Virulence-associated Genes Research Articles

Effect of traditional Chinese medicine monomers interfering with quorum-sensing on virulence factors of extensively drug-resistant Acinetobacter baumannii.

The antimicrobial resistance of Acinetobacter baumannii (A. baumannii) clinical isolates has emerged as a great threat to public health. Quorum sensing (QS) is one of the resistance mechanisms for drug-resistant A. baumannii. Interfering with QS is a promising strategy to combat infections caused by drug-resistant bacteria. This study explored the QS inhibition ability of thirty-four traditional Chinese medicine monomers (TCMMs) and assessed the effect of QS inhibitors (QSIs) on the virulence factors of twelve extensively drug-resistant A. baumannii (XDRAB) strains. Nine traditional Chinese medicine monomers, such as caffeic acid, cinnamic acid, and myricetin, were found to be able to inhibit the bacterial QS. Then, at 1/8 of the minimal inhibitory concentration, we found that these QSIs inhibited extensively drug-resistant A. baumannii adhesion and biofilm formation and downregulated the expression levels of virulence-associated genes (abaI, abaR, csuE, pgaA, and bap). In conclusion, nine traditional Chinese medicine monomers have QS inhibitory activity and may downregulate QS genes to interfere with the QS system, which could inhibit the expression of extensively drug-resistant A. baumannii virulence factors. These results suggest that traditional Chinese medicine monomers could develop as novel anti-virulence compounds to control extensively drug-resistant A. baumannii infections.

Investigation of Antibiotic Resistance Pattern and Virulence Determinants in Avian Pathogenic Escherichia coli Isolated from Broiler Chickens in Egypt

Besides its zoonotic importance, avian pathogenic Escherichia coli (APEC) causes substantial financial losses in the poultry industry globally. The progress of antimicrobial resistance in APEC is mainly associated with excessive antimicrobial use and improper sanitation. Since its beginning in the 1970s, the VITEK system has developed into the VITEK 2 system, which has used an automated system to perform all the steps required for microbial identification and antibiotic susceptibility rapidly and accurately. The present study aimed to update the available circulating data about APEC isolates by phenotypic identification, sero-grouping of APEC from broilers chickens and breeders in five governates of Egypt, investigation of their antibiotic resistance pattern by VITEK 2 system, and molecular identification of their virulence determinants. The prevalence of APEC isolated from the different internal organs (liver, lung, heart, heart blood, and spleen) was 67.5%. The most prevalent serotypes were O125, 0114, O44, O127, O142, and O78. Virulence-associated genes (iutA, fimC, and papC) were detected at rates of 84.4%, 74%, and 54.8%, respectively. The highest resistance was found against ampicillin (100%), trimethoprim-sulfamethoxazole (80%), and ampicillin-sulbactam (78.5%), which indicates that the poultry farms need a surveillance and intervention system with proper accuracy and rapidity to prevent the misuse of antibiotics and APEC outbreaks.

Whole genome characterization of thermophilic Campylobacter species isolated from dairy manure in small specialty crop farms of Northeast Ohio.

With more public interest in consuming locally grown produce, small specialty crop farms (SSCF) are a viable and growing segment of the food production chain in the United States. The goal of this study was to investigate the genomic diversity of Campylobacter isolated from dairy manure (n = 69) collected from 10 SSCF in Northeast Ohio between 2018 and 2020. A total of 56 C. jejuni and 13 C. coli isolates were sequenced. Multi-locus sequence typing (MLST) identified 22 sequence types (STs), with ST-922 (18%) and ST-61 (13%) predominant in C. jejuni and ST-829 (62%) and ST-1068 (38%) predominant in C. coli. Interestingly, isolates with similar genomic and gene contents were detected within and between SSCF over time, suggesting that Campylobacter could be transmitted between farms and may persist in a given SSCF over time. Virulence-associated genes (n = 35) involved in the uptake and utilization of potassium and organic compounds (succinate, gluconate, oxoglutarate, and malate) were detected only in the C. jejuni isolates, while 45 genes associated with increased resistance to environmental stresses (capsule production, cell envelope integrity, and iron uptake) were detected only in the C. coli isolates. Campylobacter coli isolates were also sub-divided into two distinct clusters based on the presence of unique prophages (n = 21) or IncQ conjugative plasmid/type-IV secretion system genes (n = 15). Campylobacter coli isolates harbored genes associated with resistance to streptomycin (aadE-Cc; 54%) and quinolone (gyrA-T86I; 77%), while C. jejuni had resistance genes for kanamycin (aph3'-IIIa; 20%). Both species harbored resistance genes associated with β-lactam (especially, blaOXA-193; up to 100%) and tetracycline (tetO; up to 59%). Our study demonstrated that Campylobacter genome plasticity associated with conjugative transfer might provide resistance to certain antimicrobials and viral infections via the acquisition of protein-encoding genes involved in mechanisms such as ribosomal protection and capsule modification.

Candida albicans Oropharyngeal Infection Is an Exception to Iron-Based Nutritional Immunity.

Candida albicans is a commensal of the human gastrointestinal tract and a common cause of human fungal disease, including mucosal infections, such as oropharyngeal candidiasis and disseminated infections of the bloodstream and deep organs. We directly compared the in vivo transcriptional profile of C. albicans during oral infection and disseminated infection of the kidney to identify niche specific features. Overall, 97 genes were differentially expressed between the 2 infection sites. Virulence-associated genes, such as hyphae-specific transcripts, were expressed similarly in the 2 sites. Genes expressed during growth in a poor carbon source (ACS1 and PCK1) were upregulated in oral tissue relative to kidney. Most strikingly, C. albicans in oral tissue shows the transcriptional hallmarks of an iron replete state while in the kidney it is in the expected iron starved state. Interestingly, C. albicans expresses genes associated with a low zinc environment in both niches. Consistent with these expression data, strains lacking transcription factors that regulate iron responsive genes (SEF1, HAP5) have no effect on virulence in a mouse model of oral candidiasis. During microbial infection, the host sequesters iron, zinc, and other metal nutrients to suppress growth of the pathogen in a process called nutritional immunity. Our results indicate that C. albicans is subject to iron and zinc nutritional immunity during disseminated infection but not to iron nutritional immunity during oral infection. IMPORTANCE Nutritional immunity is a response by which infected host tissue sequesters nutrients, such as iron, to prevent the microbe from efficiently replicating. Microbial pathogens subjected to iron nutritional immunity express specific genes to compensate for low iron availability. By comparing the gene expression profiles of the common human fungal pathogen Candida albicans in 2 infection sites, we found that C. albicans infecting the kidney has the transcriptional profile of iron starvation. By contrast, the C. albicans expression profile during oropharyngeal infection indicates the fungus is not iron starved. Two transcription factors that activate the transcriptional response to iron starvation are not required for C. albicans virulence during oral infection but are required for disseminated infection of the kidney. Thus, our results indicate that C. albicans is subject to nutritional iron immunity during disseminated infection but not during oropharyngeal infection, and highlight niche specific differences in the host-Candida albicans interaction.

Novel Lactotransferrin-Derived Antimicrobial Peptide LF-1 Inhibits the Cariogenic Virulence Factors of Streptococcus mutans

We previously developed a novel lactotransferrin-derived antimicrobial peptide, LF-1, with selective antibacterial activity against the characteristic cariogenic bacterium Streptococcus mutans. This study further investigated the effects of LF-1 on the cariogenic virulence factors of S. mutans and evaluated the changes in virulence-associated enzymes and genes; the viability, acidogenicity, and aciduricity of planktonic S. mutans; and initial colonisation and biofilm formation after treatment with LF-1. The method of qRT-PCR was used to evaluate S. mutans virulence-associated gene expression. LF-1 interfered with the cell viability of S. mutans within 6 h. LF-1 inhibited the acidogenicity and aciduricity of S. mutans, with reduced lactic acid production and survival in a lethal acidic environment, and inactivated lactate dehydrogenase and F1F0-ATPase activity. LF-1 decreased surface-adherent S. mutans within 60 min and inhibited S. mutans biofilm formation, where scanning electron microscopy and confocal laser scanning microscopy showed reduced extracellular matrix and bacteria. LF-1 downregulates S. mutans virulence-associated gene expression. LF-1 inhibited the growth and cariogenic virulence factors of S. mutans in vitro with a reduction in key enzymatic activity and downregulation of virulence-associated gene expression. LF-1 has promising application prospects in the fight against S. mutans and dental caries.

Antimicrobial Resistance Pattern, Pathogenicity and Molecular Properties of Hypervirulent Klebsiella pneumonia (hvKp) among Hospital-Acquired Infections in the Intensive Care Unit (ICU)

Hypervirulent Klebsiella pneumoniae (hvKp) is a new emerging variant of K. pneumoniae that is increasingly reported worldwide. The variant hvKp is known to cause severe invasive community-acquired infections such as metastatic meningitis, pyogenic liver abscesses (PLA) and endophthalmitis, but its role in hospital-acquired infections (HAIs) is little known. The aim of this study was to evaluate the prevalence of hvKp among hospital-acquired (HA) K. pneumoniae infections in the intensive care unit (ICU) and to compare between hvKp and classical K. pneumoniae (cKP) regarding antimicrobial resistance pattern, virulence and molecular characteristics. The study was cross-sectional and included 120 ICU patients suffering from HA K. pneumoniae infections between January and September 2022. K. pneumoniae isolates were subjected to antimicrobial susceptibility testing and detection of extended-spectrum-β-lactamase (ESBL) production by the Phoenix 100 automated microbiology system, string test, biofilm formation, serum resistance assay, and detection of virulence-associated genes (rmpA, rmpA2, magA, iucA) and capsular serotype-specific genes (K1, K2, K5, K20, K57) by polymerase chain reaction (PCR). Of 120 K. pneumoniae isolates, 19 (15.8%) were hvKp. The hypermucoviscous phenotype was more significantly detected in the hvKp group than in the cKP group (100% vs. 7.9%, p ≤ 0.001). The rate of resistance to different antimicrobial agents was significantly higher in the cKP group than that in the hvKp group. Fifty-three strains were identified as ESBL-producing strains, which was more frequent in the cKP group than in the hvKp group (48/101 [47.5%] vs. 5/19 [26.3%], respectively, p ≤ 0.001). The hvKP isolates were highly associated with moderate and strong biofilm formation than cKP isolates (p = 0.018 and p = 0.043 respectively). Moreover, the hvKP isolates were highly associated with intermediate sensitivity and re sistance to serum in the serum resistance assay (p = 0.043 and p = 0.016 respectively). K1, K2, rmpA, rmpA2, magA and iucA genes were significantly associated with hvKp (p ≤ 0.001, 0.004, <0.001, <0.001, 0.037 and <0.001, respectively). However, K5, K20 and K57 were not associated with hvKp. The hvKp strains have emerged as a new threat to ICU patients because of their ability to cause more severe and life-threatening infections than cKP. The string test alone as a laboratory test for screening of hvKp has become insufficient. Recently, hvKp was defined as hypermucoviscous- and aerobactin-positive. It is important to improve the awareness towards the diagnosis and management of hvKp infections.

Analysis of the role of the QseBC two-component sensory system in epinephrine-induced motility and intracellular replication of Burkholderiapseudomallei.

Burkholderia pseudomallei is a facultative intracellular bacterial pathogen that causes melioidosis, a severe invasive disease of humans. We previously reported that the stress-related catecholamine hormone epinephrine enhances motility of B. pseudomallei, transcription of flagellar genes and the production of flagellin. It has been reported that the QseBC two-component sensory system regulates motility and virulence-associated genes in other Gram-negative bacteria in response to stress-related catecholamines, albeit disparities between studies exist. We constructed and whole-genome sequenced a mutant of B. pseudomallei with a deletion spanning the predicted qseBC homologues (bpsl0806 and bpsl0807). The ΔqseBC mutant exhibited significantly reduced swimming and swarming motility and reduced transcription of fliC. It also exhibited a defect in biofilm formation and net intracellular survival in J774A.1 murine macrophage-like cells. While epinephrine enhanced bacterial motility and fliC transcription, no further reduction in these phenotypes was observed with the ΔqseBC mutant in the presence of epinephrine. Plasmid-mediated expression of qseBC suppressed bacterial growth, complicating attempts to trans-complement mutant phenotypes. Our data support a role for QseBC in motility, biofilm formation and net intracellular survival of B. pseudomallei, but indicate that it is not essential for epinephrine-induced motility per se.

Genomic characterization of beta-haemolytic streptococci isolated from prosthetic joint infections.

Prosthetic joint infection (PJI) is an increasing concern for the medical profession, as higher numbers of arthroplasty surgeries lead to rising PJI-related costs. Streptococcal PJIs constitute approximately 10% of PJIs, but their genetic features and characteristics remain largely unexplored. Little is known about antimicrobial resistance (AMR) rates, whether some sequence types (ST) dominate, and whether certain virulence-associated genes are overrepresented. We used whole-genome sequencing of Streptococcus dysgalactiae (n=22), Streptococcus agalactiae (n=10) and S.pyogenes (n=1) to elicit genomic data on 33 beta-haemolytic streptococci isolated from PJIs in Region Örebro county, Sweden. Relatedness was inferred based on single nucleotide polymorphisms in S.dysgalactiae and S.agalactiae. The genomic data were screened for virulence-associated genes available in the Virulence Factor Database. All isolates were screened for both phenotypic and genotypic resistance. The S.dysgalactiae and S.agalactiae isolates were genetically diverse, although 32% of S.dysgalactiae isolates (n=7) were ST20. The speS and PI-2A genes were less represented in these isolates among virulence-associated genes, and AMR was more frequently observed in S.agalactiae. We conclude that PJIs caused by beta-haemolytic streptococci are not dominated by genetically similar beta-haemolytic streptococci. There were distinct inter-species differences in AMR between S.agalactiae and S.dysgalactiae.

Genomic insight into Campylobacter jejuni isolated from commercial turkey flocks in Germany using whole-genome sequencing analysis.

Campylobacter (C.) jejuni is a zoonotic bacterium of public health significance. The present investigation was designed to assess the epidemiology and genetic heterogeneity of C. jejuni recovered from commercial turkey farms in Germany using whole-genome sequencing. The Illumina MiSeq® technology was used to sequence 66 C. jejuni isolates obtained between 2010 and 2011 from commercial meat turkey flocks located in ten German federal states. Phenotypic antimicrobial resistance was determined. Phylogeny, resistome, plasmidome and virulome profiles were analyzed using whole-genome sequencing data. Genetic resistance markers were identified with bioinformatics tools (AMRFinder, ResFinder, NCBI and ABRicate) and compared with the phenotypic antimicrobial resistance. The isolates were assigned to 28 different sequence types and 11 clonal complexes. The average pairwise single nucleotide-polymorphisms distance of 14,585 SNPs (range: 0-26,540 SNPs) revealed a high genetic distinction between the isolates. Thirteen virulence-associated genes were identified in C. jejuni isolates. Most of the isolates harbored the genes flaA (83.3%) and flaB (78.8%). The wlaN gene associated with the Guillain-Barré syndrome was detected in nine (13.6%) isolates. The genes for resistance to ampicillin (bla OXA), tetracycline [tet(O)], neomycin [aph(3')-IIIa], streptomycin (aadE) and streptothricin (sat4) were detected in isolated C. jejuni using WGS. A gene cluster comprising the genes sat4, aph(3')-IIIa and aadE was present in six isolates. The single point mutation T86I in the housekeeping gene gyrA conferring resistance to quinolones was retrieved in 93.6% of phenotypically fluoroquinolone-resistant isolates. Five phenotypically erythromycin-susceptible isolates carried the mutation A103V in the gene for the ribosomal protein L22 inferring macrolide resistance. An assortment of 13 β-lactam resistance genes (bla OXA variants) was detected in 58 C. jejuni isolates. Out of 66 sequenced isolates, 28 (42.4%) carried plasmid-borne contigs. Six isolates harbored a pTet-like plasmid-borne contig which carries the tet(O) gene. This study emphasized the potential of whole-genome sequencing to ameliorate the routine surveillance of C. jejuni. Whole-genome sequencing can predict antimicrobial resistance with a high degree of accuracy. However, resistance gene databases need curation and updates to revoke inaccuracy when using WGS-based analysis pipelines for AMR detection.

Whole genome characterization of a multidrug-resistant hypervirulent Pasteurella multocida with a new drug-resistant plasmid

Pasteurella multocida (P. multocida) is a zoonotic bacterium that can cause diseases in a variety of animals. It was divided into 5 serogroups, and serogroup A is mainly prevalent in avian hosts. We isolated a virulent and multidrug-resistant P. multocida strain from Guangdong duck liver and named it PMWSG-4 (GenBank accession no. CP077723.1). To understand the pathogenicity of this strain, the pathogenicity test was carried out with mice and ducks. The results showed that PMSWG-4 was highly pathogenic to ducks and mice, and the LD50 is 4.5 and 73 CFU, respectively. In order to study its genetic characteristics, pathogenicity, and relationship with the host, we performed a whole genome sequencing. The genome size of the isolated PMWSG-4 was 2.38 Mbp, with a G+C content of 40.3%, and coding 2,313 Coding DNA Sequence (CDS). The genome carries 162 potential virulence-associated genes, 32 different drug resistance phenotypes, 102 genes possibly involved in pathogen-host interaction, 2 gene island groups, and 4 prophages. In addition, we also found a new drug-resistant plasmid from strain PMWSG-4, named pXL001 (GenBank accession no. CP077724.1). After verified, the plasmid is a new plasmid carrying the floR florfenicol resistance gene. The whole genome is of great significance for further studying the pathogenesis and genetic characteristics of duck-derived P. multocida.

First study on capsular serotypes and virulence factors of Pasteurella multocida isolates from Phan Rang sheep in Vietnam.

Pasteurella multocida is considered as a main factor mediating pneumonic pasteurellosis in ruminants, including sheep. It is also a current threat to Phan Rang sheep in Vietnam. This study aimed to characterize P. multocida isolated from Phan Rang sheep, their antibiotic resistance profile, and the prevalence of some virulence-associated genes of these strains. Bacteria were isolated on brain heart infusion, 10% sheep blood agar plates, and screened by biochemical tests. The polymerase chain reaction technique was used with specific primers to identify P. multocida, the presence of virulence-associated genes, and serotypes of isolates. Antimicrobial susceptibility and biofilm formation of isolates were examined using the disk diffusion method and crystal violet-based method, respectively. A total of 41 P. multocida strains were isolated from 485 samples from clinically sick and healthy sheep. Of the isolates, 58.53% were serotype A, 9.75% were serotype B, and 31.71% were serotype D. Healthy animals were infected with serotype D only. All 15 virulence genes were identified in all strains isolated from clinically sick sheep, while strains isolated from healthy sheep carried 11/15 virulence genes tested. Among virulence-associated genes exbB, exbD, tonB, ompA, oma87, fimA, hgbA, and nanB were detected in over 90% of isolates, whereas hgbB, nanH, tbpA and pfhA were less frequent. Interestingly, pmHAS and tadD were highly prevalent in capsular type A strains, whereas the toxA gene was detected in capsular type D strains only. All of the isolated strains were fully susceptible to enrofloxacin, ciprofloxacin, neomycin, and ofloxacin. About 92.68% were susceptible to chloramphenicol and 90.24% to amikacin, but there was high resistance to erythromycin, tetracycline, and amoxicillin. Our results reveal that 53.65% of 41 isolates could produce biofilm, whereas 46.34% could not. Pasteurella multocida from Phan Rang sheep possess many virulence genes and resistance to several common antibiotics such as erythromycin, tetracycline, and amoxicillin. The results are an important warning regarding antibiotic resistance of P. multocida.

Antibacterial mechanism of mixed natural preservatives (ε-poly-lysine, cinnamon extract, and chestnut inner shell extract) against Listeria monocytogenes

This study investigated whether Listeria monocytogenes growth could be controlled using a combination of ε-poly-lysine, cinnamon extract, and chestnut inner shell extract in tryptic soy broth (TSB) and chicken meat juice. A mixed natural preservative (MNP) inhibited the growth of L. monocytogenes by approximately 1 log CFU/mL in TSB. The cell surface characteristics of L. monocytogenes, as well as hydrophobicity and auto-aggregation, were negatively impacted by treatment with MNP, and cell motility was also significantly lowered by treatment with MNP. The relative expression levels of flagella-associated and virulence-associated genes in L. monocytogenes treated with MNP were lower than those in the control treated with TSB. The antibacterial activity of MNP was clearly observed in the SEM images. L. monocytogenes treated with MNP in chicken meat juice at 4 °C completely died within 48 h, indicating that MNP exerted antibacterial effects against L. monocytogenes in chicken meat (an example of food media). Therefore, we suggest that the MNP composed of ε-poly-lysine, cinnamon extract, and chestnut inner shell extract had sufficient antibacterial effect against L. monocytogenes and may be utilized as an antibacterial agent in the food industry.

High resistance of fluoroquinolone and macrolide reported in avian pathogenic Escherichia coli isolates from the humid subtropical regions of Pakistan

ObjectivesThis study aimed to assess the antimicrobial resistance profile, virulence potential, and genetic characterization of avian pathogenic Escherichia coli (APEC) that cause colibacillosis in poultry. MethodsAntibiotic susceptibility testing (AST) was measured via the Kirby-Bauer disc diffusion method against 27 commonly used antibiotics. Phylogrouping, virulence-associated gene detection, and hybrid strain detection via multiplex polymerase chain reaction (PCR) and genetic diversity were analysed via ERIC-PCR fingertyping method. ResultsAST analysis showed 100% of isolates were multidrug-resistant (MDR) and highest resistance was against penicillin, tetracycline, and macrolide classes of antibiotics. The mcr-1 gene was present in 40% of the isolates, though only 4% of isolates were showing phenotypic resistance. Despite the scarce use of fluoroquinolone, carbapenem, and cephalosporin in the poultry sector, resistance was evident because of the high prevalence of extended-spectrum β-lactamase (ESBL) (53.7%) and other β-lactamases in APEC isolates. β-lactamase genotyping of APEC isolates revealed that 85.7% of isolates contained either blaCTX or blaTEM and around 38% of isolates were complement resistant. Growth in human urine was evident in 67.3% of isolates. Phylogroup B1 (51%) was the most prevalent group followed by phylogroups A (30.6%), D (13.61%), and B2 (4.76%). The most prevalent virulence-associated genes were fimH, iss, and tatT. Results showed that 26 isolates (17.69%) can be termed hybrid strains and APEC/EHEC (enterohemorrhagic E. coli) was the most prevalent hybrid E. coli pathotype. ERIC-PCR fingerprinting genotype analysis clustered APEC isolates in 40 groups (E1-E40). This study provides insights into the antibiotic resistance and virulence profiling of the APEC isolates in Pakistan. ConclusionsThe findings of this study provide insights into that the antibiotic resistance and virulence profiling of the APEC isolates in Pakistan. This data can inform future studies designed to better estimate the severity of the colibacillosis in poultry farms.

In vitro digestion of ESC-resistant Escherichia coli from poultry meat and evaluation of human health risk.

The spread of antimicrobial resistance (AMR) has become a threat against human and animal health. Third and fourth generation cephalosporins have been defined as critically important antimicrobials by The World Health Organization. Exposure to Extended spectrum cephalosporin-resistant E. coli may result in consumers becoming carriers if these bacteria colonize the human gut or their resistance genes spread to other bacteria in the gut microbiota. In the case that these resistant bacteria at later occasions cause disease, their resistance characteristics may lead to failure of treatment and increased mortality. We hypothesized that ESC-resistant E. coli from poultry can survive digestion and thereby cause infections and/or spread their respective resistance traits within the gastro-intestinal tract. In this study, a selection of 31 ESC-resistant E. coli isolates from retail chicken meat was exposed to a static in vitro digestion model (INFOGEST). Their survival, alteration of colonizing characteristics in addition to conjugational abilities were investigated before and after digestion. Whole genome data from all isolates were screened through a custom-made virulence database of over 1100 genes for virulence- and colonizing factors. All isolates were able to survive digestion. Most of the isolates (24/31) were able to transfer their bla CMY2-containing plasmid to E. coli DH5-á, with a general decline in conjugation frequency of digested isolates compared to non-digested. Overall, the isolates showed a higher degree of cell adhesion than cell invasion, with a slight increase after digestion compared non-digested, except for three isolates that displayed a major increase of invasion. These isolates also harbored genes facilitating invasion. In the virulence-associated gene analysis two isolates were categorized as UPEC, and one isolate was considered a hybrid pathogen. Altogether the pathogenic potential of these isolates is highly dependent on the individual isolate and its characteristics. Poultry meat may represent a reservoir and be a vehicle for dissemination of potential human pathogens and resistance determinants, and the ESC-resistance may complicate treatment in the case of an infection.

A compilation of virulence-associated genes that are frequently reported in avian pathogenic Escherichia coli (APEC) compared to other E. coli.

Escherichia coli (E. coli) survive in various hosts and environments due to their highly diversified genome. These bacteria have co-evolved with human, colonized a broad range of hosts and survive as a commensal organism or pathogen. E. coli that adopted a pathogenic lifecycle in avian hosts typically belong to phylogroups B2 and D. Phylogenic investigations discovered these E. coli are noticeably overlapped with the phylogroup of E. coli infecting humans. This overlapping is possibly due to a parallel evolution in both hosts from a common ancestor, which indicates a high zoonotic potential of avian pathogenic E. coli (APEC). However, some contrasting evidence of other phylogroups infecting the avian host has also been reported in recent studies indicating phylogroups of E. coli are not definitive, only suggestive to their virulence in chickens. Furthermore, virulence-associated genes that contribute to bacterial features necessary to establish APEC infection, are predominantly located in plasmids. Therefore, phylogenetic classification based on chromosomal markers is often inadequate to identify APEC. Moreover, E. coli can obtain virulent plasmids from other bacteria which further complicates the link between phylogenetic classification and pathotype. Previous research has reported an array of virulence-associated genes highly prevalent only in APEC isolates. Function of these genes are possibly a prerequisite to establishing APEC infections in chickens. Consequently, these genes can be used to distinguish APEC from environmental, commensal, intestinal and other extra-intestinal E. coli. Therefore, we have extensively reviewed previous literature to compile the virulence-associated genes that are highly prevalent in APEC compared to other E. coli. From this review, we have identified 10 key virulence-associated genes (iss, tsh, iroN, episomal/chromosomal ompT, iutA, cvaC, hlyF, iucD, papG allel (II/III) and papC) that are frequently reported in APEC isolates than non-pathogenic E. coli. A compilation of these research findings can be crucial to the molecular identification of APEC. Furthermore, it can serve as a guideline for future investigation and aid in formulation of intervention strategies.

Prevalence of Potential Pathogenic and Antimicrobial Resistant Escherichia coli in Danish Broilers.

Avian pathogenic Escherichia coli (APEC) are important bacteria in broiler production in terms of economy, welfare, and use of antibiotics. During a previous outbreak of APEC in the Nordic countries, it was suggested that the pathogenic clones of E. coli causing the outbreak originated from grandparent stock and were transmitted to the offspring, causing increased first week mortality. This study investigated whether the pathogenic potential of E. coli at the parent and broiler level differs in relation to pathogenic potential described by the level of virulence-associated genes and pattern of antimicrobial resistance. The hypothesis was that, due to higher biosecurity at the parent level, the E. coli population will show a lower level of antimicrobial resistance and carry fewer virulence-associated genes, as a result of fewer E. coli infections observed. From four parent flocks and eight broiler flocks, 715 E. coli were isolated from cloacal swabs of newly hatched chickens (Ross 308). The isolated E. coli were characterized by eight virulence-associated genes and phenotypic resistance against six antimicrobials. It was found that the prevalence of virulence-associated genes and phenotypic antimicrobial resistance varied significantly between flocks, and the virulence-associated genes papC and irp2 and resistance against ampicillin were significantly more prevalent in breeder flocks compared to broiler flocks.

A molecular epidemiological study on Escherichia coli in young chicks with colibacillosis identified two possible outbreaks across farms

Avian pathogenic Escherichia coli (APEC) is the cause of colibacillosis outbreaks in young poultry chicks, resulting in acute to peracute death. The high morbidity and mortality caused by colibacillosis results in poor animal welfare, reduced sustainability and economical loss worldwide. To advance the understanding of the molecular epidemiology, genomic relatedness and virulence traits of APEC, we performed systematic sampling from 45 confirmed colibacillosis broiler flocks with high first week mortality (FWM) during 2018–2021. From these flocks, 219 APEC isolates were whole genome sequenced (WGS) and bioinformatic analyses were performed. The bioinformatic analyses included sequence typing (ST), serotyping, detection of virulence-associated genes (VAGs) and phylogenetic analysis. Our results showed a high prevalence of ST23, ST429 and ST95 among APEC isolates from Norwegian broiler flocks, and identified ST23, ST429, ST117 and ST371 to cause disease more often alone, compared to ST95, ST69 and ST10. Phylogenetic analyses, together with associated metadata, identified two distinct outbreaks of colibacillosis across farms caused by ST429 and ST23 and gave insight into expected SNP distances within and between flocks identified with the same ST. Further, our results highlighted the need for combining two typing methods, such as serotyping and sequence typing, to better discriminate strains of APEC. Ultimately, systematic sampling of APEC from multiple birds in a flock, together with WGS as a diagnostic tool is important to identify the disease-causing APEC within a flock and to detect outbreaks of colibacillosis across farms.

Role of the Hypothetical Protein BB0563 during Borrelia burgdorferi Infection in Animals.

The alternative sigma factor RpoS in Borrelia burgdorferi, the etiological agent of Lyme disease, has long been postulated to regulate virulence-associated genes other than ospC and dbpA. Here, we demonstrate that bb0563, a gene encoding a hypothetical protein, is regulated by RpoS and contributes to the optimal infectivity of B. burgdorferi. When B. burgdorferi was exposed to environmental stimuli, bb0563 showed similar expression patterns as rpoS, ospC, and dbpA. Expression of bb0563 was significantly downregulated when rpoS was inactivated and was restored in the complemented strain. By using rapid amplification of cDNA ends (RACE) and luciferase reporter assays, a functional promoter was identified in the regulatory region upstream of bb0563. Gene expression from this promoter was drastically decreased in the rpoS mutant. We next investigated the role of bb0563 during animal infection. By using quantitative reverse transcription-PCR (RT-PCR), we found that bb0563 was highly expressed in mouse tissues during infection. We further created a bb0563-deficient mutant in a bioluminescent B. burgdorferi strain and examined infection dynamics using in vivo imaging. Relative to the parental and complemented strains, the mutant showed a delayed infection pattern and bacterial load was reduced. Another bb0563 deletion mutant was also created in the strain 297 background, and quantitative PCR (qPCR) analysis revealed a significantly lower spirochetal burden in tissue samples collected from animals infected with the mutant. In addition, localization studies indicate that BB0563 is not exposed on the cell surface but is associated with outer membrane. Taken together, these results suggest that bb0563 is required for optimal infectivity of B. burgdorferi during experimental infection.

Comparative Transcriptomic Analysis of MAPK-Mediated Regulation of Pathogenicity, Stress Responses, and Development in Cytospora chrysosperma.

Mitogen-activated protein kinase (MAPK) cascades are highly conserved signal transduction pathways that mediate cellular responses to various biotic and abiotic signals in plant-pathogenic fungi. Generally, there are three MAPKs in filamentous pathogenic fungi: Pmk1/Fus3/Kss1, Hog1, and Stl2. Our previous studies have shown that CcPmk1 is a core regulator of fungal pathogenicity in Cytospora chrysosperma, the causal agent of canker disease in a wide range of woody plants. Here, we identified and functionally characterized the other two MAPK genes (CcHog1 and CcSlt2) and then compared the transcriptional differences among these three MAPKs in C. chrysosperma. We found that the MAPKs shared convergent and distinct roles in fungal development, stress responses, and virulence. For example, CcHog1, CcSlt2, and CcPmk1 were all involved in conidiation and response to stresses, including hyperosmotic pressure, cell wall inhibition agents, and H2O2, but only CcPmk1 and CcSlt2 were required for hyphal growth and fungal pathogenicity. Transcriptomic analysis showed that numerous hyperosmosis- and cell wall-related genes significantly reduced their expression levels in ΔCcHog1 and ΔCcSlt2, respectively. Interestingly, RNA- and ribosome-related processes were significantly enriched in the upregulated genes of ΔCcSlt2, whereas they were significantly enriched in the downregulated genes of ΔCcPmk1. Moreover, two secondary metabolite gene clusters were significantly downregulated in ΔCcPmk1, ΔCcSlt2, and/or ΔCcHog1. Importantly, some virulence-associated genes were significantly downregulated in ΔCcPmk1 and/or ΔCcSlt2, such as candidate effector genes. Collectively, these results suggest that the similar and distinct phenotypes of each MAPK deletion mutant may result from the transcriptional regulation of a series of common or specific downstream genes, which provides a better understanding of the regulation network of MAPKs in C. chrysosperma.

Naegleria genus pangenome reveals new structural and functional insights into the versatility of these free-living amoebae.

Free-living amoebae of the Naegleria genus belong to the major protist clade Heterolobosea and are ubiquitously distributed in soil and freshwater habitats. Of the 47 Naegleria species described, N. fowleri is the only one being pathogenic to humans, causing a rare but fulminant primary amoebic meningoencephalitis. Some Naegleria genome sequences are publicly available, but the genetic basis for Naegleria diversity and ability to thrive in diverse environments (including human brain) remains unclear. Herein, we constructed a high-quality Naegleria genus pangenome to obtain a comprehensive catalog of genes encoded by these amoebae. For this, we first sequenced, assembled, and annotated six new Naegleria genomes. Genome architecture analyses revealed that Naegleria may use genome plasticity features such as ploidy/aneuploidy to modulate their behavior in different environments. When comparing 14 near-to-complete genome sequences, our results estimated the theoretical Naegleria pangenome as a closed genome, with 13,943 genes, including 3,563 core and 10,380 accessory genes. The functional annotations revealed that a large fraction of Naegleria genes show significant sequence similarity with those already described in other kingdoms, namely Animalia and Plantae. Comparative analyses highlighted a remarkable genomic heterogeneity, even for closely related strains and demonstrate that Naegleria harbors extensive genome variability, reflected in different metabolic repertoires. If Naegleria core genome was enriched in conserved genes essential for metabolic, regulatory and survival processes, the accessory genome revealed the presence of genes involved in stress response, macromolecule modifications, cell signaling and immune response. Commonly reported N. fowleri virulence-associated genes were present in both core and accessory genomes, suggesting that N. fowleri's ability to infect human brain could be related to its unique species-specific genes (mostly of unknown function) and/or to differential gene expression. The construction of Naegleria first pangenome allowed us to move away from a single reference genome (that does not necessarily represent each species as a whole) and to identify essential and dispensable genes in Naegleria evolution, diversity and biology, paving the way for further genomic and post-genomic studies.