxa and probably emerged in their most recent common ancestor. The analysis of several Pteroclava krempfi colonies from Indo-Pacific and Caribbean localities associated with several host octocorals revealed a high intra-specific genetic variability. Single- and multi-locus species delimitations resulted in three to five species hypotheses, but the statistical analysis of morphometric data showed only limited distinction among the clades of P. krempfi. However, P. krempfi clades showed differences in both host specificity, mostly at the octocoral family level, and geographic distribution, with one clade found exclusively in the Caribbean Sea and the others found in the Indo-Pacific.

Monitoring of Antimicrobial Resistance to Aminoglycosides and Macrolides in Campylobacter coli and Campylobacter jejuni From Healthy Livestock in Spain (2002–2018)

Antimicrobial resistance (AMR) in Campylobacter spp. (Campylobacter coli and Campylobacter jejuni) is a concern due to its importance in public health, particularly when it involves aminoglycosides and macrolides, drugs of choice for treatment of human cases. Co-resistance to these two antimicrobial classes involves transfer of genetic elements and/or acquisition of mutations in different genetic loci, which can in turn spread through vertical or horizontal gene transfer (HGT) phenomena, with each route having different potential implications. This study aimed at evaluating the association between the presence of phenotypic resistance to these two antimicrobial classes in C. coli and C. jejuni recovered from livestock at slaughterhouses in Spain (as part of the AMR surveillance program), and at assessing the genetic heterogeneity between resistant and susceptible isolates by analysing the “short variable region” (SVR) of the flaA gene. Over the 2002–2018 period, antimicrobial susceptibility test results from 10,965 Campylobacter isolates retrieved from fecal samples of broilers, turkeys, pigs and cattle were collected to compare the proportion of resistant isolates and the Minimum Inhibitory Concentrations (MICs) against six antimicrobials including gentamicin (GEN), streptomycin (STR), and erythromycin (ERY). AMR-associated genes were determined for a group of 51 isolates subjected to whole genome sequencing, and the flaA SVR of a subset of 168 isolates from all hosts with different resistotypes was used to build a Neighbor-Joining-based phylogenetic tree and assess the existence of groups by means of “relative synonymous codon usage” (RSCU) analysis. The proportion of antimicrobial resistant isolates to both, aminoglycosides and macrolides, varied widely for C. coli (7–91%) and less for C. jejuni (all hosts 0–11%). Across hosts, these proportions were 7–56% in poultry, 12–82% in cattle, and 22–91% in pigs for C. coli and 0–8% in poultry and 1–11% in cattle for C. jejuni. Comparison of the MIC distributions revealed significant host-specific differences only for ERY in C. jejuni (p = 0.032). A significant association in the simultaneous presentation of AMR to both antimicrobial classes was observed across hosts/bacterial species. The flaA gene analysis showed clustering of isolates sharing resistotype and to a lesser degree bacterial species and host. Several resistance markers associated with resistance to aminoglycosides and macrolides were found among the sequenced isolates. The consistent association between the simultaneous presentation of AMR to aminoglycosides and macrolides in all hosts could be due to the persistence of strains and/or resistance mechanisms in Campylobacter populations in livestock over time. Further studies based on whole genome sequencing are needed to assess the epidemiological links between hosts and bacterial strains.

Salmonella Typhimurium Triggers Extracellular Traps Release in Murine Macrophages.

Salmonella comprises two species and more than 2500 serovars with marked differences in host specificity, and is responsible for a wide spectrum of diseases, ranging from localized gastroenteritis to severe life-threatening invasive disease. The initiation of the host inflammatory response, triggered by many Pathogen-Associated Molecular Patterns (PAMPs) that Salmonella possesses, recruits innate immune cells in order to restrain the infection at the local site. Neutrophils are known for killing bacteria through oxidative burst, amid other mechanisms. Amongst those mechanisms for controlling bacteria, the release of Extracellular Traps (ETs) represents a newly described pathway of programmed cell death known as ETosis. Particularly, Neutrophil Extracellular Traps (NETs) were first described in 2004 and since then, a number of reports have demonstrated their role as a novel defense mechanism against different pathogens. This released net-like material is composed of cellular DNA decorated with histones and cellular proteins. These structures have shown ability to trap, neutralize and kill different kinds of microorganisms, ranging from viruses and bacteria to fungi and parasites. Salmonella was one of the first microorganisms that were reported to be killed by NETs and several studies have confirmed the observation and deepened into its variants. Nevertheless, much less is known about their counterparts in other immune cells, e.g. Macrophage Extracellular Traps (METs) and Salmonella-induced MET release has never been reported so far. In this work, we observed the production of METs induced by Salmonella enterica serovar Typhimurium and recorded their effect on bacteria, showing for the first time that macrophages can also release extracellular DNA traps upon encounter with Salmonella Typhimurium. Additionally we show that METs effectively immobilize and reduce Salmonella survival in a few minutes, suggesting METs as a novel immune-mediated defense mechanism against Salmonella infection. Of note, this phenomenon was confirmed in primary macrophages, since MET release was also observed in bone marrow-derived macrophages infected with Salmonella. The evidence of this peculiar mechanism provides new incipient insights into macrophages´ role against Salmonella infection and can help to design new strategies for the clinical control of this transcendental pathogen.

Mapping the functional landscape of the receptor binding domain of T7 bacteriophage by deep mutational scanning.

The interaction between a bacteriophage and its host is mediated by the phage's receptor binding protein (RBP). Despite its fundamental role in governing phage activity and host range, molecular rules of RBP function remain a mystery. Here, we systematically dissect the functional role of every residue in the tip domain of T7 phage RBP (1660 variants) by developing a high-throughput, locus-specific, phage engineering method. This rich dataset allowed us to cross compare functional profiles across hosts to precisely identify regions of functional importance, many of which were previously unknown. Substitution patterns showed host-specific differences in position and physicochemical properties of mutations, revealing molecular adaptation to individual hosts. We discovered gain-of-function variants against resistant hosts and host-constricting variants that eliminated certain hosts. To demonstrate therapeutic utility, we engineered highly active T7 variants against a urinary tract pathogen. Our approach presents a generalized framework for characterizing sequence-function relationships in many phage-bacterial systems.