OmpA is involved in the invasion of duck brain microvascular endothelial cells by Riemerella anatipestifer.

Bacterial meningitis is a major cause of morbidity and mortality. Despite advances in antimicrobial chemotherapy, the disease remains detrimental to humans, livestock, and poultry. Riemerella anatipestifer is a gram-negative bacterium causing duckling serositis and meningitis. However, the virulence factors contributing to its binding and invasion of duck brain microvascular endothelial cells (DBMECs) and penetration of the blood-brain barrier (BBB) have never been reported. In this study, immortalized DBMECs were successfully generated and used as an in vitro-model of duck BBB. Furthermore, ompA gene deletion mutant of the pathogen and multiple complemented strains carrying the complete ompA gene and its truncated forms were constructed. Bacterial growth, invasion, and adhesion assays and animal experiments were performed. The results show that the OmpA protein of R. anatipestifer had no effect on bacterial growth and adhesion ability to DBMECs. The role of OmpA in the invasion of R. anatipestifer into DBMECs and duckling BBB was confirmed. The amino acids 230-242 of OmpA represents a key domain involved in R. anatipestifer invasion. In addition, another OmpA1164 protein constituted by the amino acids 102-488 within OmpA could function as a complete OmpA. The signal peptide sequence from amino acids 1-21 had no significant effect on OmpA functions. In conclusion, this study illustrated that OmpA is an important virulence factor mediating R. anatipestifer invasion of DBMECs and penetration of the duckling BBB.

Sifting through the core-genome to identify putative cross-protective antigens against Riemerella anatipestifer.

Infectious serositis of ducks, caused by Riemerella anatipestifer, is one of the main infectious diseases that harm commercial ducks. Whole-strain-based vaccines with no or few cross-protection were observed between different serotypes of R. anatipestifer, and so far, control of infection is hampered by a lack of effective vaccines, especially subunit vaccines with cross-protection. Since the concept of reverse vaccinology was introduced, it has been widely used to screen for protective antigens in important pathogens. In this study, pan-genome binding reverse vaccinology, an emerging approach to vaccine candidate screening, was used to screen for cross-protective antigens against R. anatipestifer. Thirty proteins were identified from the core-genome as potential cross-protective antigens. Three of these proteins were recombinantly expressed, and their immunoreactivity with five antisera (anti-serotypes 1, 2, 6, 10, and 11) was demonstrated by Western blotting. Our study established a method for high-throughput screening of cross-protective antigens against R. anatipestifer in silico, which will lay the foundation for the development of a cross-protective subunit vaccine controlling R. anatipestifer infection. KEY POINTS: • Pan-genome binding reverse vaccine approach was first established in R. anatipestifer to screen for subunit vaccine candidates. • Thirty potential cross-protective antigens against R. anatipestifer were identified by this method. • The reliability of the method was verified preliminarily by the results of Western blotting of three of these potential antigens.

Riemerella anatipestifer Endonuclease I displays enzymatic activity and is associated with bacterial virulence.

Riemerella anatipestifer is an important pathogen of waterfowl, causing septicemic and exudative diseases. We previously reported that the R. anatipestifer AS87_RS02625 is a secretory protein of the type IX secretion system (T9SS). In this study, R. anatipestifer T9SS protein AS87_RS02625 was determined to be a functional Endonuclease I (EndoI), which has DNase and RNase activities. Optimal temperature and pH of the recombinant R. anatipestifer EndoI (rEndoI) to cleave λDNA were determined as 55-60 °C and 7.5 respectively. The DNase activity of the rEndoI was dependent on the presence of divalent metal ions. Presence of Mg2+ at a concentration range of 7.5-15 mM in the rEndoI reaction buffer displayed the highest DNase activity. In addition, the rEndoI displayed RNase activity to cleave MS2-RNA (ssRNA), either in the absence or presence of divalent cations Mg2+, Mn2+, Ca2+, Zn2+ and Cu2+. The DNase activity of the rEndoI was significantly enhanced by Mg2+, Mn2+ and Ca2+ but not Zn2+ and Cu2+. Moreover, we indicated that R. anatipestifer EndoI functioned on the bacterial adherence, invasion, in vivo survival and inducing inflammatory cytokines. These results indicate that the R. anatipestifer T9SS protein AS87_RS02625 is a novel EndoI, displays endonuclease activity and plays an important role in bacterial virulence.

Manganese Efflux Achieved by MetA and MetB Affects Oxidative Stress Resistance and Iron Homeostasis in Riemerella anatipestifer.

In bacteria, manganese homeostasis is controlled by import, regulation, and efflux. Here, we identified 2 Mn exporters, MetA and MetB (manganese efflux transporters A and B), in Riemerella anatipestifer CH-1, encoding a putative cation diffusion facilitator (CDF) protein and putative resistance-nodulation-division (RND) efflux pump, respectively. Compared with the wild type (WT), ΔmetA, ΔmetB, and ΔmetAΔmetB exhibited sensitivity to manganese, since they accumulated more intracellular Mn2+ than the WT under excess manganese conditions, while the amount of iron in the mutants was decreased. Moreover, ΔmetA, ΔmetB, and ΔmetAΔmetB were more sensitive to the oxidant NaOCl than the WT. Further study showed that supplementation with iron sources could alleviate manganese toxicity and that excess manganese inhibited bacterial cell division. RNA-Seq showed that manganese stress resulted in the perturbation of iron metabolism genes, further demonstrating that manganese efflux is critical for iron homeostasis. metA transcription was upregulated under excess manganese but was not activated by MetR, a DtxR family protein, although MetR was also involved in manganese detoxification, while metB transcription was downregulated under iron depletion conditions and in fur mutants. Finally, homologues of MetA and MetB were found to be mainly distributed in members of Flavobacteriaceae. Specifically, MetB represents a novel manganese exporter in Gram-negative bacteria. IMPORTANCE Manganese is required for the function of many proteins in bacteria, but in excess, manganese can mediate toxicity. Therefore, the intracellular levels of manganese must be tightly controlled. Manganese efflux transporters have been characterized in some other bacteria; however, their homologues could not be found in the genome of Riemerella anatipestifer through sequence comparison. This indicated that other types of manganese efflux transporters likely exist. In this study, we characterized 2 transporters, MetA and MetB, that mediate manganese efflux in R. anatipestifer in response to manganese overload. MetA encodes a putative cation diffusion facilitator (CDF) protein, which has been characterized as a manganese transporter in other bacteria, while this is the first observation of a putative resistance-nodulation-division (RND) transporter contributing to manganese export in Gram-negative bacteria. In addition, the mechanism of manganese toxicity was studied by observing morphological changes and by transcriptome sequencing. Taken together, these results are important for expanding our understanding of manganese transporters and revealing the mechanism of manganese toxicity.

Machine learning combined with MALDI-TOF MS has the potential ability to identify serotypes of the avian pathogen Riemerella anatipestifer.

AIM: Combining MALDI-TOF MS and machine learning to establish a new rapid method to identify two important serotypes of Rimerella anatipestifer. METHODS AND RESULTS: MALDI-TOF MS was performed on 115 R. anatipestifer strains (serotype 1, serotype 2, and other serotypes) to explore its ability to identify serotypes of R. anatipestifer. Raw spectral data were generated in diagnostic mode; these data were preprocessed, clustered, and analysed using principal component analysis. The results indicated that MALDI-TOF MS completely differentiated serotype 1 from serotype 2 of R. anatipestifer; the potential serotype-associated m/z loci are listed. Furthermore, Random Forest and Support Vector Machine were used for modelling to identify the two important serotypes, and the results of cross-validation indicated that they had ∼80% confidence to make the right classification. CONCLUSION: We proved that MALDI-TOF MS can differentiate serotype 1 from serotype 2 of R. anatipestifer. Additionally, the identification models established in this study have high confidence to screen out these two important serotypes from other serotypes.

Evaluation of the immunoprotection efficacy of Riemerella anatipestifer fur-deficient mutant as an attenuated vaccine.

Riemerella anatipestifer (R. anatipestifer, RA) is an infectious pathogen that causes septicemia and polyserositis in ducks. Our previous studies showed that RA CH-1 ∆fur was significantly attenuated in ducklings, which highlights the potential of this strain as a live attenuated vaccine. In this study, it was shown that infection with 109 CFU of the fur mutant did not cause any clinical symptoms or significant histological lesions in 3-day-old ducklings and that the bacteria were readily cleared by the host within 3 d. Compared with the nonvaccinated group, the group inoculated with the mutant strain RA CH-1 ∆fur exhibited protection of ducklings against a high-dose (2.28 × 1010 CFU) challenge with the wild-type strain RA CH-1. Moreover, the average body weights and body weight gains of the Δfur-inoculated group were not significantly affected by the challenge. Further analysis revealed that RA CH-1 ∆fur elicited higher IgY titers and that the serum antibody levels persisted for at least 49 d after immunization. Overall, our study showed that RA CH-1 ∆fur is a safe and effective vaccine candidate that is expected to play an important role in RA CH-1 infection prevention in the duck industry.

DEAD Box Protein DhR1 Is a Global Regulator Involved in the Bacterial Fitness and Virulence of Riemerella anatipestifer.

DEAD box proteins perform diverse cellular functions in bacteria. Our group previously reported that the transposon Tn4531 insertion in Riean_0395 (designated dhR1), which encodes a putative DEAD box helicase, attenuated the virulence of R. anatipestifer strain YZb1. Here, we show that, compared to the wild-type (WT) R. anatipestifer strain Yb2, the growth or survival of the ΔdhR1 mutant in tryptic soy broth (TSB) was significantly decreased in response to cold, pH, osmotic stress, ethanol, Triton X-100, and oxidative stress, and the dhR1 deletion significantly reduced biofilm formation and the adhesion capacity to Vero cells, whereas the growth of ΔdhR1 was less impaired in iron-limited TSB. Moreover, the virulence of ΔdhR1 in ducklings was attenuated by about 80-fold, compared to the WT. In addition, a transcriptome analysis showed that the dhR1 deletion in the strain Yb2 affected the expression of 58 upregulated genes and 98 downregulated genes that are responsible for various functions. Overall, our work reveals that the deletion of DhR1 results in a broad effect on the bacterial fitness, biofilm formation, iron utilization, and virulence of R. anatipestifer, which makes it a global regulator. IMPORTANCE R. anatipestifer infection has been a continued and serious problem in many duck farms, but little is known about the mechanism underlying the pathogenesis of R. anatipestifer and how R. anatipestifer adapts to the external environment and thereby persists in duck farms. The results of this study demonstrate that the DEAD box protein DhR1 is required for the tolerance of R. anatipestifer to cold, pH, and other stresses, and it is also necessary for biofilm formation, iron utilization, and virulence in ducklings, demonstrating multiple functions of DhR1.

Antibiotic resistance of Riemerella anatipestifer and comparative analysis of antibiotic-resistance gene detection methods.

Riemerella anatipestifer is an important pathogen in waterfowl, and is generally multidrug resistant. This study assessed the current status of Riemerella anatipestifer antibiotic resistance and antibiotic-resistance genes (ARGs), compared the results of different detection methods, and evaluated a new method of studying the association between antibiotic resistance and ARGs in Riemerella anatipestifer. In this study, 51 strains of Riemerella anatipestifer were isolated from ducks on several farms, their resistance to 28 antibiotics was assessed, and the isolates were subjected to whole-genome sequencing. The number of ARGs carried by Riemerella anatipestifer was predicted, compared, and analyzed, and the consistency between ARGs and antibiotic-resistance phenotypes was assessed. The potential for loss of resistance genes during the sequencing and assembly of genome-wide framework map was assessed, and a new ARG detection method was pilot tested. The 51 strains of Riemerella anatipestifer were multidrug resistant (MDR) and had high level of resistance to aminoglycosides, trimethoprim, lincosamides, polypeptides, and macrolides. Based on the genome-wide framework map of the 51 strains, 3 local databases of ABRicate software and 1 online database of CARD website were used to detect ARGs, and a mean of 4 to 5 ARGs were identified per isolate. Although the detection results differed according to the database used, the general performance was consistent. The online website detected more types of ARGs than the ABRicate software. The association between ARGs and antibiotic-resistance phenotypes was assessed, and the ermF gene was identified as a possible key ARGs regulating macrolide resistance of Riemerella anatipestifer. The method used to investigate and detect Riemerella anatipestifer ARGs was convenient and rapid, and had strong accuracy and pertinence. The ARGs detection method reported here combined the advantages of PCR and genome detection, and could greatly reduce workload and detect ARGs more precisely.

Riemerella anatipestifer GldG is necessary for secretion of effectors by type IX secretion system.

Riemerella anatipestifer secretes proteins through the type IX secretion system (T9SS). Recent studies have shown that the R. anatipestifer T9SS component proteins GldM and GldK also act as crucial virulence factors. In our previous study, the disruption of AS87_RS00460 gene, which encodes the predicted protein GldG, significantly reduced the bacterial virulence of R. anatipestifer wild-type strain Yb2, but the mechanism was unclear. In this study, we investigated the function of the GldG in bacterial virulence and protein secretion using the mutant strain Yb2ΔgldG and complementation strain cYb2ΔgldG. Our results demonstrate that the gldG gene encodes a gliding-motility-associated ABC transporter substrate-binding protein GldG, which was localized to the bacterial membrane in an immunoblotting analysis, and functions in the bacterium's adherence to and invasion of host cells and its survival in host blood. The resistance of mutant strain Yb2ΔgldG to complement-dependent killing was significantly reduced. Yb2ΔgldG displayed reduced gliding motility and deficient protein secretion. Label-free quantification (LFQ) with liquid chromatography-mass spectrometry (LC-MS) showed that 10 proteins with a conserved T9SS C-terminal domain were differentially secreted by Yb2ΔgldG and Yb2. The secretion levels of those 10 proteins were determined with immunoblotting, and the results were consistent with the LFQ LC-MS data. All of these effects were rescued by complementation with a plasmid encoding Yb2 gldG. Our results demonstrate that the R. anatipestifer gldG gene encodes the protein GldG, which is involved in bacterial virulence and protein secretion.

Riemerella anatipestifer AS87_RS02955 Acts as a Virulence Factor and Displays Endonuclease Activity.

Riemerella anatipestifer is an important bacterial pathogen in the global duck industry and causes heavy economic losses. In our previous study, we demonstrated that R. anatipestifer type IX secretion system components GldK and GldM, and the secretion protein metallophosphoesterase, acted as virulence factors. In this study, R. anatipestifer AS87_RS02955 was investigated for virulence and enzymatic activity properties. We constructed AS87_RS02955 mutation and complementation strains to assess bacterial virulence. In vivo bacterial loads showed a significantly reduced bacterial loads in the blood of ducks infected with mutant strain Yb2Δ02955, which was recovered in the blood of ducks infected with the complementation strain cYb2Δ02955, demonstrating that AS87_RS02955 was associated with virulence. Further studies showed AS87_RS02955 was a novel nonspecific endonuclease with no functionally conserved domain, but enzymatic activity toward DNA and RNA was indicated. DNase activity was activated by Zn2+, Cu2+, Mg2+, Ca2+, and Mn2+ ions but inhibited by ethylenediaminetetraacetic acid. RNase activity was independent of metal cations, but stimulated by Mg2+, Ca2+, and Mn2+. RAS87_RS02955 enzymatic activity was active across a broad pH and temperature range. Moreover, we identified four sites in rAS87_RS02955, F39, F92, I134, and F145, which were critical for enzymatic activity. In summary, we showed that R. anatipestifer AS87_RS02955 encoded a novel endonuclease with important roles in bacterial virulence. IMPORTANCE R. anatipestifer AS87_RS02955 was identified as a novel T9SS effector and displayed a nonspecific endonuclease activity in this study. The protein did not contain a conserved His-Asn-His motif structure, which is similar to the endonuclease from Prevotella sp. Its mutant strain Yb2Δ02955 demonstrated significantly attenuated virulence, suggesting AS87_RS02955 is an important virulence factor. Moreover, AS87_RS02955 displayed nonspecific endonuclease activity to cleave λ DNA and MS2 RNA, while four protein sites were critical for endonuclease activity. In conclusion, R. anatipestifer AS87_RS02955 plays important roles in bacterial virulence.

Emergence of plasmid-mediated tigecycline, ß-lactam and florfenicol resistance genes tet(X), blaOXA-347 and floR in Riemerella anatipestifer isolated in China.

Bacterial antimicrobial resistance (AMR) continues to develop, with the horizontal transfer of antibiotic resistance genes (ARGs) through plasmids playing a major role. Recently, the antimicrobial resistance of R. anatipestifer has become increasingly severe, jeopardizing the development of the poultry industry. In this study, we used PromethION to determine the whole genome sequence of R. anatipestifer RCAD0416, a multidrug-resistant isolate from China. We detected a plasmid in the isolate. We named the plasmid pRCAD0416RA-1; the plasmid was 37356 bp in size with 36 putative open reading frames and included the blaOXA-347, floR, tet(X), ermF, ereD, and AadS resistance genes. Most resistance genes might be obtained from R. anatipestifer HXb2. Mobile elements and floR might be transmitted by plasmid pB18-2 from Acinetobacter indicus, and the ICEPg6Chn1 mobile elements can be transmitted from Proteus genomosp. The plasmid pRCAD0416RA-1 was transferred to Escherichia coli K-12 × 7232 via electroporation. Subsequent antimicrobial sensitivity tests (AST) showed a noticeable levels of antimicrobial resistance to ß-lactams (4-8 fold), tigecycline (8 fold), and florfenicol (8 fold). These types of antibiotics are in common clinical use. The purpose of this article is to elucidate the basic characteristics of pRCAD0416RA-1 and the level of resistance mediated by blaOXA-347, floR, and tet(X).

Identification, serotyping, and antimicrobial susceptibility of Riemerella anatipestifer isolated from ducks in Vietnam.

Background: Septicemia caused by Riemerella anatipestifer (R. anatipestifer) is a serious problem in the duck industry worldwide, and it is currently one of the major concerns for duck farming in Vietnam.. Aim: This study was conducted to identify the causative agent of septicemia in ducks in Vietnam. The antimicrobial susceptibility and serotypes of R. anatipestifer isolates were also determined to provide valuable information for disease treatment and vaccine development. Methods: Riemerella anatipestifer was isolated using blood agar and chocolate agar media. The commercial API 20NE microtest system and the partial nucleotide sequence analysis of the 16s rRNA were used to identify R. anatipestifer strains. Serotypes were determined by slide agglutination test using standard antisera against R. anatipestifer. The disk diffusion method was utilized to investigate the antimicrobial susceptibility of R. anatipestifer isolated strains. Results: A total of 408 samples were collected from ducks with typical symptoms of septicemia for R. anatipestifer isolation. Sixty-nine R. anatipestifer strains were identified. Serotyping results showed that 30 out of 69 bacterial strains were classified as serotypes 1, 6, 8, 10, and 20, with serotype 10 being the most prevalent. The antimicrobial susceptibility test revealed that 100% of the bacterial isolates were susceptible to Amoxicillin/clavulanic acid and Imipenem. On the contrary, the majority of R. anatipestifer strains were resistant to Nalidixic acid (89.9%), Streptomycin (75.4%), and Norfloxacin (72.5%). Conclusion: This is the first ever report in terms of identification, serotyping, and antimicrobial susceptibility tests of R. anatipestifer causing septicemia in ducks of Vietnam, providing useful scientific information for treatment as well as vaccine development to control the disease.

Genome-Wide Analysis and Characterization of the Riemerella anatipestifer Putative T9SS Secretory Proteins with a Conserved C-Terminal Domain.

Riemerella anatipestifer is a major pathogenic agent of duck septicemic and exudative diseases. Recent studies have shown that the R. anatipestifer type IX secretion system (T9SS) acts as a crucial virulence factor. We previously identified two T9SS component proteins, GldK and GldM, and one T9SS effector metallophosphoesterase, which play important roles in bacterial virulence. In this study, 19 T9SS-secreted proteins that contained a conserved T9SS C-terminal domain (CTD) were predicted in R. anatipestifer strain Yb2 by searching for CTD-encoding sequences in the whole genome. The proteins were confirmed with a liquid chromatography-tandem mass spectrometry analysis of the bacterial culture supernatant. Nine of them were reported in our previous study. We generated recombinant proteins and mouse antisera for the 19 predicted proteins to confirm their expression in the bacterial culture supernatant and in bacterial cells. Western blotting indicated that the levels of 14 proteins were significantly reduced in the T9SS mutant Yb2ΔgldM culture medium but were increased in the bacterial cells. RT-qPCR indicated that the expression of these genes did not differ between the wild-type strain Yb2 and the T9SS mutant Yb2ΔgldM. Nineteen mutant strains were successfully constructed to determine their virulence and proteolytic activity, which indicated that seven proteins are associated with bacterial virulence, and two proteins, AS87_RS04190 and AS87_RS07295, are protease-activity-associated virulence factors. In summary, we have identified at least 19 genes encoding T9SS-secreted proteins in the R. anatipestifer strain Yb2 genome, which encode multiple functions associated with the bacterium's virulence and proteolytic activity. IMPORTANCE Riemerella anatipestifer T9SS plays an important role in bacterial virulence. We have previously reported nine R. anatipestifer T9SS-secreted proteins and clarified the function of the metallophosphoesterase. In this study, we identified 10 more secreted proteins associated with the R. anatipestifer T9SS, in addition to the nine previously reported. Of these, 14 proteins showed significantly reduced secretion into the bacterial culture medium but increased expression in the bacterial cells of the T9SS mutant Yb2ΔgldM; seven proteins were shown to be associated with bacterial virulence; and two proteins, AS87_RS04190 and AS87_RS07295, were shown to be protease-activity-associated virulence factors. Thus, we have demonstrated that multiple R. anatipestifer T9SS-secreted proteins function in virulence and proteolytic activity.

Florfenicol/Chlortetracycline Effect on Pharmacodynamic Indices for Mutant Selection of Riemerella anatipestifer in Ducks.

Riemerella anatipestifer can cause septicemia and death in ducks and geese, leading to significant economic losses to animal farms. The emergence of resistance of R. anatipestifer to commonly used antibiotics increases the difficulty of treating R. anatipestifer infection. The aim of this study was to evaluate the utility of antibiotic combination to restrict mutant selection of multidrug-resistant (MDR) R. anatipestifer isolates. Pharmacokinetics of florfenicol and chlortetracycline in Pekin ducks were evaluated using both noncompartmental analysis and population pharmacokinetic models. The areas under the curve of florfenicol and chlortetracycline after single 20 and 10 mg/kg oral administration were 49.3 and 6.84 mg*h/L, respectively. Chlortetracycline exhibited high apparent clearance and low systemic exposure. Minimum inhibitory concentration (MIC) and mutant prevention concentration (MPC) values of the two antibiotics were determined in 10 and 2 MDR R. anatipestifer isolates, respectively, to derive fTMSW (the fraction of time over 24 hours wherein the free drug concentration was within the mutant selection window [MSW]) and fT>MPC (the fraction of time that the free drug concentration was above the MPC). Both fTMSW and fT>MPC were estimated from simulated concentration-time profiles relative to MIC and MPC. Florfenicol and chlortetracycline combination have additive activities against R. anatipestifer in majority of isolates and could significantly decrease monotherapy MPC of florfenicol and chlortetracycline, as well as optimize both fTMSW and fT>MPC parameters, provided that the bioavailability of chlortetracycline is improved. The application of pharmacokinetic/pharmacodynamic analyses to MPC concepts to restrict selection of mutant bacterial strains can help improve short- and long-term outcomes of antibiotic treatment in animal farms.

Riemerella anatipestifer T9SS Effector SspA Functions in Bacterial Virulence and Defending Natural Host Immunity.

Riemerella anatipestifer is a major pathogenic agent of duck septicemic and exudative diseases. Recent studies have shown that the R. anatipestifer type IX secretion system (T9SS) is a crucial factor in bacterial virulence. The AS87_RS04190 protein was obviously missing from the secreted proteins of the T9SS mutant strain Yb2ΔgldM. A bioinformatic analysis indicated that the AS87_RS04190 protein contains a T9SS C-terminal domain sequence and encodes a putative subtilisin-like serine protease (SspA). To determine the role of the putative SspA protein in R. anatipestifer pathogenesis and proteolysis, we constructed two strains with an sspA mutation and complementation, respectively, and determined their median lethal doses, their bacterial loads in infected duck blood, and their adherence to and invasion of cells. Our results demonstrate that the SspA protein functions in bacterial virulence. It is also associated with the bacterial protease activity and has a conserved catalytic triad structure (Asp126, His158, and Ser410), which is necessary for protein function. The optimal reactive pH and temperature were determined to be 7.0 and 50°C, respectively, and Km and Vmax were determined to be 10.15 mM and 246.96 U/mg, respectively. The enzymatic activity of SspA is activated by Ca2+, Mg2+, and Mn2+ and inhibited by Cu2+ and EDTA. SspA degrades gelatin, fibrinogen, and bacitracin LL-37. These results demonstrate that SspA is an effector protein of T9SS and functions in R. anatipestifer virulence and its proteolysis of gelatin, fibrinogen, and bacitracin LL-37. IMPORTANCE In recent years, Riemerella anatipestifer T9SS has been reported to act as a virulence factor. However, the functions of the proteins secreted by R. anatipestifer T9SS are not entirely clear. In this study, a secreted subtilisin-like serine protease SspA was shown to be associated with R. anatipestifer virulence, host complement evasion, and degradation of gelatin, fibrinogen, and LL-37. The enzymatic activity of recombinant SspA was determined, and its Km and Vmax were 10.15 mM and 246.96 U/mg, respectively. Three conserved sites (Asp126, His158, and Ser410) are necessary for the protein's function. The median lethal dose of the sspA-deleted mutant strain was reduced >10,000-fold, indicating that SspA is an important virulence factor. In summary, we demonstrate that the R. anatipestifer AS87_RS04190 gene encodes an important T9SS effector, SspA, which plays an important role in bacterial virulence.

Characterization of RASA-1, a novel class A extended-spectrum beta-lactamase from Riemerella anatipestifer.

A novel chromosomally-located ß-lactamase gene, blaRASA-1, was identified in Riemerella anatipestifer RA-CH-1. The RASA-1, encoded by blaRASA-1, was a class A extended-spectrum ß-lactamase (ESBL), which shared 42.7% and 40.5% identities with the RAA-1 and CGA-1 ß-lactamase, respectively. Overexpression of RASA-1 in Escherichia coli confers broad resistance to ß-lactams and the purified native RASA-1 revealed ESBL-like hydrolysis activity. Blasting in GenBank showed that blaRASA-1 was exclusively detected in Riemerella anatipestifer. Moreover, sequence analysis revealed that this gene was located within the multi-resistance region of Riemerella anatipestifer genome.

Phenotypic and genomic analysis reveals Riemerella anatipestifer as the potential reservoir of tet(X) variants.

BACKGROUND: Tigecycline is regarded as one of the last-resort antimicrobials clinically. Emergence of plasmid-mediated tet(X) undermines such an important drug. However, the origins of tet(X) remain largely unexplored. METHODS: Riemerella anatipestifer strains were characterized by PCR, antimicrobial susceptibility testing, WGS and bioinformatics analysis. Functional analysis of tet(X) was verified by cloning experiments. Genomic structures of chromosome- and plasmid-mediated tet(X) were analysed. RESULTS: Thirty-eight R. anatipestifer strains were collected and found to be positive for tet(X). These strains were resistant to multiple antimicrobials; 55.3% (21/38) of the strains were resistant to tigecycline and all of the strains demonstrated resistance to tetracycline. The complete genome sequences of 18 representative strains were obtained. WGS analysis of 38 genomes identified 13 tet(X) variants located on chromosomes, which increased MICs of tigecycline (16-256-fold) for Escherichia coli, although most of them could not confer high-level resistance to tigecycline in the original R. anatipestifer hosts. Genomic environment analysis indicated that the occurrence of multiple tet(X) variants is common and other resistance genes, such as catB, tet(Q), floR, blaOXA, ereD and ermF, could be located in the same chromosomal regions. Two types of tet(X)-bearing segments were identified, one of which was floR-ISCR2-tet(X). This indicates that tet(X) variants were not conserved in chromosomal structures, but in regions with potential transferability. Furthermore, an MDR plasmid carrying tet(X18) was found in R. anatipestifer 20190305E2-2, different from the chromosomal tet(X21). CONCLUSIONS: This study confirmed that tet(X) is highly prevalent in R. anatipestifer. The transfer risk of tet(X) across R. anatipestifer to other clinical pathogens warrants further investigations.

RAA Enzyme Is a New Family of Class A Extended-Spectrum ß-Lactamase from Riemerella anatipestifer Strain RCAD0122.

Whole-genome sequencing of Riemerella anatipestifer isolate RCAD0122 revealed a chromosomally located ß-lactamase gene, blaRAA-1, which encoded a novel class A extended-spectrum ß-lactamase (ESBL), RAA-1. RAA-1 shared ≤65% amino acid sequence identity with other characterized ß-lactamases. The kinetic assay of native purified RAA-1 revealed ESBL-like hydrolysis activity. Furthermore, blaRAA-1 could be transferred to a homologous strain by natural transformation. However, an epidemiological study showed that the blaRAA-1 gene is not prevalent currently.

Source Tracking and Global Distribution of the Tigecycline Non-Susceptible tet(X).

The emergence of tet(X) genes has compromised the clinical use of the last-line antibiotic tigecycline. We identified 322 (1.21%) tet(X) positive samples from 12,829 human microbiome samples distributed in four continents (Asia, Europe, North America, and South America) using retrospective data from worldwide. These tet(X) genes were dominated by tet(X2)-like orthologs but we also identified 12 samples carrying novel tet(X) genes, designed tet(X45), tet(X46), and tet(X47), were resistant to tigecycline. The metagenomic analysis indicated these tet(X) genes distributed in anaerobes dominated by Bacteroidaceae (78.89%) of human-gut origin. Two mobile elements ISBf11 and IS4351 were most likely to promote the transmission of these tet(X2)-like orthologs between Bacteroidaceae and Riemerella anatipestifer. tet(X2)-like orthologs was also developed during transmission by mutation to high-level tigecycline resistant genes tet(X45), tet(X46), and tet(X47). Further tracing these tet(X) in single bacterial isolate from public repository indicated tet(X) genes were present as early as 1960s in R. anatipestifer that was the primary tet(X) carrier at early stage (before 2000). The tet(X2) and non-tet(X2) orthologs were primarily distributed in humans and food animals respectively, and non-tet(X2) were dominated by tet(X3) and tet(X4). Genomic comparison indicated these tet(X) genes were likely to be generated during tet(X) transmission between Flavobacteriaceae and E. coli/Acinetobacter spp., and ISCR2 played a key role in the transmission. These results suggest R. anatipestifer was the potential ancestral source of tet(X). In addition, Bacteroidaceae of human-gut origin was an important hidden reservoir and mutational incubator for the mobile tet(X) genes that enabled spread to facultative anaerobes and aerobes. IMPORTANCE The emergence of the tigecycline resistance gene tet(X) has posed a severe threat to public health. However, reports of its origin and distribution in human remain rare. Here, we explore the origin and distribution of tet(X) from large-scale metagenomic data of human-gut origin and public repository. This study revealed the emergency of tet(X) gene in 1960s, which has refreshed a previous standpoint that the earliest presence of tet(X) was in 1980s. The metagenomic analysis from data mining covered the unculturable bacteria, which has overcome the traditional bacteria isolating and purificating technologies, and the analysis indicated that the Bacteroidaceae of human-gut origin was an important hidden reservoir for tet(X) that enabled spread to facultative anaerobes and aerobes. The continuous monitoring of mobile tigecycline resistance determinants from both culturable and unculturable microorganisms is imperative for understanding and tackling the dissemination of tet(X) genes in both the health care and agricultural sectors.

Duck Interleukin-22: Identification and Expression Analysis in Riemerella anatipestifer Infection.

Riemerella anatipestifer is one of the most devastating pathogens affecting the global duck farms. Infection is involved in secretion of proinflammatory cytokines, including interleukin- (IL-) 17A. During the immune response to infection, IL-22 and IL-17A are often produced concurrently and at high levels in inflamed tissues. Little is known about duck IL-22 (duIL-22) during R. anatipestifer infection. We describe the characterization of duIL-22 and its mRNA expression analysis in splenic lymphocytes and macrophages treated with heat-killed R. anatipestifer and in the spleens and livers of R. anatipestifer-infected ducks. Full-length cDNA of duIL-22 encoded 197 amino acids. The deduced amino acid sequence of duIL-22 shared a 30.4-40.5% similarity with piscine counterparts, 57.4-60.1% with mammalian homologs, and 93.4% similarity to the chicken. Duck IL-22 mRNA expression level was relatively high in the skin of normal ducks. It was increased in mitogen-stimulated splenic lymphocytes and in killed R. anatipestifer-activated splenic lymphocytes and macrophages. Compared with healthy ducks, IL-22 transcript expression was significantly upregulated in the livers and spleens on days 1 and 4 postinfection, but not on day 7. IL-17A was significantly increased in the spleens only on day 4 postinfection and in the livers at all time points. When splenic lymphocytes were stimulated with heat-killed R. anatipestifer, CD4+ cells predominantly produced IL-22 while IL-17A was expressed both by CD4+ and CD4- cells. These results suggested that IL-22 and IL-17A are likely expressed in different cell types during R. anatipestifer infection.