Iron efflux by IetA enhances ß-lactam aztreonam resistance and is linked to oxidative stress through cellular respiration in Riemerella anatipestifer.

BACKGROUND: Riemerella anatipestifer encodes an iron acquisition system, but whether it encodes the iron efflux pump and its role in antibiotic resistance are largely unknown. OBJECTIVES: To screen and identify an iron efflux gene in R. anatipestifer and determine whether and how the iron efflux gene is involved in antibiotic resistance. METHODS: In this study, gene knockout, streptonigrin susceptibility assay and inductively coupled plasma mass spectrometry were used to screen for the iron efflux gene ietA. The MIC measurements, scanning electron microscopy and reactive oxygen species (ROS) detection were used to verify the role of IetA in aztreonam resistance and its mechanism. Mortality and colonization assay were used to investigate the role of IetA in virulence. RESULTS: The deletion mutant ΔietA showed heightened susceptibility to streptonigrin, and prominent intracellular iron accumulation was observed in ΔfurΔietA under excess iron conditions. Additionally, ΔietA exhibited increased sensitivity to H2O2-produced oxidative stress. Under aerobic conditions with abundant iron, ΔietA displayed increased susceptibility to the ß-lactam antibiotic aztreonam due to heightened ROS production. However, the killing efficacy of aztreonam was diminished in both WT and ΔietA under anaerobic or iron restriction conditions. Further experiments demonstrated that the efficiency of aztreonam against ΔietA was dependent on respiratory complexes Ⅰ and Ⅱ. Finally, in a duckling model, ΔietA had reduced virulence compared with the WT. CONCLUSION: Iron efflux is critical to alleviate oxidative stress damage and ß-lactam aztreonam killing in R. anatipestifer, which is linked by cellular respiration.

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.

Anti-inflammatory activity of diindolylmethane alleviates Riemerella anatipestifer infection in ducks.

3,3'-Diindolylmethane (DIM) is found in cruciferous vegetables and is used to treat various inflammatory diseases because of its potential anti-inflammatory effects. To investigate effects of DIM in Riemerella anatipestifer-infected ducks which induce upregulation of inflammatory cytokines, ducks were treated orally with DIM at dose of 200 mg/kg/day and infected the following day with R. anatipestifer. Infected and DIM-treated ducks exhibited 14% increased survival rate and significantly decreased bacterial burden compared to infected untreated ducks. Next, the effect on the expression level of inflammatory cytokines (interleukin [IL]-17A, IL-17F, IL-6, IL-1ß) of both in vitro and in vivo DIM-treated groups was monitored by quantitative reverse-transcription PCR (qRT-PCR). Generally, the expression levels of the cytokines were significantly reduced in DIM-treated splenic lymphocytes stimulated with killed R. anatipestifer compared to stimulated untreated splenic lymphocytes. Similarly, the expression levels of the cytokines were significantly reduced in the spleens and livers of DIM-treated R. anatipestifer-infected ducks compared to infected untreated ducks. This study demonstrated the ameliorative effects of DIM in ducks infected with R. anatipestifer. Thus, DIM can potentially be used to prevent and/or treat R. anatipestifer infection via inhibition of inflammatory cytokine expression.

Characterization of RaeE-RaeF-RopN, a putative RND efflux pump system in Riemerella anatipestifer.

Resistance-nodulation-division (RND) efflux systems are ubiquitous in Gram-negative bacteria and play a predominant role in antimicrobial resistance and other diverse phenotypes, but the knowledges of RND efflux systems are poorly understood so far in Riemerella anatipestifer. According to the sequence annotation, RIA_1117-RIA_1118-RIA_1119 operon in RA-GD strain encodes a putative tripartite RND efflux system. RIA_1117, RIA_1118 and RIA_1119 genes encode an outer member protein (OMP), an inner membrane pump protein (pump transporter), and a periplasmic membrane fusion protein (MFP), respectively. Furthermore, RIA_1119 protein is annotated as a MexE component. In this work, the biological functions of RIA_1117-RIA_1118-RIA_1119 proteins were studied. The antibiotic susceptibility testing showed that the inactivation of RIA_1117, RIA_1118 and RIA_1119 genes all raised susceptibility to amikacin, streptomycin and SDS. By induction with the above antimicrobial agents, the transcription levels of RIA_1117 and RIA_1118 genes were up-regulated significantly using qRT-PCR detection, but no significance difference was observed for the transcription level of RIA_1119 gene. CCCP inhibitor assay confirmed that RIA_1117, RIA_1118 and RIA_1119 proteins mediated amikacin, streptomycin and SDS resistance depending on proton motive force (PMF). Spot assay and streptomycin accumulation assay confirmed that RIA_1117, RIA_1118 and RIA_1119 proteins contributed to export streptomycin, and CCCP increased the accumulation of streptomycin. Furthermore, RIA_1117, RIA_1118 and RIA_1119 proteins also were involved in the fitness and virulence of RA-GD strain. These results showed that RIA_1117-RIA_1118-RIA_1119 operon encoded a RND efflux system, which has the substrate specificity for streptomycin, amikacin and SDS and contributed to the growth and virulence of RA-GD. RIA_1117-RIA_1118-RIA_1119 was designated RaeE-RaeF-RopN efflux system. Based on the above results and structural analysis, RIA_1117, RIA_1118 and RIA_1119 proteins corresponded to RopN (OMP), RaeF (pump transporter) and RaeE (MFP), respectively.

The functional identification of Dps in oxidative stress resistance and virulence of Riemerella anatipestifer CH-1 using a new unmarked gene deletion strategy.

Excessive iron in the bacterial cytoplasm can potentiate the production of harmful reactive oxygen species (ROS). Riemerella anatipestifer (R. anatipestifer, RA), a gram-negative bacterium, encodes an iron uptake system, but its iron detoxification mechanism is unknown. Here, the dps gene of R. anatipestifer CH-1 (RA-CH-1) was deleted using sacB as a counterselection marker. The dps mutant was more sensitive to H2O2 than the wild type in iron-rich conditions but not in iron-limited conditions, suggesting that Dps prevents H2O2-induced damage through iron binding. However, the dps mutant and wild type were identically sensitive to bactericidal antibiotics, and antibiotic treatment did not enhance RA-CH-1 ROS production. Furthermore, Dps prevents DNA damage by binding DNA. The RA-CH-1 dps transcript level was higher in the stationary phase than in the early and exponential phases and was increased by OxyR in the presence of H2O2. Finally, duckling colonization by the dps mutant was similar to that by the wild type at 48 h postinfection but significantly lower at 60 h postinfection, suggesting that RA-CH-1 Dps is not involved in host invasion but increases resistance to host clearance. Dps thus likely plays an important role in R. anatipestifer physiology and pathogenesis through protecting against oxidative stress.

RanB, a putative ABC-type multidrug efflux transporter contributes to aminoglycosides resistance and organic solvents tolerance in Riemerella anatipestifer.

Riemerella anatipestifer is a Gram-negative bacterium, which is an important pathogen infecting ducks and resistant to various antibiotics. The efflux pump is an important resistance mechanism of Gram-negative bacteria, but little research has been done in R. anatipestifer. In this study, the drug resistance mediated by RIA_1614 gene of R. anatipestifer RA-GD strain was studied, because the gene was presumed to be an efflux pump component of ABC. Firstly, the deletion strain RA-GD△RIA_1614 and complemented strain RA-GD△RIA_1614 pCPRA::RIA_1614 were constructed. Then, MICs of various antimicrobial agents to parent and deletion strains and the tolerance of the strains to organic solvents were detected to screen the substrates for RIA_1614 gene. Moreover, the transcription levels of RIA_1614 gene in the parent and the complemented strains exposed to the substrates were detected by quantitative real-time RT-PCR. Furthermore, the efflux abilities of parent, deletion and complemented strains to substrates were determined by antibiotic accumulation test. In addition, in vitro competition ability and virulence of the strains were also detected. The results showed that the deletion strain was more sensitive to aminoglycosides and organic solvents than parental strain RA-GD. When RA-GD and complemented strain were exposed to sub-repression levels of aminoglycosides and organic solvents, the transcription levels of RIA_1614 gene were significantly up-regulated. Sodium o-vanadate inhibitor assay confirmed that RIA_1614 protein contributed to amikacin and streptomycin resistance and organic solvent tolerance. Streptomycin accumulation test showed that the RIA_1614 protein was able to export streptomycin, and the addition of ATPase inhibitor sodium o-vanadate increased the accumulation of streptomycin, indicating that RIA_1614 protein was an ATP-dependent efflux transporter. Growth and competition experiments revealed that RIA_1614 protein had no significant effect on growth of RA-GD, but decreased in vitro competition ability of the strain. Furthermore, pathogenicity tests showed that RIA_1614 protein involved in the virulence of the strain. Based on the results and amino acid sequence analysis, it was determined that RIA_1614 protein was a member of ABC efflux pumps, and the protein was named RanB.

Prevalence of fluoroquinolone resistance and mutations in the gyrA, parC and parE genes of Riemerella anatipestifer isolated from ducks in China.

BACKGROUND: Riemerella anatipestifer is one of the most serious infectious disease-causing pathogens in the duck industry. Drug administration is an important method for prevention and treatment of infection in duck production, leading to widespread drug resistance in R. anatipestifer. METHODS: For a total of 162 isolates of R. anatipestifer, the MICs were determined for a quinolone antimicrobial agent, namely, nalidixic acid, and three fluoroquinolones, namely, ciprofloxacin, enrofloxacin and ofloxacin. The gyrA, parC, and parE gene fragments were amplified by PCR to identify the mutation sites in these strains. Site-directed mutants with mutations that were detected at a high frequency in vivo were constructed (hereafter referred to as site-directed in vivo mutants), and the MICs of these four drugs for these strains were determined. RESULTS: In total, 100, 97.8, 99.3 and 97.8% of the 137 R. anatipestifer strains isolated between 2013 and 2018 showed resistance to nalidixic acid, ciprofloxacin, enrofloxacin, and ofloxacin, respectively. The high-frequency mutation sites were detected in a total of 162 R. anatipestifer strains, such as Ser83Ile and Ser83Arg, which are two types of substitution mutations of amino acid 83 in GyrA; Val799Ala and Ile811Val in ParC; and Val357Ile, His358Tyr, and Arg541Lys in ParE. MIC analysis results for the site-directed in vivo mutants showed that the strains with only the Ser83Ile mutation in GyrA exhibited an 8-16-fold increase in MIC values, and all mutants showed resistance to ampicillin and ceftiofur. CONCLUSIONS: The resistance of R. anatipestifer to quinolone agents is a serious problem. Amino acid 83 in GyrA is the major target mutation site for the fluoroquinolone resistance mechanism of R. anatipestifer.

New Perspectives on Galleria mellonella Larvae as a Host Model Using Riemerella anatipestifer as a Proof of Concept.

Galleria mellonella larvae have been used as a host model to study interactions between pathogens and hosts for several years. However, whether the model is useful to interrogate Riemerella anatipestifer infection biology remained unknown. This study aimed to exploit the potential of G. mellonella larvae and reveal their limitations as a host model for R. anatipestifer infection. G. mellonella larvae were shown to be effective for virulence evaluations of different R. anatipestifer strains. Furthermore, the virulent strain R. anatipestifer CH-1 had a stronger ability to proliferate than the attenuated strain R. anatipestifer ATCC 11845 in both G. mellonella larvae and ducklings. Unconventionally it was shown that G. mellonella larvae cannot be used to evaluate the efficacy of antimicrobials and their combinations. Additionally, it was shown that certain virulence factors, such as OmpA (B739_0861), B739_1208, B739_1343, and Wza (B739_1124), were specific only for ducklings, suggesting that G. mellonella larvae must be cautiously used to identify virulence factors of R. anatipestifer Evaluation of heme uptake-related virulence genes, such as tonB1 and tonB2, required preincubating the strains with hemoglobin before infection of G. mellonella larvae since R. anatipestifer cannot obtain a heme source from G. mellonella larvae. In conclusion, this study revealed the applicability and limitations of G. mellonella as a model with which to study the pathogen-host interaction, particularly in the context of R. anatipestifer infection.

Rifampin resistance and its fitness cost in Riemerella anatipestifer.

BACKGROUND: Riemerella anatipestifer (R. anatipestifer) is one of the most important poultry pathogens worldwide, with associated infections causing significant economic losses. Rifampin Resistance is an important mechanism of drug resistance. However, there is no information about rpoB mutations conferring rifampin resistance and its fitness cost in Riemerella anatipestifer. RESULTS: Comparative analysis of 18 R.anatipestifer rpoB sequences and the determination of rifampin minimum inhibitory concentrations showed that five point mutations, V382I, H491N, G502K, R494K and S539Y, were related to rifampin resistance. Five overexpression strains were constructed using site-directed mutagenesis to validate these sites. To investigate the origin and fitness costs of the rpoB mutations, 15 types of rpoB mutations were isolated from R. anatipestifer ATCC 11845 by using spontaneous mutation in which R494K was identical to the type of mutation detected in the isolates. The mutation frequency of the rpoB gene was calculated to be 10- 8. A total of 98.8% (247/250) of the obtained mutants were located in cluster I of the rifampin resistance-determining region of the rpoB gene. With the exception of D481Y, I537N and S539F, the rifampin minimum inhibitory concentrations of the remaining mutants were at least 64 µg/mL. The growth performance and competitive experiments of the mutant strains in vitro showed that H491D and 485::TAA exhibit growth delay and severely impaired fitness. Finally, the colonization abilities and sensitivities of the R494K and H491D mutants were investigated. The sensitivity of the two mutants to hydrogen peroxide (H2O2) and sodium nitroprusside (SNP) increased compared to the parental strain. The number of live colonies colonized by the two mutants in the duckling brain and trachea were lower than that of the parental strain within 24 h. CONCLUSIONS: Mutations of rpoB gene in R. anatipestifer mediate rifampin resistance and result in fitness costs. And different single mutations confer different levels of fitness costs. Our study provides, to our knowledge, the first estimates of the fitness cost associated with the R. anatipestifer rifampin resistance in vitro and in vivo.

The drug tolerant persisters of Riemerella anatipestifer can be eradicated by a combination of two or three antibiotics.

BACKGROUND: Riemerella anatipestifer (RA), the causative agent of duck infectious serositis, leads to high mortality in duck flocks and great economic losses in duck industry. Previous studies on RA are largely focused on its detection, virulence factors, serology, epidemiology as well as antibiotic resistance. Neither drug tolerant persisters nor the persister level under the treatment of antibiotics has been revealed. The persisters are non-growing or dormant cells within an isogenic bacterial population; they play important roles in recurrent infection and formation of drug resistant mutants. The aim of this study is to detect the drug tolerant persisters from the exponentially grown population of RA reference strain (RA 11845) or RA clinical isolate (RA TQ3), and address whether a single antibiotic or a combination of two or three antimicrobials can eradicate the persisters at respective maximum serum/plasma concentration (Cmax). RESULT: With the concentration of a test antibiotic increased, a small fraction of cells in the exponentially grown culture of RA reference strain (RA 11845) or RA clinical isolate (RA TQ3) always survived, irrespective of treatment time, indicating the presence of drug tolerant presisters. A single antibiotic cannot eradicate the persisters of both RA strains at respective Cmax, except that the Cmax of ceftiofur wiped out the population of the reference strain (RA 11845). Besides, the clinical isolate RA TQ3 presented a higher tolerance to ceftiofur in comparison to that of the reference strain (RA 11845). Combination of any two or three antimicrobials eliminated the drug tolerant persisters of RA TQ3 completely at respective Cmax. CONCLUSION: A sub-community of drug tolerant persisters was present in RA population. Persisters of RA TQ3 are single drug tolerant and not multidrug tolerant persisters.

A novel resistance gene, lnu(H), conferring resistance to lincosamides in Riemerella anatipestifer CH-2.

The Gram-negative bacterium Riemerella anatipestifer CH-2 is resistant to lincosamides, having a lincomycin (LCM) minimum inhibitory concentration (MIC) of 128 µg/mL. The G148_1775 gene of R. anatipestifer CH-2, designated lnu(H), encodes a 260-amino acid protein with ≤41% identity to other reported lincosamide nucleotidylyltransferases. Escherichia coli RosettaTM (DE3) containing the pBAD24-lnu(H) plasmid showed four- and two-fold increases in the MICs of LCM and clindamycin (CLI), respectively. A kinetic assay of the purified Lnu(H) enzyme for LCM and CLI showed that the protein could inactive lincosamides. Mass spectrometry analysis demonstrated that the Lnu(H) enzyme catalysed adenylylation of lincosamides. In addition, an lnu(H) gene deletion strain exhibited 512- and 32-fold decreases in LCM and CLI MICs, respectively. The wild-type level of lincosamide resistance could be restored by complementation with a shuttle plasmid carrying the lnu(H) gene. The transformant R. anatipestifer ATCC 11845 [lnu(H)] acquired by natural transformation also exhibited high-level lincosamide resistance. Moreover, among 175 R. anatipestifer field isolates, 56 (32.0%) were positive for the lnu(H) gene by PCR. In conclusion, Lnu(H) is a novel lincosamide nucleotidylyltransferase that inactivates LCM and CLI by nucleotidylylation, thus conferring high-level lincosamide resistance to R. anatipestifer CH-2.

Antimicrobial susceptibility of Riemerella anatipestifer strains isolated from geese and ducks in Hungary.

Riemerella anatipestifer causes anatipestifer disease in many avian species. A total of 185 R. anatipestifer strains isolated in Hungary between 2000 and 2014 from geese and ducks were tested against 13 antibiotics (ampicillin, doxycycline, enrofloxacin, erythromycin, florfenicol, flumequine, gentamicin, penicillin, spectinomycin, streptomycin, sulphamethoxazole-trimethoprim, sulphonamide compounds, and tetracycline) by the Kirby-Bauer disk diffusion method. The majority of the strains were susceptible to florfenicol (97.9%), ampicillin (95.1%), penicillin (93%), sulphamethoxazole-trimethoprim (92.4%), and spectinomycin (86.5%). The highest resistance rates were observed for flumequine, tetracycline, erythromycin and streptomycin (94%, 91.4%, 75.1% and 71.4% resistance, respectively). The resistance patterns showed some variation depending on the geographical origin of the strains. The average rate of extensive drug resistance was 30.3%, and its proportion tended to increase in the period examined.

A strain of pathogenic Bacillus subtilis results in brain damage in ducklings when co-infected with Riemerella anatipestifer.

Bacillus subtilis is a Gram-positive bacterium widely used in medicine and agriculture. So far, little is known about its pathogenicity in animals. In this study, a strain of Bacillus subtilis, HFBF-B11 isolated from brain tissue of ducklings co-infected with Riemerella anatipestifer was characterized. The strain demonstrated consistent characteristics of B. subtilis in staining and morphological, biochemical and physiological analyses. Moreover, its DNA sequence, which was obtained via PCR sequencing of 16S rRNA, exhibited 99% homology with the B. subtilis reference strain. In in vitro cultures HFBF-B11 exhibited ß-hemolysis. The results of experiments showed that a single infection of HFBF-B11 in 9-day-old ducklings did not result in clear clinical symptoms. However, following co-infection with HFBF-B11 and R. anatipestifer, the animals demonstrated liver injury and blood-brain barrier disruption leading to infection and brain damage with a mortality rate of 100%. These results suggest that the HFBF-B11 strain of B. subtilis is an opportunistic pathogen of ducklings. This is the first report about the isolation of a B. subtilis strain with pathogenicity in ducklings.

In vitro susceptibility of four antimicrobials against Riemerella anatipestifer isolates: a comparison of minimum inhibitory concentrations and mutant prevention concentrations for ceftiofur, cefquinome, florfenicol, and tilmicosin.

BACKGROUND: Mutant prevention concentration (MPC) is an alternative pharmacodynamic parameter that has been used to measure antimicrobial activity and represents the propensities of antimicrobial agents to select resistant mutants. The concentration range between minimum inhibitory concentration (MIC) and MPC is defined as mutant selection window (MSW). The MPC and MSW parameters represent the ability of antimicrobial agents to inhibit the bacterial mutants selected. This study was conducted to determine the MIC and MPC values of four antimicrobials including ceftiofur, cefquinome, florfenicol and tilmicosin against 105 Riemerella anatipestifer isolates. RESULTS: The MIC50/MIC90 values of clinical isolates tested in our study for ceftiofur, cefquinome, florfenicol and tilmicosin were 0.063/0.5、0.031/0.5、1/4、1/4 µg/mL, respectively; MPC50/ MPC90 values were 4/64、8/64、4/32、16/256 µg/mL, respectively. These results provided information on the use of these compounds in treating the R. anatipestifer infection; however, additional studies are needed to demonstrate their therapeutic efficacy. CONCLUSION: Based on the MSW theory, the hierarchy of these tested antimicrobial agents with respect to selecting resistant subpopulations was as follows: cefquinome > ceftiofur > tilmicosin > florfenicol. Cefquinome was the drug that presented the highest risk of selecting resistant mutant among the four antimicrobial agents.

In vivo activity of cefquinome against Riemerella anatipestifer using the pericarditis model in the duck.

Cefquinome is a fourth-generation cephalosporin with broad-spectrum antibacterial activity, including activity against enteric gram-negative bacilli such as Riemerella anatipestifer. The pericarditis model was used to examine the pharmacodynamic characteristics of cefquinome against R. anatipestifer. Serum levels of cefquinome following the administration of different doses were determined by LC-MS/MS. Ducks with ca. 10(6) CFU/mL at the initiation of therapy were treated with cefquinome at doses that ranged from 0.0156 to 2 mg/kg of body weight/day (in 3, 6, 12, or 24 divided doses) for 24 h. The percentage of a 24-h dosing interval that the unbound serum cefquinome concentrations exceeded the MIC (fT > MIC) were the pharmacokinetic (PK)-pharmacodynamic (PD) parameter that best correlated with efficacy (R(2) 86.3% for R. anatipestifer, compared with 58.9% for the area under the concentration-time curve/MIC and 10.6% for peak/MIC). A sigmoid Emax model was used to estimate the magnitudes of the %fT > MIC associated with net bacterial stasis, a 1-log10 CFU reduction from baseline, and a 2-log10 CFU reduction from baseline; the corresponding values were (22.5 ± 1.3) %, (35.2 ± 4.5) %, and (42.4 ± 2.7) %. These data showed that treatment with cefquinome results in marked antibacterial effects in vivo against R. anatipestifer and that the host's immunity may also play a key role in the anti-infective therapy process.

Identification of a gene in Riemerella anatipestifer CH-1 (B739-2187) that contributes to resistance to polymyxin B and evaluation of its mutant as a live attenuated vaccine.

Riemerella anatipestifer (R. anatipestifer) causes severe perihepatitis, pericarditis, airsacculitis and meningitis in the duck, leading to great economic losses worldwide. Given the increased prevalence of drug-resistance strains, vaccination is the best strategy to prevent R. anatipestifer infection in ducklings. In this study, we identified a gene in R. anatipestifer (B739-2187) that can restore the resistance of the Salmonella phoP mutant to polymyxin B using genetic complementation. Furthermore, the deletion of B739-2187 in R. anatipestifer resulted in a mutant exhibiting increased sensitivity to polymyxin B. The R. anatipestifer B739-2187 mutant did not exhibit phenotypic defects, as indicated by its growth curve, lipopolysaccharide and outer membrane protein profiles, and attachment and invasion of duck embryo fibroblast cells. The duck animal experiments demonstrated that the deletion of B739-2187 significantly decreased the virulence of R. anatipestifer, and the B739-2187 mutant provided 100% protection against challenge with wild-type R. anatipestifer, exhibiting the characteristics of an ideal live vaccine.

Antibacterial, anti-inflammatory, and antioxidant effects of Yinzhihuang injection.

The Yinzhihuang injection, a traditional Chinese medicine, has been the recent target of increasing interest due to its anti-inflammatory properties. The molecular basis by which Yinzhihuang injection could cure Riemerella anatipestifer (RA) serositis in ducks is unclear. This study evaluated the antibacterial, anti-inflammatory and antioxidant effects of Yinzhihuang injection, using disease models of RA-induced infectious serositis in ducks and heptane-induced inflammation in mice and rats. The duck mortality rate was reduced from 60% to 20% and both the inflammatory response and histological damage were ameliorated by treatment with Yinzhihuang injection (0.02 g/kg). Further studies indicated that superoxide dismutase (SOD), nitric oxide synthase (NOS), and inducible nitric oxide synthase (iNOS) were elevated while malondialdehyde (MDA), nitric oxide (NO) and RA growth were inhibited when the ducks were treated by Yinzhihuang injection. In addition, Yinzhihuang injection (0.04 g/ml) effectively inhibited xylene-induced auricle swelling in mice, (demonstrating an inhibition rate of 35.21%), egg albumen-induced paw metatarsus swelling in rats, (demonstrating an inhibition rate of 22.30%), and agar-induced formation of granulation tissue. These results suggest that Yinzhihuang injection ameliorates RA-induced infectious serositis in ducks by modulation of inflammatory mediators and antioxidation.

Riemerella anatipestifer lacks luxS, but can uptake exogenous autoinducer-2 to regulate biofilm formation.

Riemerella anatipestifer (RA) causes major economic losses to the duck industry. Autoinducer-2 (AI-2) is a quorum-sensing signal that regulates bacterial physiology. The luxS and pfs genes are required for AI-2 synthesis in many bacterial species. pfs encodes Pfs, which functions upstream of LuxS in the biosynthesis of AI-2. In this study, we investigated the AI-2 activity of RA using an AI-2 bioassay, which showed that RA does not produce AI-2. Bioinformatic analysis indicated that the RA genome has a pfs, but not a luxS, homologue. To investigate the function of RA pfs, an avian pathogenic Escherichia coli (APEC) pfs mutant was constructed, which was subsequently transformed with a recombinant plasmid carrying RA pfs. An AI-2 bioassay demonstrated that RA pfs restored AI-2 production to the APEC pfs mutant, suggesting that RA pfs functions in AI-2 synthesis. Furthermore, we found that RA utilizes exogenous AI-2 to regulate biofilm formation. RA biofilm formation decreased significantly upon addition of exogenous AI-2. Real-time quantitative PCR results showed that expression of 13 genes related to RA biofilm formation decreased significantly when exogenous AI-2 was added to the RA culture media. These findings will benefit future studies on AI-2 regulation in RA.

ErmF and ereD are responsible for erythromycin resistance in Riemerella anatipestifer.

To investigate the genetic basis of erythromycin resistance in Riemerella anatipestifer, the MIC to erythromycin of 79 R. anatipestifer isolates from China and one typed strain, ATCC11845, were evaluated. The results showed that 43 of 80 (53.8%) of the tested R. anatipestifer strains showed resistance to erythromycin, and 30 of 43 erythromycin-resistant R. anatipestifer strains carried ermF or ermFU with an MIC in the range of 32-2048 µg/ml, while the other 13 strains carrying the ereD gene exhibited an MIC of 4-16 µg/ml. Of 30 ermF + R. anatipestifer strains, 27 (90.0%) carried the ermFU gene which may have been derived from the CTnDOT-like element, while three other strains carried ermF from transposon Tn4351. Moreover, sequence analysis revealed that ermF, ermFU, and ereD were located within the multiresistance region of the R. anatipestifer genome.

Identification of ribosomal RNA methyltransferase gene ermF in Riemerella anatipestifer.

Riemerella anatipestifer is a major bacterial pathogen of waterfowl, globally responsible for avian septicaemic disease. As chemotherapy is the predominant method for the prevention and treatment of R. anatipestifer infection in poultry, the widespread use of antibiotics has favoured the emergence of antibiotic-resistant strains. However, little is known about R. anatipestifer susceptibility to macrolide antibiotics and its resistance mechanism. We report for the first time the identification of a macrolide resistance mechanism in R. anatipestifer that is mediated by the ribosomal RNA methyltransferase ermF. We identified the presence of the ermF gene in 64/206 (31%) R. anatipestifer isolates from different regions in China. An ermF deletion strain was constructed to investigate the function of the ermF gene on the resistance to high levels of macrolides. The ermF mutant strain showed significantly decreased resistance to macrolide and lincosamide, exhibiting 1024-, 1024-, 4- and >2048-fold reduction in the minimum inhibitory concentrations for erythromycin, azithromycin, tylosin and lincomycin, respectively. Furthermore, functional analysis of ermF expression in E. coli XL1-blue showed that the R. anatipestifer ermF gene was functional in E. coli XL1-blue and conferred resistance to high levels of erythromycin (100 µg/ml), supporting the hypothesis that the ermF gene is associated with high-level macrolide resistance. Our work suggests that ribosomal RNA modification mediated by the ermF methyltransferase is the predominant mechanism of resistance to erythromycin in R. anatipestifer isolates.