Campylobacter species of poultry origin have been associated with up to 80% of human campylobacteriosis cases. Layer chickens have received less attention as possible reservoirs of Campylobacter species. Initially, the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of two archived Campylobacter isolates (Campylobacter jejuni strain 200605 and Campylobacter coli strain 200606) from layer chickens to five antimicrobials  were determined using broth microdilution while the presence of selected antimicrobial resistance genes was performed by polymerase chain reaction (PCR) using specific primers. Whole-genome sequencing (WGS) was performed by the Illumina HiSeq X platform. The analysis involved antimicrobial resistance genes, virulome, multilocus sequence typing (MLST), and phylogeny. Both isolates were phenotypically resistant to ciprofloxacin (MIC: 32 vs. 32 µg/mL), nalidixic acid (MIC: 128 vs. 64 µg/mL), and tetracycline (MIC: 64 vs. 64 µg/mL), but sensitive to erythromycin (MIC: 1 vs. 2 µg/mL) and gentamicin (MIC: 0.25 vs. 1 µg/mL) for C. jejuni strain 200605 and C. coli strain 200606, respectively. WGS confirmed C257T mutation in the gyrA gene and the presence of cmeABC complex conferring resistance to FQs in both strains. Both strains also exhibited tet(O) genes associated with tetracycline resistance. Various virulence genes associated with motility, chemotaxis, and capsule formation were found in both isolates. However, the analysis of virulence genes showed that C. jejuni strain 200605 is more virulent than C. coli strain 200606. The MLST showed that C. jejuni strain 200605 belongs to sequence type ST-5229 while C. coli strain 200606 belongs to ST-5935, and both STs are less common. The phylogenetic analysis clustered C. jejuni strain 200605 along with other strains reported in Korea (CP028933 from chicken and CP014344 from human) while C. coli strain 200606 formed a separate cluster with C. coli (CP007181) from turkey. The WGS confirmed FQ-resistance in both strains and showed potential virulence of both strains. Further studies are recommended to understand the reasons behind the regional distribution  of such rare STs.Thermophilic  C. jejuni and C. coli are considered the leading etiologies of human campylobacteriosis , PCR was performed using specific primers  with settings of a threshold of 85% identity and a minimum length of 60% .https://tygs.dsmz.de (accessed on 19 February 2021), for a whole genome-based taxonomic analysis [The genome sequences (from our study) and those collected from public databases .The MIC values were interpreted using epidemiological cut-off values of the European Committee for Antimicrobial Susceptibility Testing  was used to edit, align, and analyze the DNA chromatograms [gyrA and tet(O)) with those from the GenBank database. Standard sensitive strains (L04566.1 and U63413.1) and resistant strains (KX982339.1 and MT176401.1) for gyrA were used for comparison. For the gyrA gene, the comparison was performed with Clustal Omega [BioEdit software (version 7.2.6.1)  and NCBI’s AMRFinderPlus database [C. jejuni and C. coli, respectively, were given after submission.For bioinformatics analysis, the filtered reads were mapped to reference genomes (NCTC11168 and NCTC11366) using Burrows-Wheeler Aligner (BWA-MEM), followed by variants identification and annotation. Produced mass sequence data were used to search for genetic variation based on the NCBI reference genome. After removing duplicates with Sambamba (v0.6.7)  and idendatabase . Virulendatabase . The gendatabase . GenBankC. jejuni strain 200605 and C. coli strain 200606 to ciprofloxacin (CIP), nalidixic acid (NAL), and tetracycline (TET) with MIC values ranging between 32 μg/mL and 128 μg/mL. Also, C. jejuni strain 200605 and C. coli strain 200606 were sensitive to erythromycin (MIC: 1 vs. 2 µg/mL), and gentamicin (MIC: 0.25 vs. 1 µg/mL), respectively.The phenotypic AMR results revealed high-level resistance of gyrA and tet(O) genes, but no band was seen for cmeB. WGS confirmed the presence of the C257T point mutation in the quinolone resistance-determining region of the gyrA gene of both strains. Abricate and Resfinder [tet(O/32/O) and tet(O) genes associated with resistance to doxycycline, tetracycline, and minocycline were found in both isolates by the WGS. Apart from blaOXA-452 found in both isolates, C. jejuni strain 200605 also showed the blaOXA-521 and blaOXA-193 genes. The detection of PointFinder genes returned mutations in gyrA and 23S rRNA genes, but no mutations were found in cmeR and rpsL for C. jejuni strain 200605. Conversely, cmeR was not detected, while rpsL was found but without a mutation for C. coli strain 200606. The latter also showed 12 point mutations in 23S rRNA. Mass screening of contigs of both isolates using ABRicate also showed resistance to cephalosporin, penam, and the presence of cmeB (efflux pump) conferring resistance to different antimicrobials.PCR confirmed the presence of DNA of esfinder  confirmeC. jejuni strain 200605 genome with PGAP returned 116 contigs: 1808 genes, of which 1688 were CDSs (with protein), 41 were RNAs , and 79 were pseudogenes . The annotation of the C. coli strain 200606 genome returned 29 contigs: 1,865 genes, of which 1743 were CDSs (with protein), 42 were RNAs , and 80 were pseudogenes . Additional details of both strains are given in The annotation of the C. jejuni strain 200605 and C. coli strain 200606, respectively.Of the called variants , SNPs, iC. jejuni strain 200605 and C. coli strain 200606 showed 87 and 57 virulence genes, respectively , a cytolethal distending toxin (cdtABC), invasion genes , and a biofilm formation gene (eptC) were only found in C. jejuni strain 200605 and not in C. coli strain 200606. However, both strains harbor genes coding for lipooligosaccharides (LOS), lipopolysaccharides (LPS), capsular , chemotaxis , and motility  factors.ectively . AdhesioC. jejuni strain 200605 forms a cluster with CP014344, which was isolated from a human in South Africa. It is also closely related to other strains of chicken origin from several countries including South Korea (CP028933), the USA , and China . However, it is separated from another cluster of CP059964 (chicken) and CP048756 (duck), both from China (C. jejuni strains used to generate the tree except for the C. jejuni (CP010502) strain that was isolated from human blood in Finland. The genome size was slightly higher compared to isolates from Type (Strain) Genome Server (TYGS), and it varied from 1.48–1.94 Mbp.The Genome BLAST Distance Phylogeny (GBDP) approach used to generate a phylogenomic tree  shows thom China . There wC. coli strain 200606 formed a separate cluster (species and subspecies) along with C. coli (CP007181) that was isolated from turkey and belongs to the same ST-1150 as the isolate of this study. Also, δ values were lower (0.181–0.175) than values for the cluster at the top of the tree (>0.2) (The GBDP phylogenomic tree  shows the (>0.2) . The oveC. jejuni strain 200605 belongs to ST-5229. So far, ST-5229 has not been assigned to a given clonal complex (CC). C. coli strain 200606 belongs to ST-5935, which belongs to CC-1150.Campylobacter spp. in table eggs is low, there is limited knowledge of their prevalence and ecology in layer chickens. Also, studies on the antimicrobial resistance profiles of layer chicken-derived Campylobacter isolates are limited [Campylobacter from layers important for epidemiological studies are also scanty.Although the prevalence of  limited ,32. ThisgyrA gene, while the second factor is the cmeABC operon coding for an efflux pump [Campylobacter strains to FQs has been previously reported in Korea [Campylobacter strains and may explain the increasing resistance trend [Campylobacter strains have been classified by the World Health Organization (WHO) as high-priority antibiotic-resistant pathogens for which new drugs are required [This study highlights the genomic characterization and phylogenetic analysis of two FQ-resistant strains from layers in Gangneung. The isolates showed increased resistance to FQs. The resistance to ciprofloxacin has been attributed to two loci that were found in our isolates. The first one is the C257T point mutation in the lux pump ,58. Incrin Korea ,60 and win Korea ,61, but in Korea ,63. The ce trend ,65. FQ-rrequired ,67.Campylobacter infections [Campylobacter strains from poultry may be associated with the limited use of macrolides in poultry production. Tylosin is used in swine or cattle, but not in poultry [Campylobacter strains that are ciprofloxacin-resistant compared to erythromycin-resistant ones [Ciprofloxacin and erythromycin have been used as the drugs of choice for treating fections . The glofections . Both stfections . The red poultry ,71. Howeant ones ,72.tet(O) gene coding for the ribosomal protection protein TetO [Phenotypic and genomic data showed resistance to tetracycline, which concurs with previous findings all over the world ,73,74. Hein TetO  found inein TetO . Moreoveein TetO . It is wein TetO .Campylobacter spp. are known to be inherently resistant to β-lactams including ampicillin [blaOXA-452, 521, and 193 genes which are inherent to Campylobacter. Ampicillin resistance is mainly due to enzymatic inactivation by blaOXA-61, but other factors like porins and reduced affinity to penicillin-binding protein (PBP) have also been reported [C. coli strains [rpsL was found but without mutation. Surveillance of gentamicin-resistant strains should be performed in response to the increasing number of resistant strains as reported in the USA and China [picillin , and we reported ,77. The reported ,79,80. H strains . The lim strains ,82 and icadF, pebA, and jlpA), invasion (ciaBC), toxin (cdtABC), flgSR two-component system, and biofilm formation (eptC) factors were only found in the C. jejuni strain 200605 genome and not in the C. coli strain 200606 genome. These factors highlight the virulent nature of the C. jejuni strain compared to C. coli which concurs with the literature [cheA, V, W), LOS, LPS, and capsule formation . The mentioned genes contribute to the pathogenicity of Campylobacter strains while infecting humans, as they are all required for successful colonization and survival [Campylobacter strains were negatively affected in absence of some important genes [Campylobacter strains lacking cdtB and cdtC were not cytotoxic, had reduced colonization, and had extra-intestinal invasiveness [flaA, flaB, flgB, flgE, and flaC) are involved in various cell functions, like motility and biofilm formation [kps D, E, F, C, S, T) and LPS associated gene (hldE) in both strains underline their virulence potential. The role of the capsule in the pathogenesis of Campylobacter has not been well defined, but it is suspected to interact with the mucus layer during adhesion, and it helps with intracellular survival [HldE is involved in protein glycosylation and correct LPS configuration [C. coli strain 200606 harbored additional genes  involved in capsule biosynthesis [C. jejuni, suggesting an exchange of some genes between C. jejuni and C. coli species. However, the introgression of C. coli by C. jejuni is not new [This study revealed that adhesion , but it was distantly related to another strain of human origin also reported in Korea (CP017229). This suggests some host preference and adaptation in Campylobacter. A study in Japan highlighted a distant relationship between C. jejuni from wild crows and poultry, showing the possibility of divergence due to host adaptation [C. jejuni strain 200605 clustered with CP014344 collected from humans in South Africa, which could not be justified by the current study. We speculate that travel may be a predisposing factor in the occurrence of such a phenomenon. However, the phylogenetic tree (C. jejuni strains used to build the tree. C. coli strain 200606 clustered with C. coli (CP007181) isolated from turkey, and this cluster was distantly related to other C. coli strains used to build the tree. Both chickens and turkeys are domestic poultry, and it seems common to find both strains clustering together. Also, introgression of CP007181 by C. jejuni would explain the clustering together with C. coli strain 200606 of this study in which some C. jejuni genes were found. Furthermore, the analysis showed that other factors like the species, subspecies, percent G+C, and δ statistics were different from the values of other C. coli strains used to build the tree  of the same ST have been previously collected from chickens in Korea [C. coli strain 200606 belongs to ST-5935, which is part of the CC-1150. This ST is not common, but it has been reported in C. coli of chicken origin in Vietnam [C. coli from chickens in China [Campylobacter species to the environment.in China . FurtherCampylobacter strains from various hosts and countries to indicate the taxonomic features of isolates used in this study.A limitation in our study was a low number of sequenced strains due to limited resources. However, the phylogenetic trees included C. jejuni strain 200605 and C. coli strain 200606 from layer chickens in Korea. Both strains showed C257T point mutation in gyrA and cmeABC operon often associated with quinolone resistance. The two strains also carry tet(O) genes associated with tetracycline resistance. The presence of various virulence factors involved in motility, adhesion, invasion, toxin production, and chemotaxis shows the pathogenic potential of the studied strains. Phylogenomics revealed that the two strains resemble other strains of poultry and human origins. C. jejuni strain 200605 and C. coli strain 200606 belong to less common STs and this warrants further investigation. To the best of our knowledge, this is the first report of WGS data from Campylobacter species from layer chickens in Korea. Special attention should be paid to FQ-resistant strains due to a limited number of available alternative treatments.The current study describes the WGS of


Oryza meridionalis  and an Oryza rufipogon like taxa that have been shown to have a chloroplast genome sequence that is closer to that of O. meridionalis than to O. rufipogon from Asia. Rare plants of intermediate morphology have been observed in the wild despite a reported reproductive barrier between these two species. We now report the resequencing of plants from 26 populations including both taxa and putative hybrids. A comparison of chloroplast and nuclear genome sequences indicated re-combinations that demonstrated hybridisation in both directions. Individuals with intermediate morphology had high nuclear genome heterozygosity consistent with a hybrid origin. An examination of specific genes  revealed the presence of heterozygotes with alleles from both parents suggesting that some wild plants were early generation hybrids. These plants may have low cross-fertility preserving the continuation of the two distinct species. Repeated backcrossing of these rare hybrids to one parent would explain the plants exhibiting chloroplast capture. These observations suggest that reticulate evolution is continuing in wild Oryza populations and may have been a key process in rice evolution and domestication.The wild rice gene pool, i.e., AA-genome, in Australia is geographically and genetically distinct from that in Asia. Two distinct taxa are found growing together in northern Australia,  Pisum (Opuntia (Elymus)  ; baobabs(Elymus) ; and Adeenanthos . Much efenanthos . Morpholenanthos . Geneticenanthos , polymorenanthos , and incenanthos . Next-geenanthos .Oryza) belongs to the tribe Oryzeae—a member of the grass subfamily Ehrhartoideae. The genus Oryza comprises 11 genome groups of which the AA-genome group (with eight diploid species) is the most diverged group, distributed throughout the world, except for, Antarctica (Oryza species), BBCC (three Oryza species), CC (three Oryza species), CCDD (three Oryza species), EE (one Oryza species), FF (one Oryza species), GG (three Oryza species), HHJJ (two Oryza species), HHKK (one Oryza species), and KKLL (one Oryza species)  . Vicariaspecies) . The estears ago . Four Orustralia . Of thesr length . More real forms . O. rufiustralia . Australrom Asia . This inrom Asia .Oryza species, especially in crosses between O. sativa and its wild relatives , resulting in hybrid sterility  has been observed in these wild rice populations, which were assumed to be due to hybridisation between the ype taxa . These wWe have now reexamined the origins of these hybrids by re-sequencing and analysing the nuclear genomes of these wild rice populations including wild plants of hybrid appearance.Australian wild rice is widely distributed from the south of Townsville to the northern parts of Cape York Peninsula, Queensland, Australia. Fresh leaves of wild rice from 26 different geographical sites were collected in the two consecutive years 2015 and 2016 by 1 to identify any sequencing error before performing further analysis. Sequences were trimmed at both 0.05 and 0.01 quality scores to truncate low-quality reads. Based on the percentage of loss of reads and bases, 0.01 trimmed reads were considered for further analysis. The total Illumina raw reads for the 26 samples ranged from 56,349,078 to 104,325,654 bp. The total raw nucleotides ranged between 8,416,673,405 and 15,628,960,467 bp. After trimming, the total 0.01 trimmed reads ranged from 54,631,067 to 98,924,768 and nucleotides from 7,925,007,230 to 14,260,593,039 bp . Samples were kept in liquid nitrogen to prevent thawing throughout the pulverisation process. DNA was extracted from pulverised leaf tissue of wild rice samples following the CTAB method . The qua3,039 bp .O. rufipogon type taxa (= Taxon A) and O. meridionalis (= Taxon B) was 384.8 and 354.9 Mb, respectively . Sequence data for the Australian O. rufipogon like taxon is reported in Prior to final mapping, the best mapping parameters were evaluated with the three different alignment quality thresholds: 1.0 length fraction (LF) and 0.95 similarity fraction (SF), 1.0 LF and 0.90 SF and 1.0 LF and 0.85 SF. Illumina paired-end reads were then mapped to the reference genome Os-Nipponbare-Reference-IRGSP-1, which was downloaded at Rice Annotation Project Database  with theectively . Other dO. meridionalis, and the reference chloroplast genome of O. rufipogon type taxa obtained from O. meridionalis and O. rufipogon type taxa  = 1.0604; R(b) = 2.5331, R(c) = 0.5698; R(d) = 0.9914; R(e) = 2.5331; R(f) = 1; gamma = 0.587. Phylogenetic reconstruction was performed using PAUP* version 4 software  were investigated to test for nuclear introgression from the putative parental populations. Among these, WR44, WR52, and WR153 were reported by ype taxa , and Ory ex Watt  were ali ex Watt  with defdelTest2  on the Esoftware  with maxseed shattering 4 (sh4) on chromosome 4, four starch synthesis related genes on chromosome 6  and granule bound starch synthase II (GBSSII) on chromosome 7. Plausible hybridisation generations were determined based on the level of heterozygosity across the whole genome of these hybrids.Single nucleotide polymorphism (SNP) genotypes of six genes located on chromosomes 4, 6, and 7 from hybrids and putative parental taxa were aligned, with respect to Os-Nipponbare-Reference-IRGSP-1 with CLC-GWB to screen for contributing donor parents in genotypes of hybrid populations. These genes include GBSSI, SSI, SBEI, and ALK) from two putative parental populations (WR24 and WR81), one hybrid and outgroup species were first aligned with MAFFT version 7.407 (O. sativa spp. japonica cv. Nipponbare was considered an outgroup species (A). We simulated a total of 5 four-taxon tests to determine biparental nuclear geneflow between hybrid and two putative parental taxa. We estimated Patterson’s D-statistic based on the random distribution of ABBA-BABA polymorphisms in a four-taxon test .Four taxon (ABBA-BABA) test as a measure of geneflow was performed with Hybrid Check version 1 software . Four geon 7.407  with defxon test . The D-sr2 = 0.2 (which recursively eliminates SNPs within the sliding window if r2 is greater than 0.2). To investigate the relationship of population structure with geographic distance, a subset of genome-wide SNP data  from 26 wild rice genotypes  were used to perform isolation by distance (IBD) analysis using Mantel test  to assess the genetic variation in wild rice populations using plink version 1.9 . To ensutel test  with thetel test . Nei  contained an O. rufipogon type chloroplast genome and an O. meridionalis type nuclear genome.The analysis of phylogenetic relationships based on the whole chloroplast and nuclear genome had suggested that chloroplast capture was present in Australian wild rice populations . The wholis type  while thype taxa . While Wt genome . This reO. sativa spp. japonica cv. Nipponbare reference genome. Of the six genes, one gene (sh4) on chromosome 4 is associated with seed shattering while the other five genes are starch synthesis-related genes located on chromosome 6  and chromosome 7 (GBSSII). From SNP data, it was clearly shown that the six genes of WR62 received alleles from both O. rufipogon type taxa and O. meridionalis parents. SBEI and GBSSII had heterozygous SNP genotypes with both O. rufipogon type taxa and O. meridionalis alleles in all of five hybrids  also had heterozygous loci for ALK and SSI, respectively. Sequence alignment images of SNP genotypes of sh4 gene , ALK gene , and GBSSII   was found in WR44, followed by 5,917,274 in WR62, 5,291,708 in WR52, 5,295,508 in WR161 and 4,764,266 in WR153 (O. rufipogon type taxa was applied to the above-mentioned formula (2) for WR62, which exhibited O. rufipogon type nuclear genome while the genome size of O. meridionalis was used for the other hybrids. A high level of genetic variation was observed in both WR44 (0.69%) and WR62 (0.6%) across the whole genome followed by WR161 (0.34%), WR52 (0.32%), and WR153 (0.31%) in relation to O. sativa spp. japonica cv. Nipponbare .SNP were analysed for six genes on chromosomes 4, 6, and 7 were analysed to assess the genetic contribution of the putative parents to the natural hybrid populations in relation to  hybrids . However,202 bp)  showed h,202 bp)  while WR,202 bp) . In all in WR153 . Similarpponbare . The levD-statistic) was performed to test for biallelic mutation patterns (ABBA-BABA) to determine geneflow between the hybrids and two putative perennial parental populations in a topology {, O. sativa spp. japonica cv. Nipponbare}. The length of a MAFFT aligned sequence was 4,473 bp with a total of four blocks: ; ; ; and . The D-statistic value based on the distribution of AABB and BABA on each block showed significant deviation for all four-taxon trees  and hybrid while the positive value suggests geneflow between O. meridionalis (WR81) and hybrid. In all cases, O. sativa spp. japonica cv. Nipponbare was an outgroup. The Z-score for all four-taxon tests was significant (p < 0.001).A four-taxon test . The number of allelic positions where the variants were located was determined. The total number of variants ranged from 1,918,262 in WR24 to 7,310,928 in WR81 , and the least heterozygosity was found in O. rufipogon type taxa (0.16%). The SNPs that were uniquely present in individuals were determined to define the genetic relationships between individuals. The rare or unique heterozygous SNP position (%) ranged from 4 to 48%. The highest number of unique heterozygous SNP positions was in O. rufipogon type taxa and the lowest number in O. meridionalis, indicating O. rufipogon type taxa might be subject to more genetic drift compared to O. meridionalis populations due to their smaller population size.To estimate intra- and interspecific variations in the wild rice gene pool, variants comprised of five different types: SNV, insertions, deletions, MNV, and replacement were assessed across the whole genome of twenty-one putative parental taxa  formed another group  of 26 wild rice genotypes were first processed to remove linkage for PCA analysis. A total of 490,655 SNP sites were retained that fell below the linkage threshold. PCA was performed on linkage-pruned sites to determine the genetic groupings of wild rice populations. PCA analysis showed two clear clusters: (1) one group formed with hybrids  and one putative parental taxon (WR24) and (2) all er group . The firR2 = −0.008, p-value = 0.42) between Nei’s pairwise genetic distance and geographic distances (R2) was 0.008 with a p-value of 0.42. This indicated genetic differentiation was not significantly increased with geographic distance, indicating genetic connectivity in wild rice populations across a wide range.Isolation by distance analysis based on a Monte-Carlo test with 999 permutations clearly showed that there was no significant relationship  differed from each other by 34 variants  and O. sativa spp. Nipponbare cv. japonica (125 variants). In the Australian O. rufipogon, ancient chloroplast capture has occurred from O. meridionalis  due to recent bidirectional chloroplast capture from these parents. The recent introgressive hybridisation demonstrated here may be an explanation of both the apparent chloroplast capture in the Australia O. rufipogon populations and the wild hybrids.This study confirms two types of the chloroplast genome in hybrid populations introgressed from one of their parental taxa. A total of five hybrids were studied of which only one hybrid (WR62) exhibited an  origins . Chlorop origins . Unidire origins  or nucle origins  from one origins . Unlike  origins . In this origins . Brozynstic tree . Despitevariants . The O. dionalis , howeverO. meridionalis. These observations clearly demonstrate an ongoing hybridisation and introgression persisting in the wild rice populations evidenced by the presence of viable hybrids between O. rufipogon type taxa and O. meridionalis being found at very low frequency. Earlier findings postulated that interspecific crossability between an O. meridionalis and a perennial Asian O rufipogon was modest but asymmetric  and O. rufipogon produce low fertility pollen (<10%) as found with putative annual strains (<20–40%)  and panicle fertility (66.9%), however, geographical distance seems to be the reason for the development of reproductive barriers  . Intra-sbarriers . They obocations .D-statistic values from the four-taxon test indicate a nuclear geneflow from both putative parents into hybrids. Early generation hybrids exhibited higher heterozygosity and polymorphic variations than that of the later generations of hybrids. Repeated backcrossing between F1/F2 offspring and O. meridionalis may lead to comparatively low heterozygous SNP variation in the later-generation of hybrids. PCA analysis also reveal that early generation hybrids were clustered with O. rufipogon type taxa and later generation hybrids (except for WR161) form a group with O. meridionalis. Reproductive barriers underpin a termination of hybridisation and thereby, introduce introgression (SEED DEVELOPMENT 1) on chromosome 6 is detected in O. meridionalis and O. sativa (SEED DEVELOPMENT 1 gene (sdv1-m) in wild rice strain from the Jpn2 site  appears to evoke segregation distortion (1:2:0) in a BC4F2 progeny. Therefore, offspring carrying homozygous recessive alleles (sdv1-m) cause seed abortion at the seed development stage. O. sativa and O. meridionalis, then backcrossed with O. sativa to obtain backcrossing progeny. Despite homozygous recessive alleles, offspring bear seed if they are self-fertilised. O. rufipogon from Asia or Australia does not have this abortive gene. Backcrossing with O. meridionalis along with selfing may reduce seed abortion in later-generation hybrids.The positive and negative significant gression . An inte. sativa . A recesO. rufipogon type taxa are distinguished with open panicles and short awns from O meridionalis, which exhibits long awns with closed panicles. However, awn lengths sometimes overlapped  were searched to discover extremely rare apparent hybrids based upon their intermediate morphology. This study has confirmed the hybrid identity of these extremely rare plants. The Asian O. rufipogon samples that have been sequenced all show the presence of an O. rufipogon chloroplast that is distinct from that of O. meridionalis and the Australian O. rufipogon like taxa  remain as distinct populations despite the extremely rare hybridisation events between these two taxa confirmed in this study. The reproductive barrier between them resulting in low pollen fertility requires the backcrossing of the hybrids in subsequent generations.ike taxa . The twoO. sativa ssp. japonica cv. Nipponbare. This finding is, in agreement with the result reported by O. meridionalis and 2,564,013 SNPs in O. rufipogon, compared to Asian O. rufipogon  and cultivated O. sativa ssp. indica , with respect to O. sativa ssp. japonica cv. Nipponbare. A molecular study using restriction fragment length polymorphism (RFLP) markers revealed that cultivated rice retained 40% of the wild alleles during rice domestication  had a more heterozygous and repetitive genome than O. rufipogon (Taxon A), resulting in a higher number of scaffolds in the egnoem of this taxon. Mating systems play an important role in acquiring genetic variations. Perennials exhibit outcrossing mating system, which results in a higher degree of genetic variation than annuals while selfing is a feature of annuals  in dispersing Oryza species over large distances is quite evident  can be found below: RH conceived the project. AF and RH collected field samples. SH, AF, and RH analysed the data. SH wrote the first draft of the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Apis cerana abansis, which lives in the southeastern margin of the Titan Plateau, by using PacBio, Illumina and high-throughput chromatin conformation capture (Hi-C) sequencing technologies. With a more comprehensive annotation pipeline, we obtained an ampler and more accurate Apis cerana genome than previous studies. Comparative genomic analysis was performed to identify the divergence among different A. cerana genomes by studying two aspects: the differential content of repeat content and the gene loss/gain events occurred in chemosensory receptors and immune-related proteins. Our results show that the content of repetitive sequences differ in types and quantity among four A. cerana strains; the gene loss/gain events in chemoreceptor- and immune-related proteins occur in different A. cerana strains, especially in A. cerana abansis (Aba strain). Specifically, while compared with the other three published genomes, the Aba strain contains the largest number of repeat contents and loses the largest number of both chemosensory-receptor- and immune-related proteins, as well as subfamilies, whereas the Baisha strain contains the largest number of chemoreceptor- and immune-related proteins. We hypothesized that gene loss/gain may be evolutionary strategies used by the different A. cerana strains to adapt to their respective environments.In this study, we obtained a chromosome-scale assembly genome of Apis cerana abansis, widely distributed in the southeastern margin of the Qinghai-Tibet Plateau, is considered an excellent model to study the phenotype and genetic variation for highland adaptation of Asian honeybee. Herein, we assembled and annotated the chromosome-scale assembly genome of A. cerana abansis with the help of PacBio, Illumina and Hi-C sequencing technologies in order to identify the genome differences between the A. cerana abansis and the published genomes of different A. cerana strains. The sequencing methods, assembly and annotation strategies of A. cerana abansis were more comprehensive than previously published A. cerana genomes. Then, the intraspecific genetic diversity of A. cerana was revealed at the genomic level. We re-identified the repeat content in the genome of A. cerana abansis, as well as the other three A. cerana strains. The chemosensory and immune-related proteins in different A. cerana strains were carefully re-identified, so that 132 odorant receptor subfamilies, 12 gustatory receptor subfamilies and 22 immune-related pathways were found. We also discovered that, compared with other published genomes, the A. ceranaabansis lost the largest number of chemoreceptors compared to other strains, and hypothesized that gene loss/gain might help different A. cerana strains to adapt to their respective environments. Our work contains more complete and precise assembly and annotation results for the A. cerana genome, thus providing a resource for subsequent in-depth related studies. Apis cerana is one of the ten Apis species wildly distributed throughout the Asian region [A. cerana to the varied habitats [Apis cerana abansis [Apis cerana abansis  presents distinguishable morphological characteristics compared to other ecological types of Asian honeybees. The morphological indexes of the Aba strain, such as proboscis length, the area of the forewing and the third and the fourth notum are larger than other A. cerana strains [Apis cerana abansis is considered to be an excellent model to study the phenotype and genetic variation for the highland adaptation of Asian honeybee.Asian honeybee n region . The geohabitats ,3,4. The abansis ,6,7, is  strains ,3,7,8. HA complete and accurate genome assembly is a crucial basis for studying the connection between gene function and the characteristics of organisms and is necessary for functional genomics and population genomics studies . Second-A. cerana strains, GenBank assembly accession as GCA_001442555.1, GCA_002290385.1 and GCA_011100585.1 , which were respectively collected from Korea; Wufu Mountain of Jiangxi, China; and Baisha of Hainan Island, China, have been sequenced [Apis mellifera genomes, A. cerana has fewer odorant receptors, as well as a more effective social defense system, such as physiological resistance to the parasitic mite Varroa [A. cerana, Baisha strain genome is 215.67 Mb in size (examined using PacBio and Hi-C sequencing technology) with a contig N50 of 4.49 Mb, which provides high-quality data for genomic comparison with other Apis species [A. mellifera, a high-quality genome was assembled by first generated contigs based on PacBio sequencing, which were then merged with linked-read 10× Chromium data, followed by scaffolding using a BioNano optical genomic map and a Hi-C chromatin interaction map, complemented by a genetic linkage map [So far, three equenced ,17,18. Te Varroa ,17. A chkage map .A. cerana, repeat content has not been systematically identified. The current large variation in repeat content contained in different genomes (from 4.2% to 9.65%) [A. cerana.Repeat content can occupy a large proportion of the eukaryotic genome , and itso 9.65%) ,17,18 isA. mellifera, a total of 170 olfactory receptors and 10 taste receptors were identified [The odorant receptor is an important basis and prerequisite for the olfactory recognition system of insects, and it is an essential element for the mediation of the conversion of chemical signals such as external odorants into electrical signals , while gentified .A. cerana contains 12 mainly immune-related pathways [The honeybee has been widely used as a model animal for studying individual and population immunity . In earlpathways ,17. The pathways .A. cerana have been sequenced, thus giving us a better understanding of the genomic organization of this species. However, genomic diversity and variation within different ecological types of this species remain poorly understood. According to previous studies, Korea, Wufu and Baisha strains collected from different geographic locations can be approximately considered representative for three ecological types of Changbaishan, Central China and Hainan Island. Therefore, complete genomic sequences of those three strains give a good opportunity to delineate the intra-species ecological genomics of the Asian honeybee, A. cerana.Several genomes of A. cerana Aba strain from the Western Plateau of Sichuan and obtain a high-quality highland A. cerana reference genome. We demonstrated the divergence of different A. cerana genomes by studying two aspects: the differential content of repeat content, the gene loss/gain events that occurred in chemosensory receptors and immune-related proteins. Gene loss/gain can contribute to evolutionary novelties and can be positively selected [A. cerana, gene loss/gain may contribute to environmental adaptation and could result from a positive selection.In this study, we used third-generation sequencing technology, second-generation sequencing and Hi-C sequencing technology to sequence the collected selected ,27. ThusA. cerana workers from the plateau region in Western Sichuan were collected from Mosudu Village, Songgang Town, Markang City, Aba Prefecture, Sichuan Province .The The genomic DNAs were extracted from the bee’s thorax through conventional the CTAB (cetyltrimethylammonium bromide) ,28 methoThree different genomic DNA libraries were constructed and sequenced according to the manufacturer’s instructions to generate a high-quality chromosome-scale assembly genome: (i) 20-kb library using a PacBio Sequel platform, (ii) Hi-C reads sequencing by phase genomics, and (iii) paired-end library using Illumina NovaSeq PE150 platform (150 bp sequencing length with insert size of 350 bp). Two different cDNA libraries were constructed and sequenced according to the manufacturer’s instructions to generate a complete and accurate cDNA data that greatly contributed to genomic annotation: (i) 4-kb library using a PacBio Sequel platform, (ii) paired-end library using Illumina NovaSeq PE150 platform (150 bp sequencing length with insert size of 400 bp). All sequencing was conducted by the Novogene Bioinformatics Institute, Beijing, China.The PacBio reads were assembled into high-quality consensus sequences by Wtdbg2 v2.5  with theThe quality of the assembled genome was assessed by BUSCO v3.0.2  and CEGMA combined strategy based on homology alignment and de novo search to identify the whole genome repeats was applied in our repeat annotation pipeline. Ab initio prediction was used to build de novo repetitive elements database by RepeatModeler v1.73  with defApis mellifera (ncbi.GCF_003254395.2), Apis cerana (ncbi.GCF_001442555.1), Nasonia vitripennis (ensembl.metazoa.v32), Polistes dominula (ncbi.GCF_001465965.1), Cephus cinctus (ncbi.GCF_000341935.1), Athalia rosae (ncbi.GCF_000344095.1), Apis dorsata (ncbi.GCF_000469605.1) and Apis florea (ncbi.GCF_000184785.3) were aligned to the assembly genome using TblastN v2.9.0+ [Homologous proteins for homolog prediction were downloaded from Ensembl and NCBI. The protein sequences of For ab initio gene prediction, Augustus v3.2.3 , Geneid The RNAseq reads from Illumina sequencing were assembled by Trinity v2.8.5  for geneThe non-redundant reference gene set was generated by merging genes predicted by three methods with EvidenceModeler v1.1.1 , using PGene functions were assigned according to the best match by aligning the protein sequences to the Swiss-Prot using Blastp . The motifs and domains were annotated using InterProScan v5.31  by searcDrosophila melanogaster were collected from FlyBase [Bombyx mori, and mice were downloaded from NCBI [A. florea and A. mellifera were obtained, referring to another past study [A. cerana strains. Ultimately, a total of 6535 predicted odorant receptor proteins from our collected protein sets were used as a database for the prediction of odorant receptor proteins of different A. cerana strains in this study. Almost 101 gustatory receptor proteins were obtained and set as a database to identify the gustatory receptor in different A. cerana strains. These gustatory receptors were from the FlyBase [Drosophila melanogaster, Bombyx mori, A. mellifera and different A. cerana strains that were predicted and reported in previous studies [A. mellifera, Drosophila melanogaster and Bombyx mori were collected through the KEGG [A. cerana strains. The different A. cerana strains were predicted by the protein2genome  module of Exonerate v 2.4.0. We discarded the proteins with translation errors due to the presence of stop codons. The longest protein located in the same gene loci was taken as the prediction result; the putative gene-containing regions recognized from the BLAST hits but not through good Exonerate hits were separately re-examined.All odorant receptors of  FlyBase . The odorom NCBI . The odost study ,56, as w FlyBase , while N studies . A totalthe KEGG  databaseThe predicted odorant receptor needed to contain the 7tm_6 domain to be considered an odorant receptor protein . The gusA. cerana strains as the foreground branch. Likelihood ratio test (LRT) indicated that the model allowing sites to experience specific adaptations provided a significantly better fit to the data than the null model, in which sites could under positive selection. The positively selected genes must contain the positive selection sites.We use Orthofinder v2.3.8  to get 1Genome sequencing of the Aba strain using PacBio provided a total data size of 128.98G (515.92X), reads number of 8,451,522, an average length of 15,262 bp, N50 with 21,185 bp. Genome sequencing using Illumina sequencing provided a total size of 36.54G (151.76X). Transcriptome data through PacBio sequencing were: total data size of 31.51G, reads number of 28,246,937, the average length of 1116 bp, N50 with 1350 bp; Illumina sequencing: total data size of 25G . In ordeA. cerana abansis  . The conDe novo prediction, homology-based and transcriptome-based strategies were combined to identify and annotate protein-coding genes . FinallyA. mellifera. However, the number of scaffolds not mounted was higher in Aba (867) than in Baisha (110). Baisha has the smallest genome size (215.67 Mb) in A. cerana, and Wufu (228.79 Mb) has the largest genome size in A. cerana. Aba predicted the greatest number of genes at 11,240.The Aba genome has a contig N50 of 7.91 Mb and a scaffold N50 of 13.27 Mb, which are larger than contig N50 (3.89 Mb) and the scaffold N50 (13.17 Mb) of Baisha . MeanwhiA. cerana strains, 832 new proteins were identified in Aba compared with the other three A. cerana strains, including 785 proteins of unknown function. Baisha showed 775 newly identified proteins compared with the other three A. cerana strains, of which 748 were of unknown function. Korea showed 215 new proteins not predicted in other A. cerana strains, of which 192 proteins were of unknown function. Wufu had 130 new proteins not predicted in the protein set of other A. cerana strains, of which 119 proteins were of unknown function , which confirms that at least one structural variant is presented between these two species, A. cerana and A. mellifera.We identified one common chromosomal structural variant among Aba and Baisha, as well as analysis . A previellifera . HoweverA. cerana had less repeat content than A. mellifera [A. mellifera . As this was unexpected, we re-identified the repeat content in Baisha, Korea and Wufu.Through our repeat annotation pipeline, 31,320,299 bp of Aba genomic sequences were identified as repetitive sequences, accounting for 13.79% of the total genomic length. In a previous study, a total length of 19.73 Mb (9.15%) , 14.79 MA. cerana strains contain more repetitive sequences than A. mellifera and have fewer repetitive sequences compared to B. terrestris  and B. impatiens  [We found that Aba contained the highest repeat content, followed by Baisha , Wufu  and Korea  . All A. 44.6 Mb) .A. cerana, and the repeat classes of DNA, long interspersed nuclear element (LINE), low complexity and short interspersed nuclear element (SINE), were similar in amount in each genome (The repeat classes of Simple repeat and Unknown occupied a larger proportion of repetitive sequences in h genome . Among tWe found that some repeat classes were more abundant in the genomes based on second-generation sequencing technologies (Korea or Wufu) than in the genomes based on third-generation sequencing technologies (Aba or Baisha), such as L1, L2 in LINE and acro in Satellite.Interestingly, one PIF-Harbinger of DNA transposon and two centers of Satellite were only presented in Korea. With regards to LTR class, we found that ERVL-MaLR only existed in Baisha and Korea, and there were 1 and 4 copies, respectively. In the SINE class, we found that one tRNA-RTE element was only present in Baisha and Wufu. All the detailed information can be seen in A. cerana strains was consistent, and in the areas with higher repetition content, GC content was lower.We showed the distribution of the repeat content on the genomes of different strains . We founA. cerana , while Wufu missed the immune-related proteins annotated as K06061 (mastermind) and K05700 (vinculin) B. K03009A. cerana abansis genome. Based on a more comprehensive annotation pipeline, more proteins were identified in A. cerana compared to previous studies [A. cerana genome than previous studies, and our results are useful for the in-depth comparative genomic analysis of honeybees.In this study, PacBio and Illumina sequencing technology were used to assemble and annotate the  studies ,17,18; wA. cerana and found no direct correlation between genome size and repeat content in A. cerana. Baisha showed the smallest genome size among the different A. cerana strains and more repeat content, while Wufu showed the largest genome size but lower repeat content. Additionally, our results showed that the number of repeat contents in A. cerana (from 9.02% to 13.79%) was higher compared to A. mellifera (7.5%) [A. mellifera [We identified more repeat contents in a (7.5%) . It is wellifera .In this study, strains of Baisha and Wufu come from tropical and subtropical regions ,18, respA mutually beneficial synergy has developed between bees, which take in nutrients by feeding on plant pollen and nectar, and plants, which pollinate through bees . As planA. cerana strains derived from a cold and harsh environment unlike Baisha and probably were subjected to less immune pressure than Baisha, which caused their immune-associated proteins to be lost over time. Among the immune-related protein loss events, we found that Wufu lost two immune-related KEGG orthologs and Aba lost one immune-related KEGG ortholog. We still need more evidence to prove whether the lost events can influence resistance against diseases. In addition, we also found ten new immune-related pathways of A. cerana that were not reported previously, which will help us deepen our study of the immune system of A. cerana.Temperature and moisture can affect microbial species diversity ,74. The A. cerana strains may have resulted in an “adaptive gene loss/gain” event. Several studies have mentioned that gene loss/gain may help organisms to adapt to their specific environment [Drosophila, the loss of chemoreceptors such as odorant receptors and gustatory receptors is increased when the diet changes, while some of the chemoreceptors that contribute to the current diet undergo gene duplication and are subject to positive selection [C. elegans [D. melanogaster [In term of chemosensory receptors and immune-related proteins identified in this study, we found that Baisha had the highest number of proteins (373), followed by Wufu (350), Korea (349) and Aba (332). So, we propose that the gene loss/gain that occurred among different ironment ,77,78,79election . Large-s elegans ,82 and Dnogaster  revealednogaster . The mosnogaster ,86.A. cerana has directly affected the corresponding function, such as olfaction, still needs to be proven.In summary, whether the gene loss/gain in A. cerana strains by the site model and the branching site model by PAML. Our results showed that no genes were subjected to under selection, thus suggesting that the phenotypic difference between Aba and other strains does not occur due to selection pressure on the coding region. Accordingly, future studies need to consider the differential expression of genes, the degree of methylation differences, etc. to resolve the reasons for this phenotypic difference.We calculated the selection pressure between different A. cerana genome for honeybees. We found differences in genomic structural features among different A. cerana strains, such as the number of chemoreceptors, immune-related proteins and repeat content, which is probably related to adaptation to the local environment. This comparative genomic analysis identified new features of the A. cerana genome, revealing that the intraspecific genetic diversity of different A. cerana strains and gene loss events may promote the adaptation of different A. cerana strains to their respective environments. Strains surviving in different environments may provide an excellent biological model for studying the effects of different environments.Our study provided an ampler and more precise


Alexandrium minutum and larger A. catenella, globally distributed toxic red tide dinoflagellates, were studied under a matrix of photoperiods  and temperatures . Under continuous growth light condition (L:D 24:0), the growth rate (µ) of small A. minutum increased from low to medium temperature, then decreased to high temperature, while the µ of large A. catenella continuously decreased with increasing temperatures. Shortened photoperiods reduced the µ of A. minutum, but enhanced that of A. catenella. As temperature increased, cellular Chl a content increased in both A. minutum and A. catenella, while the temperature-induced effect on RubisCO content was limited. Shortened photoperiods enhanced the Chl a but reduced RubisCO contents across temperatures. Moreover, shortened photoperiods enhanced photosynthetic capacities of both A. minutum and A. catenella, i.e., promoting the PSII photochemical quantum yield , saturation irradiance (EK), and maximum relative electron transfer rate (rETRmax). Shortened photoperiods also enhanced dark respiration of A. minutum across temperatures, but reduced that of A. catenella, as well as the antioxidant activities of both species. Overall, A. minutum and A. catenella showed differential growth responses to photoperiods across temperatures, probably with cell size.Shift of phytoplankton niches from low to high latitudes has altered their experienced light exposure durations and temperatures. To explore this interactive effect, the growth, physiology, and cell compositions of smaller  After centrifugation  again, the supernatant was used to measure RubisCO with an enzyme-linked immunosorbent assay kit (CUSABIO CSB-E16686Pl Quant Kit) [To measure the RubisCO content, 15 mL culture was collected, centrifuged (7000× ant Kit) .Data were presented as mean and standard deviations (mean ± SD). Paired t-tests, one-way ANOVA with Bonferroni post-tests , and comparisons of linear curve fit were used to detect significant differences among cultures of each diel L:D cycle and temperature combination for each species. The confidence level for statistical tests was set at 0.05.Alexandrium species to a matrix of photoperiods and temperatures. Small A. minutum grew faster under continuous growth light, while large A. catenella grew faster under short photoperiods. Shortened photoperiods enhanced cellular Chl a content and photosynthetic capacity, but reduced RubisCO content and antioxidant activity. The optimal growth temperature of A. minutum was higher than that of A. catenella. Our results complement others [Alexandrium species compositions in particular in the East China Sea, because different taxa show differential, even opposite, growth responses to photoperiod and temperature changes, as well as different responses of different strains within species.Large differences were observed in growth and physiological responses of differently cell-sized t others ,17 to sh


Serratia marcescens, particularly in microbial competition through its antimicrobial activity, towards the growth and secreted virulence factors of four clinical pathogenic bacteria (methicillin-resistant Staphylococcus aureus (MRSA), Enterococcus faecalis, Salmonella enterica serovar Typhimurium and Pseudomonas aeruginosa) as well as Staphylococcus aureus and Escherichia coli. Prodigiosin was first extracted from S. marcescens and its purity confirmed by absorption spectrum, high performance liquid chromatography (HPLC) and liquid chromatography-tandem mass spectrophotometry (LC-MS/MS). The extracted prodigiosin was antagonistic towards all the tested bacteria. A disc-diffusion assay showed that prodigiosin is more selective towards Gram-positive bacteria and inhibited the growth of MRSA, S. aureus and E. faecalis and Gram-negative E. coli. A minimum inhibitory concentration of 10 μg/μL of prodigiosin was required to inhibit the growth of S. aureus, E. coli and E. faecalis whereas > 10 μg/μL was required to inhibit MRSA growth. We further assessed the effect of prodigiosin towards bacterial virulence factors such as haemolysin and production of protease as well as on biofilm formation. Prodigiosin did not inhibit haemolysis activity of clinically associated bacteria but was able to reduce protease activity for MRSA, E. coli and E. faecalis as well as decrease E. faecalis, Salmonella Typhimurium and E. coli biofilm formation. Results of this study show that in addition to its role in inhibiting bacterial growth, prodigiosin also inhibits the bacterial virulence factor protease production and biofilm formation, two strategies employed by bacteria in response to microbial competition. As clinical pathogens were more resistant to prodigiosin, we propose that prodigiosin is physiologically important for S. marcescens to compete against other bacteria in its natural soil and surface water environments.Prodigiosin, a red linear tripyrrole pigment, has long been recognised for its antimicrobial property. However, the physiological contribution of prodigiosin to the survival of its producing hosts still remains undefined. Hence, the aim of this study was to investigate the biological role of prodigiosin from  Serratia marcescens, as a secondary metabolite during the bacterial idiophase )+ with a retention time of 13.5 minutes. Taken together, these results confirm that prodigiosin is present as a predominant compound in the crude sample. Hence, the prodigiosin within the crude extract is of a relatively high purity and no further purification steps were carried out for the sample.To further identify and confirm the presence of prodigiosin in the extracted sample, HPLC and LC-MS/MS were carried out. Prodigiosin was detected by the HPLC where only a single peak was observed at 535 nm with a retention time of ~4.75 minutes . In the 65 g/mol , correspS. aureus and E. faecalis) and three Gram-negative  bacteria were selected to test the effectiveness of prodigiosin in inhibiting bacterial growth. Chloramphenicol  successfully inhibited the growth of all the tested bacteria while no inhibition zones were observed with the negative controls. Of the six selected bacteria treated with prodigiosin, three Gram-positive  and one Gram-negative (E. coli) bacteria were inhibited by prodigiosin at 250 μg/μL and 500 μg/μL. The growth of both P. aeruginosa and Salmonella Typhimurium was not negatively affected by prodigiosin at these concentrations  tested and suggests that prodigiosin has a role in thwarting S. marcescens’ competitors that are generally found in the same environment.In the disc-diffusion assay, three Gram-positive , incomplete (α-haemolysis) and no destruction (γ-haemolysis) of erythrocytes . BacteriE. faecalis cultures did not cause haemolysis, indicating that E. faecalis V583 belongs to the γ-haemolysis group even though other strains of E. faecalis are known to exhibit β-haemolytic activity [E. coli presented α-haemolysis [Salmonella Typhimurium demonstrated α-haemolysis which is similar to the Salmonella Pathogenicity Island (SPI)-1 type III secretion-dependent haemolytic activity exhibited by Salmonella Typhimurium SL1344 [S. aureus [P. aeruginosa [S. aureus exhibiting double haemolysis due to the presence of both α- and β-haemolysins [S. marcescens in the environment.We noted that clear zones were observed for both untreated bacterial cultures and solvent control over the period of the assay  indicatiactivity . E. coliemolysis  in both m SL1344 . Previou. aureus  and P. aruginosa  exhibit molysins  Fig 5)..E. faecaS. aureus, E. faecalis, E. coli and P. aeruginosa treated with prodigiosin were smaller compared to the untreated bacterial cultures, indicating that prodigiosin interfered with proteolysis or protease secretion by these bacteria. The anti-proteolytic effect of prodigiosin was most significant for both MRSA and E. coli (p<0.05) followed by E. faecalis, P. aeruginosa and S. aureus. No inhibition zones were observed for treated and untreated Salmonella Typhimurium suggesting that this strain is incapable of secreting proteases . We noted a significant reduction in biofilm formation for prodigiosin-treated E. faecalis and Salmonella Typhimurium (p<0.05) when compared to the respective untreated bacteria. Surprisingly, S. aureus (p<0.05) and P. aeruginosa responded to the presence of prodigiosin by increasing biofilm formation. The amount of biofilm formed by MRSA did not change in the presence of prodigiosin  and two standard laboratory strains (S. aureus and E. coli). We were also interested to investigate if prodigiosin is advantageous to the survival of S. marcescens by inhibiting the production of known Gram-positive and Gram-negative bacterial virulence factors involved in microbial competition such as proteases [S. marcescens Sma 274 was cultured in PGB (pH 7) at 28°C for 96 hours [Ecological competition among microorganisms results from nutrient and antibiotic stress as well as space limitations. This competition is a powerful selection pressure that is often mediated through the synthesis and secretion of bioactive metabolites by the competing bacteria . These sroteases  and the roteases . To obta96 hours . Sma 274E. coli is resistant to prodigiosin as is E. coli KCTC 1116 [E. coli laboratory strain, E. coli OP50, is susceptible to prodigiosin, which is consistent with E. coli MG1655 susceptibility towards prodigiosin [P. aeruginosa PA14 and Salmonella Typhimurium SL1344 are resistant towards prodigiosin at both tested concentrations. While P. aeruginosa [P. aeruginosa UFPEDA 39 [P. aeruginosa MCTC 1688 [P. aeruginosa KCCM 11266 [Salmonella Typhimurium resistance to prodigiosin appears to be more consistent. Salmonella Typhimurium SL1344 (this study), Salmonella Typhimurium KCTC 1926 [Salmonella Typhimurium KCCM 40253 [Prodigiosin has a tripyrrole ring structure  and cyclCTC 1116 . Howeverdigiosin . We alsoruginosa  and P. aFPEDA 39  are alsoCTC 1688  and P. aCM 11266  are suscCTC 1926  and SalmCM 40253  are all P. aeruginosa resistance towards prodigiosin could be attributed to the known production of rhamnolipids, a biosurfactant [P. aeruginosa rhamnolipids could have emulsified prodigiosin and blocked its antimicrobial activity. Alternatively, multidrug resistance of P. aeruginosa clinical isolates is attributed to the presence of several efflux pumps such as MexAB-OprM, MexCD-OprJ, MexEF-OprN and MexXY-OprM [mexCD-oprJ operon is induced by disinfectants and cytotoxic agents [P. aeruginosa. Additionally, mexEF-oprN and mexXY-oprM are induced by nitroaromatic antimicrobial agents [P. aeruginosa MexAB-OprM pump confers antibiotic resistance to most β-lactams except imipenem as well as other antimicrobial agents, including chloramphenicol, tetracycline, quinolones and macrolides [P. aeruginosa efflux pumps and virulence factors may have obstructed the antimicrobial effects of prodigiosin. Similarly, the resistance of Salmonella Typhimurium towards prodigiosin could also be due to the bacterial intrinsic multidrug resistance, including secretion of enzymes to inactivate antimicrobial drugs, decreasing cell permeability to antibiotics, modifying cellular drug targets and activating efflux pumps to remove drugs [rfactant . Microbirfactant . As prodxXY-OprM . Among tc agents . Since pc agents , it is pl agents  and aminl agents  respecticrolides . As MexAcrolides , the comve drugs . NeverthS. marcescens co-exists with other Gram-positive and Gram-negative bacteria in its natural habitat, in the presence of these bacteria under stressful conditions, prodigiosin may have a greater effect on the survival of competing Gram-positive bacteria due to the lipophilic characteristic of prodigiosin from the presence of the chain of the monopyrrole C-ring in prodigiosin  against eighteen oxacillin-resistant S. aureus (ORSA) clinical strains [A limitation of our study is the use of crude prodigiosin extract which resulted in an MIC generally higher than that previously reported for purified prodigiosin. For example, the MIC of prodigiosin purified from .3 μg/mL , purifienst MRSA  and puri strains . AlthougS. aureus also promotes skin colonisation by damaging keratinocytes [Bacterial virulence factors reported to be important in microbial dominance include production of proteases  and forminocytes . In the inocytes .P. aeruginosa LasR mutant secretes many virulence factors including proteases that are toxic to other microbes in addition to eukaryotic cells [Pseudomonas fluorescens CHA0 secretes extracellular proteases to suppress the nematode Meloidogyne incognita [S. marcescens secretes prodigiosin to inhibit proteases secreted by other environmental bacteria in order to survive in competitive multispecies niches such as soil and surface water. We treated the different bacteria cultures with prodigiosin and monitored proteolysis of casein present in skim milk. Prodigiosin significantly inhibited proteolytic activity of proteases secreted by MRSA, E. faecalis, E. coli and P. aeruginosa. As we had noted above, prodigiosin inhibits the growth of MRSA, S. aureus, E. facealis and E. coli used in the current study, hence, the reduction in proteolytic activity of prodigiosin-treated bacteria may be due to lower bacterial cell density and therefore, reduced protease secretion as compared to the untreated cultures. On the other hand, the prodigiosin anti-proteolytic effect could directly target proteases. This is likely true for P. aeruginosa whereby prodigiosin failed to inhibit growth Click here for additional data file.


Escherichia coli, three strains of Pseudomonas aeruginosa and two strains of Klebsiella pneumoniae, with or without specific resistance to gentamicin or fosfomycin. We reported that ALBC containing gentamicin and clindamycin (COPAL G+C) seems to be the most interesting ALBC of our tested panel for the prevention of biofilm formation by gentamicin-susceptible strains, even if clindamycin is not effective against Gram-negative bacteria. However, gentamicin-resistant strains are still a problem, and further studies are needed to identify an antibiotic to associate with gentamicin for an efficient dual ALBC against Gram-negative bacteria.Gram-negative bacilli can be responsible for prosthetic joint infection (PJI) even if staphylococci are the main involved pathogens. Gram-negative PJIs (GN-PJI) are considered difficult-to-treat infections due to the increase in antimicrobial resistance and biofilm formation. To minimize the risk of infection in cases of arthroplasties with cemented prosthesis, bone cement can be loaded with antibiotics, especially gentamicin. In this study, we aimed to compare the prophylactic antibiofilm activity of ready-to-use antibiotic-loaded bone cements (ALBC), already commercialized or new prototypes. We compared ALBCs containing gentamicin alone, gentamicin plus vancomycin, gentamicin plus clindamycin, gentamicin plus Fosfomycin, and fosfomycin alone, to plain cement (no antibiotic); these comparisons were conducted to investigate the biofilm formation of three strains of  Staphylococcus aureus and Staphylococcus epidermidis [Pseudomonas aeruginosa, Escherichia coli and Klebsiella pneumoniae [Prosthetic joint infection (PJI) is one of the most serious complications of total hip or knee joint arthroplasties and requires a multidisciplinary approach for successful management ,2. The mdermidis ,4. Howevdermidis ,6 and cadermidis . Even ifdermidis . Monomiceumoniae ,6. Theseeumoniae ,9. Biofieumoniae .To minimize the risk of infection in cases of arthroplasties with cemented prosthesis, the bone cement can be loaded with antibiotics . The objS. aureus and coagulase-negative staphylococci (CoNS) are used as model microorganisms in in vitro studies exploring the potential of ALBCs for the prevention of PJIs [S. aureus and CoNS [The interest in using ALBCs for prosthesis fixation during revision arthroplasties has been highlighted in several studies, even if its use is still debated for primary arthroplasties ,15,16,17 of PJIs ,19,20. Wand CoNS . We repoand CoNS .P. aeruginosa and E. coli [Few studies have been performed regarding the prophylactic effect of ALBCs against Gram-negative bacteria. In 2013, Chang et al. tested various ALBCs and reported that gentamicin had good antibacterial activity against various bacteria, including  E. coli . However E. coli ,24. CombE. coli and P. aeruginosa [In this study, we aimed to test the prophylactic antibiofilm effect of commercialized ready-to-use ALBCs  against Gram-negative bacteria. We also proposed testing the antibiofilm effect of new ALBC prototypes loaded with gentamicin and/or fosfomycin, directly provided as ready-to-use ALBCs by the manufacturers. This antibiotic has a broad antibiotic spectrum and shows an interesting synergism with gentamicin against the biofilm of ruginosa .Pseudomonas aeruginosa, 3 strains of Escherichia coli and 2 strains of Klebsiella pneumoniae. These clinical strains were isolated during routine work performed at the Bacteriology Department of Hôpital de la Croix-Rousse, Hospices Civils de Lyon, where identification was performed with MALDI-TOF . Resistance profiles were determined with Vitek 2 . All the strains were tested with the Crystal Violet method beforehand, to ensure that they can form at least a moderate biofilm regarding Stepanovic’s classification [Eight clinical strains of Gram-negative bacteria with specific antibiotic resistances (gentamicin or fosfomycin) were used in this study: 3 strains of fication . AntibioSix different bone cements were used in this study. Four of them were already commercialized by Heraeus Medical: plain cement , cement loaded with gentamicin alone , cement loaded with gentamicin plus vancomycin  and cement loaded with gentamicin plus clindamycin . Two other cements loaded with gentamicin plus fosfomycin  or with fosfomycin  were specially prepared for this study. Disk-like specimens  were used. Specific antibiotic loads for each cement are presented in We used the same protocol as in Cara et al. . BrieflyOvernight bacterial cultures in liquid Brain–Heart Infusion  were standardized to OD600 = 1 ± 0.05 before being diluted at 1:100 in ALBCs elution solutions , and 100 µL was added in a 96-well plate . Plates were incubated for 24 h at 37 °C in a humid atmosphere. Then, the supernatant was removed, and biofilms were washed for 45 min using BiofilmCare, with a smooth washing method that favors the preservation of the biofilm . BiofilmThree independent experiments in technical triplicate (3 wells for each condition for each experiment) were performed. Results were presented as the number of viable bacteria inside the biofilm after 24 h of incubation. Data  were presented as histograms (mean with SD). We compared the data from each day using Kruskal–Wallis tests and Dunn’s multiple comparisons. We performed a first test to compare G, G+V, G+C, G+F and F to the control condition (plain cement). Then, we performed a second test to compare G, G+V, G+C, G+F and F with each other. All analyses were performed using Prism 8 software .E. coli, K. pneumoniae and P. aeruginosa  was observed for G+C when compared to plain cement (A similar pattern was observed for cement F B. On Daycement F B. Howeven cement B. Concercement F C. On Dayn cement C.Globally, G+V and G+F had similar effects when compared to G. Adding vancomycin did not seem to modify the activity of gentamicin in G+V and adding fosfomycin to gentamicin did not provide additional antibiofilm effect to G+F. The remaining effect of G+C observed on Day 9, compared to other ALBCs, could be explained by the highest concentration of gentamicin in this ALBC.E. coli, K. pneumoniae and P. aeruginosa (E. coli, only G+C, G+F and F showed a significant decrease in the number of viable bacteria inside the biofilm on Day 1 when compared to plain cement, G and G+V (p values (Next, we tested the antibiofilm prophylactic effect of ALBCs against gentamicin-resistant strains of ruginosa . For the and G+V A. In thip values A. In thiK. pneumoniae on Day 1, but in this case, G+C achieved the best antibiofilm prophylactic effect (A similar pattern was observed for the gentamicin-resistant c effect C. Howevec effect A,B.P. aeruginosa, resistance to gentamicin seemed to have less of an impact on the biofilm formation. Indeed, all ALBCs containing gentamicin had similarly decreased biofilm formation, with no differences between them (For een them C.E. coli and the K. pneumoniae on Day 1. The highest concentration of gentamicin in G+C improved the antibiofilm effect of the ALBCs, only on Day 1, for the gentamicin-resistant E. coli and K. pneumoniae.Globally, adding fosfomycin to gentamicin partially rescued the capacity of ALBCs to prevent biofilm formation by gentamicin-resistant strains, but only for the P. aeruginosa and E. coli as we met some difficulties in finding a fosfomycin-resistant K. pneumoniae in our collection of clinical strains. As expected, fosfomycin alone was not efficient against these fosfomycin-resistant strains (In this part, we only tested fosfomycin-resistant  strains .E. coli, we observed that all ALBCs containing gentamicin had a strong effect on the decrease in biofilm formation on Day 1 (Concerning the fosfomycin-resistant on Day 1 A. On Dayon Day 1 A. G+C keP. aerugionosa, a similar pattern was observed (P. aeruginosa.Regarding fosfomycin-resistant observed B. All ALobserved B. For G+Gentamicin-containing ALBCs globally kept a good antibiofilm effect against fosfomycin-resistant strains. However, the use of G+C provided higher decreases regarding biofilm formation for the two tested strains.E. coli, K. pneumoniae and P. aeruginosa, which are three species often identified in GN-PJIs [In this study, we investigated the in vitro prophylactic antibiofilm activity of commercialized ready-to-use ALBCs against several strains of  GN-PJIs ,6. We us GN-PJIs . ALBCs wGlobally, we observed that all gentamicin-containing ALBCs have the same behavior: a good, but not strong, activity against gentamicin-susceptible strains , but an E. coli strain and the gentamicin-resistant E. coli strain (E. coli [Regarding fosfomycin, it did not seem interesting in our conditions. Indeed, results for the ALBC containing only fosfomycin were significantly different from those for plain cement, only on Day 1, for the gentamicin-susceptible i strain A. Regard(E. coli . HoweverOther interesting approaches have been reported as alternatives to antibiotics for inclusion in PMMA cement. Robu et al. showed that peppermint oil and silver nanoparticles could be incorporated into bone cement, with good biocompatibility and promising antimicrobial effects . AnotherTo conclude, COPAL G+C seems to be the most interesting ALBC of our tested panel for the prevention of biofilm formation by gentamicin-susceptible Gram-negative bacteria. Further studies will be performed to support and complete our results, especially microscopic fluorescent imaging to provide more insights regarding biofilm viability in the presence of G+C cement. However, gentamicin-resistant strains are still a problem, and further studies are needed to identify an antibiotic/antimicrobial to associate with gentamicin for an efficient dual ALBC against Gram-negative bacteria.


Neovison vison) and ferret (Mustela putorius furo) respiratory tracts showed substantial differences, demonstrating that ACE2 is present in the lower respiratory tract of mink but not ferrets. The presence of ACE2 in the respiratory tract in some species was much more restricted as indicated by limited immunolabelling in the nasal turbinate, trachea and lungs of cats (Felis catus) and only the nasal turbinate in the golden Syrian hamster (Mesocricetus auratus). In the lungs of other species, ACE2 could be detected on the bronchiolar epithelium of the sheep (Ovis aries), cattle (Bos taurus), European badger (Meles meles), cheetah (Acinonyx jubatus), tiger and lion (Panthera spp.). In addition, ACE2 was present in the nasal mucosa epithelium of the serotine bat (Eptesicus serotinus) but not in pig (Sus scrofa domestica), cattle or sheep. In the intestine, ACE2 immunolabelling was seen on the microvillus of enterocytes (surface of intestine) across various taxa. These results provide anatomical evidence of ACE2 expression in a number of species which will enable further understanding of host susceptibility and tissue tropism of ACE2 receptor‐mediated viral infection.Angiotensin converting enzyme 2 (ACE2) is a host cell membrane protein (receptor) that mediates the binding of coronavirus, most notably SARS coronaviruses in the respiratory and gastrointestinal tracts. Although SARS‐CoV‐2 infection is mainly confined to humans, there have been numerous incidents of spillback (reverse zoonoses) to domestic and captive animals. An absence of information on the spatial distribution of ACE2 in animal tissues limits our understanding of host species susceptibility. Here, we describe the distribution of ACE2 using immunohistochemistry (IHC) on histological sections derived from carnivores, ungulates, primates and chiroptera. Comparison of mink ( Agarose‐embedded cell pellets were then processed into formalin‐fixed paraffin embedded sections by routine histology methods  was derived from the manufacturer's datasheet. Amino acid sequence alignment was conducted using Protein BLAST available on blast.ncbi.nlm.nih.gov and visualised using MEGA7 , little brown bat (Myotis lucifugus) and least horseshoe bat (Rhinolophus pusillus) (Supporting information Table Ovis aries), horse , cheetah (Acinonyx jubatus), Sumatran , Asiatic and African lion , European lynx (Lynx lynx), Western gorilla (Gorilla sp.), American mink, ferret, European badger (Meles meles), golden headed lion tamarin (Leontopithecus chrysomelas) and common marmoset  , dog and sheep, 83% for cattle, 78% for pig and common marmoset, and 67% for hamster , but with no evidence of virus‐mediated pneumonia   Figure .4This study demonstrates the expression of ACE2 in the lungs of cattle, sheep, mink, European badgers, domestic cats, tigers and lions. The availability of ACE2 in the lungs of mink, cat, tigers and lions can be linked to the susceptibility of these species to develop pneumonia following SARS‐CoV‐2 infection , Siberian tigers and African lions infected with SARS‐CoV‐2, supported by radiographic evidence of peri‐bronchial consolidation and cytological detection of SARS‐CoV‐2‐infected tracheal epithelial cells  and the wild cousin of the domestic ferret, the European polecat (Mustela putorius), live predominantly solitary lives, coming together only for breeding. Such circumstances would limit opportunities for viral transmission in the wild and are in stark contrast to the high‐density housing of mink on fur farms across Europe, North America and Asia. However, other species of mustelids including weasels, ferret badgers  and hog badgers (Arctonyx collaris) are consumed in parts of East Asia and may be found together in close proximity to other species in live animal markets , yellow‐bellied weasel (Mustela kathiah) and Siberian weasel (Mustela sibirica)  was identified as the source of human infection during the 2003–2004 outbreak (Andersen et al., in vivo susceptibility of pigs, cattle and sheep (Ulrich et al., The emergence of SARS‐CoV‐2 is likely a consequence of virus evolution and positive selection of an ancestral bat coronavirus in an intermediate animal host which subsequently infected a human host (Andersen et al., A major concern is the potential for transmission of SARS‐CoV‐2 to free‐living wildlife and the subsequent establishment of a reservoir of infection providing the conditions for viral mutation, recombination and reintroduction to the human population (Hoffmann et al., No ethical approval was required as tissue blocks were derived from histology archives.F.Z.X.L., S.A., C.C., N.T. performed the experiments. F.Z.X.L., A.N., S.S., S.L.P., S.V., A.S.B., A.M.P.B. conducted formal analysis. F.Z.X.L., D.B., E.W., J.J., A.M.P.B., S.A. provided the methodology. A.N., R.J.D., W.H.M.P., I.H.B., A.R.F., S.M.B. provided with resources. F.Z.X.L. wrote the original draft. All authors reviewed and edited the manuscript.The authors declare no conflict of interest.Supporting informationClick here for additional data file.Supporting informationClick here for additional data file.Supporting informationClick here for additional data file.Supporting informationClick here for additional data file.


Escherichia coli deserves attention for its broad repercussions on public health. Moreover, wild host species are of interest, particularly wild boar. Indeed, the constantly increasing population densities and the limited data on AMR in this species lead to health risks where spatial overlap with humans and domestic animals occurs. Therefore, 1504 wild boar fecal samples were analyzed to investigate ESBL/AmpC-producing E. coli and the effects of host-related factors and of human population density on their spread. A high prevalence of ESBL/AmpC-producing E. coli emerged in wild boar, species not treated with antibiotics, supporting that infection may be acquired through environmental contamination, whether of human or animal origin. Young animals were more colonized than older ones, demonstrating higher susceptibility as seen in domestic animals. Moreover, a positive association recorded between frequency of the TEM resistance gene and human population density suggests that spatial overlap may influence the infection in wild boar. Further analyses would be desirable to investigate the origin of the recorded environmental contamination, although a role of wild boar as a maintenance host of AMR strains emerged.Antimicrobial resistance (AMR) represents a complex global issue due to the many factors involved. Extended-spectrum β-lactamase and AmpC (ESBL/AmpC)-producing Escherichia coli are among the most important strains. Moreover, wildlife hosts are of interest as they are likely antibiotics free and are assumed as environmental indicators of AMR contamination. Particularly, wild boar (Sus scrofa) deserves attention because of its increased population densities, with consequent health risks at the wildlife–domestic–human interface, and the limited data available on AMR. Here, 1504 wild boar fecal samples were microbiologically and molecularly analyzed to investigate ESBL/AmpC-producing E. coli and, through generalized linear models, the effects of host-related factors and of human population density on their spread. A prevalence of 15.96% of ESBL/AmpC-producing E. coli, supported by blaCTX-M (12.3%), blaTEM (6.98%), blaCMY (0.86%) and blaSHV (0.47%) gene detection, emerged. Young animals were more colonized by ESBL/AmpC strains than older subjects, as observed in domestic animals. Increased human population density leads to increased blaTEM prevalence in wild boar, suggesting that spatial overlap may favor this transmission. Our results show a high level of AMR contamination in the study area that should be further investigated. However, a role of wild boar as a maintenance host of AMR strains emerged.The complex health problem of antimicrobial resistance (AMR) involves many host species, numerous bacteria and several routes of transmission. Extended-spectrum β-lactamase and AmpC (ESBL/AmpC)-producing  Antimicrobial resistance (AMR) represents a global health problem that involves humans, animal species and ecosystems ,2,3. AniEscherichia coli is one of the most important bacteria that contributes to the complexity of AMR, and among its strains, those producing β-lactamases, extended-spectrum β-lactamases (ESBLs) and other β-lactamases such as AmpCs encoded by plasmid-located genes are of particular interest. Indeed, resistance to most beta-lactam antibiotics, including third and fourth-generation cephalosporins, can limit treatment options in case of infection [E. coli resistant to third-generation cephalosporins in the European Union and European Economic Area during 2015, and comparing these data with others from 2007, an approximately 4-fold increase was detected [E. coli ESBL and AmpC has been reported in several animal species [nfection  and leadnfection . Authors species ,9, but,  species .Wild species are generally not treated with antimicrobials and may acquire AMR bacteria or antimicrobial residues just through food and water in environments contaminated by domestic animals or humans . Indeed,Sus scrofa) has been of high interest because of its role of an ‘invasive species’, the very close phylogenetical similarity to pigs (Sus scrofa domesticus) and the great increase of its population densities [E. coli. Indeed, the use of antibiotics in both veterinary and human medicine and the known relationship between usage of antimicrobials and the occurrence of resistance in bacterial isolates in manure or manure contaminated surface water leads to AMR risks for wild boars in anthropized environments and highlights the role of drug resistance “conductor” of the environment [E. coli is constantly present both in the digestive tract and in the environment, e.g., in water and soil [E. coli in wild boar are still limited, especially with respect to factors that may influence its spread or the potential health risks related to the contact of these populations with humans or domestic animals.Among free-range species, wild boar  the prevalence within the population and (ii) the effects of host-related factors and of human population density on the spread of these pathogenic bacteria.Therefore, we carried out an epidemiological investigation of ESBL/AmpC-producing 2 . The territory includes four wild boar hunting areas . From th2 . For eac2 , wild boar fecal samples were collected through the regional health monitoring program on free-ranging animals in the Lombardy region (D.d.g. 5 December 2012—no. 11358). By hunting season, 34.91% of wild boars were hunted in 2017–2018, 25.33% in 2018–2019 and 39.76% in 2019–2020. For each wild boar, gender, age and the hunting area (HA) were registered. Of the hunted wild boars, 53.32% were females and 46.68% males. The age of wild boar was evaluated based on tooth eruption patterns [E. coli was performed through a double synergy diagnostic method. Specifically, 1 g of feces was diluted in 9 mL (1:10 dilution) of brain–heart infusion (BHI) broth supplemented with 1 mg/L cefotaxime for a pre-enrichment phase. After an overnight incubation, a drop of the BHI broth was used to inoculate MacConkey agar supplemented with 1 mg/L cefotaxime [E. coli was conducted by PCR phylogenetic group analysis according to Clermont et al. [E. coli phylogroups A, B1, B2, C, D, E, F and clade I and can exclude other strains with typical Escherichia phenotype, i.e., E. albertii and E. fergusonii.The identification of β-lactamase-producing fotaxime . Positivt et al. . This isblaCTX-M [blaSHV [blaTEM [blaCMY [E. coli are described in blaCTX-M, blaSHV, blaTEM and blaCMY, is shown. All the amplicons found to be positive for blaTEM and blaSHV were sequenced [The detection of resistance genes was performed with a panel of reactions. Specifically, multiple PCR was used for the identification of blaCTX-M ; single  [blaSHV , blaTEM  [blaTEM  and blaC [blaCMY . All theequenced .E. coli. To investigate the effects of the same explanatory variables on AMR genes spread, we modeled each gene of microbiological positive subjects using a Binomial GLM. The models were compared using the likelihood ratio test (LRT). A post-hoc Tukey test was used to perform pairwise comparisons when statistically significant factors were detected in the models. Statistical analyses were carried out using R software version 4.0.3 [p-value (p) was less than 0.05.The analysis can be split into three components: descriptive analysis of hunted wild boar, estimation of the apparent prevalence (p) with the corresponding confidence intervals estimated using an exact method and analysis of factors associated with prevalence. We used logistic regression models (GLM)  to definon 4.0.3 , specifiE. coli distributed among all the eight phylo-groups.The phylogenetic analysis showed the presence of 240 n = 63, 26.25%), A , F , C , B2 , E , D , clade I . The overall microbiological prevalence of ESBL/AmpC-producing E. coli in wild boars was 15.96%  (E. coli ESBL/AmpC than adults (p = 0.0207) and sub-adults (p = 0.0098). In wild boars hunted during 2017–2018, the probability of being positive was significantly higher compared to in 2018–2019 (p = 0.0232) and 2019–2020 (p = 0.0015). Wild boars hunted in HA 3 were more colonized by E. coli ESBL/AmpC than those of HA 4 (p = 0.0033) (p > 0.05).Specifically, the distribution of the studied strains in the different phylogenetic groups was as follows: B1 (–17.91%) . Statist 0.0033) . No otheblaCTX-M in E. coli isolates was 12.3% . Statistical models showed the effects of hunting season and age class on the presence of this gene (blaCTX-M was higher than in E. coli of sub-adults (p = 0.0197) and adults (p = 0.0245) (p = 0.0005). No other statistically significant differences emerged (p > 0.05).According to AMR resistance genes, the overall prevalence of his gene . Indeed, 0.0245) . Wild boblaCMY resistance gene, the overall prevalence in E. coli isolates was of 0.86% . Statistical analysis did not reveal any significant effect . Sequence analysis of the amplicons revealed the presence of SHV-12 (7/7). Sequences were deposited in NCBI GenBank with accession numbers from MZ026052 to MZ026058. No significant effects emerged from the statistical models . Analysis of the amplicon sequences  revealed the presence of TEM-1 and its variants (91/98), TEM-33 (1/98), TEM-135 (2/98) and TEM-176 (3/98). Additionally, a novel blaTEM allele, named TEM-244 was detected in one isolate (1/98). Sequences were deposited in NCBI GenBank with accession numbers from MZ026059 to MZ026156. Hunting season and human population density influence the probability of detecting the gene: animals hunted during 2017–2018 had a significantly higher prevalence of blaTEM than those of 2018–2019 (p = 0.0186) and 2019–2020 (p = 0.0368) . No other statistically significant differences emerged (p > 0.05).Concerning  0.0368) . A statiE. coli in free-range wild boar. Young animals were more colonized by ESBL/AmpC strains, and particularly by the blaCTX-M resistance gene, than older subjects. Wild boar hunted during hunting season 2017–2018 showed a higher prevalence of ESBL/AmpC strains, blaCTX-M and blaTEM than animals of 2018–2019 and 2019–2020. The increasing of human population density leads to an increase of prevalence of blaTEM in wild boar.The present study revealed a high prevalence of ESBL/AmpC-producing E. coli phylogenetic groups were B1  and A , in accordance with Holtmann et al. [E. coli and AMR genes recorded are consistent with previous reports for this species [E. coli  appears higher than those previously recorded in wild boars by Wasyl et al. [blaCTX-M  was confirmed as the most frequent among AMR genes as reported by Literak et al. [blaTEM and blaSHV are consistent with the results of Holtmann et al. [E. coli or of AMR genes occurs through direct contact with the environment, soils or surface water (feeding and drinking) contaminated with livestock/pig manure [In this study the most frequent ESBL/AmpC-producing n et al.   and blaCTX-M (16.52%) in young animals compared to sub-adults  and adults . To the best of our knowledge, in the only available reference in which an age comparison of prevalence of these strains was performed for wild boar, no significant differences were registered [E coli strains, which can cause severe enteritis especially when they are only a few weeks old [E. coli that occurred in young wild boars, an in-depth analyses of virulence factors, i.e., adhesins , of isolated E. coli would be useful to determine whether these strains were part of the normal microflora or could potentially cause diseases in domestic pigs.Statistical models showed a higher prevalence of eeks old , suggesteeks old . In ordeblaCTX-M  and blaTEM  recorded between hunting seasons could be associated with both the role of the environmental indicator of wild boar and that of the “conductor” in the transmission of pathogenic strains/resistance genes of the environment [blaTEM in E. coli of wild boar being positively related to human population density suggests that this spatial overlap, particularly due to feeding with anthropogenic food sources and waste, can expose wild boar to traces of antimicrobial substances or directly to resistant bacteria of human origin [blaTEM was the most frequent isolated among nosocomial strains [blaCTX-M genes became the dominant enzymes in human populations [blaTEM still occurs in the most clinically relevant enterobacterial species [blaCTX-M in the human population of our study area, the hypothesis that blaCTX-M may not yet be as widespread in the human population and therefore does not have a direct effect on wild boar could be suggested.The difference in prevalence of ESBL/AmpC strains , n origin , blaTEM  strains , being a strains . From thulations . Indeed,ulations  reported species  with the species , and givE. coli recorded in wild boar in this study leads to strategies for reducing and preventing the spread of resistant bacteria in the environment and between species. As the main concern of AMR is related to the over-use of antimicrobials [The high prevalence of ESBL/AmpC-producing crobials , the redE. coli recorded in this study shows the potential role of wild boar as a maintenance host of AMR strains. However, the creation of historical data series would be desirable in order to monitor and better evaluate these temporal trends in wild boar. In addition, the role of this species as indicator of environmental contamination, supported by the recorded positive association between blaTEM and human population density, leads to an in-depth analysis that also includes domestic animals. Indeed, through a “One Health” approach, further specific sampling should be carried out at both farm and human/environmental levels within hunting areas to assess prevalence of these strains/AMR genes and to evaluate whether their presence actually influences their spread in wild boar.The high prevalence of ESBL/AmpC-producing


The editorial introduces a special section of the Journal of Biomedical Optics on Advances in Terahertz Biomedical Science and Applications. This special section includes one review and ten research papers addressing the complex challenges of terahertz biophotonics and related areas of biomedical optics. Moreover, a few of the papers in this special section are dedicated to similar biomedical applications of novel optical tools from the neighboring infrared (IR) range.With rapid progress in terahertz (THz) technology, quite portable and ergonomic THz spectroscopy and imaging systems emerge, thus, pushing research and engineering efforts into applying THz technology in various demanded branches of applied physics and optics, including biomedical optics.https://doi.org/10.1117/1.JBO.26.4.043005), modern research results in the area of THz-wave–skin interactions are summarized, considering both diagnostic and therapeutic applications. Indeed, due to quite a limited depth of THz-wave penetration in tissues along with the simplicity of THz measurements of the skin, it remains the most studied localization in the THz range, with a numerous emerging clinical applications.In the review paper by A.I. Nikitkina et al., entitled “Terahertz radiation and the skin: a review” (URL: https://doi.org/10.1117/1.JBO.25.12.123002), T. Zhang et al. introduced a new type of a water-free tissue-mimicking phantom for THz biophotonics. This phantom is based on graphite powders embedded into a polyvinyl chloride plastisol matrix. The effective THz optical properties of such a phantom  can be managed in a wide range by changing its composition, thus allowing to mimic the THz optical properties of various biological tissues. Obviously, development of such novel low-cost and reproducible tissue-mimicking phantoms is an important problem of THz biophotonics. Second, capabilities of mid-IR interband cascade laser in surgical thermal ablation of normal fibroblasts and primary undifferentiated pleomorphic sarcoma tumor cells were demonstrated by E. Larson and co-authors in the paper “Mid-infrared absorption by soft tissue sarcoma and cell ablation utilizing a mid-infrared interband cascade laser” (URL: https://doi.org/10.1117/1.JBO.26.4.043012).Two papers of the special section consider modern problems of oncodiagnosis. In the research article “Development of oral cancer tissue-mimicking phantom based on polyvinyl chloride plastisol and graphite for terahertz frequencies” (URL: https://doi.org/10.1117/1.JBO.26.4.043006), an ability to differentiate between lyophilized (dehydrated) pellets of diabetic and non-diabetic human blood plasma using the THz pulsed spectroscopy was demonstrated. In another paper by A.A. Lykina et al., entitled “Terahertz high resolution spectroscopy of thermal decomposition gas products of blood plasma and kidney tissue pellets” (URL: https://doi.org/10.1117/1.JBO.26.4.043008), content of the thermal-decomposition gas products of diabetic and non-diabetic dried blood plasma and kidney tissues was studied, involving methods of high-resolution gas spectroscopy at THz frequencies. The authors show that such gas spectroscopy in the THz range also yields useful information for the differentiation between diabetic and non-diabetic samples, where acetone serves as a main marker of diabetic blood and tissues.Two research works consider applications of THz technology in studying glycated tissues and blood, aimed at diabetes mellitus diagnosis. In one paper by A.A. Lykina et al., entitled “Terahertz spectroscopy of diabetic and non-diabetic human blood plasma pellets” . Using quite different THz dielectric responses of intact tissues (either in vivo or freshly excised ex vivo) and frozen ones,Completely novel applications of THz pulsed spectroscopy , the high sensitivity of the THz-wavelength to the content of water in tissues was exploited for studying pathological changes in the cornea caused by ultraviolet light exposure. The observed results of the rabbit cornea measurements in the THz range were verified by optical coherence tomography. They highlighted an ability for in vivo contactless estimation of the cornea hydration  based on its THz reflectivity. In turn, in a paper by L. Ke et al., entitled “Terahertz spectroscopy analysis of human corneal sublayers” (URL: https://doi.org/10.1117/1.JBO.26.4.043011), a method for sensing human corneal composition at different depths  was proposed, relying on high-sensitivity THz spectroscopy. Using novel approaches to collecting and processing data, the authors studied THz responses of human corneas at different edema stages. The observed results demonstrated that the epithelium layer of cornea acts as a barrier that maintains the hydration level of the stroma; while at the final stages of edema, the epithelium lost its barrier properties. The epithelium quality can be used to predict the level of corneal swelling.Two papers of the special section are dedicated to the THz sensing of corneal properties. In a research article by E.N. Iomdina and co-authors, entitled “Terahertz scanning of the rabbit cornea with experimental UVB-induced damage: https://doi.org/10.1117/1.JBO.26.4.043009), addressed a problem of human temperature measurement using IR thermal imaging systems, with an emphasis on diagnosis of possible infectious disease transmission, including COVID-19. A systematic study of the IR spectra of insulin involving model insulin specimens was carried out by S. Delbeck and H.M. Heise, in their article “Systematic stability testing of insulins as representative biopharmaceuticals by using ATR FTIR-spectroscopy with focus on quality assurance” (URL: https://doi.org/10.1117/1.JBO.26.4.043007). Insulin specimens were stored at different temperatures  for up to three months, while their weekly IR spectroscopic measurements yielded monitoring of the hormonal secondary structural changes, which correlated negatively with the insulin bioactivity. The observed results revealed that IR spectroscopy holds potential for rapid and reliable analysis of the secondary structural changes within insulin molecules and, thus, is capable of insulin quality control. Finally, Y. Zhu and co-authors, in their article “Dual short wavelength infrared transillumination/reflectance mode imaging for caries detection” (URL: https://doi.org/10.1117/1.JBO.26.4.043004), developed a system for simultaneous acquisition of both the short-wavelength infrared reflectance and occlusal transillumination images of lesions on tooth proximal and occlusal surfaces, which is aimed at differentiating between shallow and deep occlusal lesions.New applications of spectroscopic and imaging systems from the IR range, which is neighboring to THz, are also considered in this special section. C. Healy and co-authors, in their paper “Globally deployed COVID-19 fever screening devices using infrared thermographs consistently normalize high readings to afebrile range” ,