Epidemiology of Haemophilus parasuis isolates from pigs in China using serotyping, antimicrobial susceptibility, biofilm formation and ERIC-PCR genotyping

Background Haemophilus parasuis is a commensal organism of the upper respiratory tract of healthy pigs and causes high morbidity and mortality in piglets. The aim of this study was to investigate the epidemiology of H. parasuis in China from 2014 to 2017. Methods We characterized 143 H. parasuis isolates by serotyping, antimicrobial susceptibility, biofilm formation and with enterobacterial repetitive intergenic consensus-polymerase chain reaction (ERIC-PCR) assays. Results Serotyping revealed serovar 5 as the most prevalent (26.6%) followed by serovars 4 (22.4%), 7 (9.1 %), 13 (6.3%), 12 (5.6 %), and non-typeable (8.4%). In a panel of 23 antimicrobials, the minimum inhibitory concentration 50% (MIC50) were in the range of 0.25–16 μg/mL and MIC90 were 2–>512 μg/mL. A total of 99 isolates of H. parasuis (69.2%) were able to form biofilms and 59.6% (59/99) performed weak biofilm-forming ability. ERIC-PCR revealed a very heterogeneous pattern with 87 clusters. Discussion These H. parasuis isolates showed a high serovar and genotypic lineage diversity, different abilities to form biofilms and a high degree of genetic diversity. Biofilm formation was related to antimicrobial susceptibility but there were no statistically significant associations between the antimicrobial susceptibility and either the serovars or the ERIC-PCR clusters. This study showed a high prevalence of high-MIC H. parasuis strains and suggests the need for a continuous surveillance of clinical isolates of H. parasuis.


INTRODUCTION
The Gram-negative bacterium Haemophilus parasuis is the causative agent of Glässer's disease in pigs and is one of the most important pathogen in the modern swine industry. The disease is characterized by pneumonia, meningitis, arthritis, polyserositis, and

MATERIALS AND METHODS Serotyping
A total of 143 H. parasuis strains were studied (Table 1; Fig. 1). The information of the 143 H. parasuis strains has been reported (Zhao et al., 2018). The South China Agriculture University Animal ethics committee approved to carry out the study (No. 2014-025). Serotypes of all 143 H. parasuis field isolates were identified using the gel diffusion (GD) test against 15 reference strain antiserums as previously described   (Morozumi & Nicolet, 1986;Turni & Blackall, 2005) and the GD methodology, subjected to indirect hemagglutination (IHA) testing (Turni & Blackall, 2005). Non-typed strains were defined as isolates whose antigens did not react with antiserum against the 15 reference serotypes.

Antimicrobial susceptibility testing
The minimum inhibitory concentration (MIC) values of antimicrobials for the H. parasuis were determined by a micro double-dilution method according to protocols by the Clinical and Laboratory Standards Institute (CLSI, 2013) and a previous report (Pruller et al., 2017).

Biofilm formation
Biofilm formation experiments were performed in 96-well microtiter plates as previously described with minor modifications (Jin et al., 2006;Stepanovic et al., 2000). Briefly, all H. parasuis isolates from overnight agar cultures were suspended in 5 mL tryptic soy broth (TSB) (Becton Dickinson, Owings Mills, MD, USA) containing 10 mg/mL NAD and 5% bovine serum (Gibco Ò , Auckland, New Zealand) and 200 mL of this culture suspension was aliquoted in triplicate into flat-bottom 96 well polystyrene plates and incubated for 24 h at 37 C. The wells were washed three times with 200 mL of sterile phosphate-buffered saline to remove loosely adherent cells. The remaining attached bacteria were fixed with 200 mL of absolute methanol per well for 15 min before the plates were emptied and left to dry at room temperature. Biofilms were stained with 200 mL of 1% (w/v) crystal violet for 5 min. Excess stain was removed with three washes of distilled water and the plates were dried at 37 C for 15 min. The stain was then released by adding 100 mL of 33% (v/v) glacial acetic acid per well. The amount of released stain was quantified by measuring the absorbance at 570 nm with an automated Elx800 Universal Microplate Reader (Bio-Tek Instruments Inc., Winooski, VT, USA).

Molecular typing by ERIC-PCR
Haemophilus parasuis isolates were grown on tryptic soy agar (Becton Dickinson, Owings Mills, MD, USA) plates and several colonies of each isolate were inoculated into 5 mL TSB containing 10 mg/mL NAD and 5% bovine serum and incubated for 24 h at 37 C. Total DNA was extracted from these cultures using the TIANamp Bacteria DNA Kit (Tiangen Biotech, Beijing, China). The isolates were characterized by ERIC-PCR following published guidelines (Rafiee et al., 2000). PCR reactions were carried out with the following cycling conditions: degeneration 5 min at 94 C, 35 cycles of 30 s at 94 C, 60 sec at 50 C and 2 min at 72 C and a final step of 10 min at 72 C. The resulting band profiles were visually assessed using BioNumerics 6.6 software. A 90% similarity cut-off was used to analyze the genotypes generated by ERIC-PCR technique according to Oliveira, Blackall & Pijoan (2003).

Antimicrobial susceptibility testing
Minimum inhibitory concentration values for the reference strains were within the acceptable quality control ranges (CLSI, 2015). The results of the susceptibility testing of the 23 antimicrobials against 143 isolates of H. parasuis (See Supplemental Material S1) were tabulated as distribution of the MIC values, MIC 50 , MIC 90 . The susceptibility tests indicated that the MIC 50 values of all 23 tested antimicrobials were in the range of 0.25-16 mg/mL and MIC 90 values were 2->512 mg/mL (Table 3).

Enterobacterial repetitive intergenic consensus-polymerase chain reaction
All 143 H. parasuis strains were examined using ERIC with a 90% similarity cutoff. This analysis revealed 87 distinct groups that displayed heterogeneous ERIC patterns. Some isolates possessed 100% similarity with each other such as HP002 and HP048, HP008 and HP019, HP022 and HP076. Others such as HP049 and HP130 and five other pairs were almost perfect matches (Fig. 3). This indicated that the ERIC-PCR technique was more discriminative than serotyping and a broad genetic array was observed within serovars.
Among the 143 H. parasuis field isolates representing 15 serovars and nontypeable strains, 99 out of 143 strains were able to form some sort of biofilm. The distribution of this ability among the 15 serovars and nontypeable H. parasuis was diverse. Almost all the multiple antibiotic resistant isolates tested positive for biofilm formation. However, biofilm producers possessed higher resistance rates to more antimicrobials than nonproducers. This included resistance to lincomycin, ampicillin, penicillin, gentamicin, kanamycin, florfenicol, polymyxin B, cephalexin, lomefloxacin, cefepime, tetracycline, tulathromycin, doxycycline, ceftiofur, amoxicillin, enrofloxacin, ciprofloxacin, and tilmicosin. Moreover, biofilm producers possessed lower resistance rates to trimethoprim/ sulfamethoxazole, oxacillin, spectinomycin and when compared with non-biofilm producers (Table 5).
Enterobacterial repetitive intergenic consensus-polymerase chain reaction was employed to evaluate the genetic relationship between the H. parasuis clinical strains with different biofilm-forming abilities. The 68 genotypes of the biofilm-forming isolates were heterogeneous and lacked any dominant clones so no correlation was identified ( Fig. S1 is listed in Supplemental Material S2).

DISCUSSION
Haemophilus parasuis is one of the most common pathogen in pigs. However, the distribution and prevalence of serotypes and genotypes can vary considerably from region to region and over time within a given region. In this study, we analyzed serotypes, antibiotic resistance, biofilm formation and ERIC-PCR to characterize H. parasuis strains isolated from pigs.
Other three studies in China indicated the documented serotypes were 4, 5, 14, 13 in 20154, 5, 14, 13 in and 5, 4, 7, 13 in 20164, 5, 14, 13 in and 4, 5, 7, 1 in 20174, 5, 14, 13 in (Chen et al., 2015Ma et al., 2016;Wang et al., 2017). In addition to these studies, high NT isolation rates (10-40%) were reported in all previously described studies, which maybe become a dominant serotype in Germany, USA/Canada, Denmark and Brazil (Angen, Svensmark & Mittal, 2004;Kielstein & Rapp-Gabrielson, 1992;, and even became the most serotype in Brazil (Macedo et al., 2011;Moreno et al., 2011). In this work, serotypes 5, 4, 7, and 13 were the most frequently detected and 8.4% were NT. These showed identical serotype profiles as a previous study reporting serotypes 4, 5, and 13 as dominant (Cai et al., 2005;Zhang et al., 2011). Treatment of infections with H. parasuis commonly includes broad-spectrum antimicrobials (Aarestrup et al., 2004;de la Fuente et al., 2007;Wissing, Nicolet & Boerlin, 2001;Zhou et al., 2010). Nevertheless, the level of antimicrobial susceptibility varied a lot The sensitivities for the animal specific antimicrobials gamithromycin and tildipirosin (new macrolide antimicrobials for respiratory diseases) have not been reported until the current work. The high MIC values we found suggested that the use of these drugs should be regulated. Furthermore, these results indicated that there has been a rapid increase in the rate of resistance. This attributable at least in part to the use of antibiotic additives in animal feeds and the extensive use of antimicrobial agents in veterinary medicine. Additionally, there is a limit that there are currently no approved clinical breakpoints available for H. parasuis. Therefore, we should strictly control the use of antimicrobial agents in food animals and need a continuous surveillance of antimicrobial susceptibility to minimize the emergence of resistance in future. In a previous study, we have detected the antimicrobial resistance genes for the 143 H. parasuis (Zhao et al., 2018), combined with the antimicrobial susceptibility results in this study, demonstrated that the high MIC values of H. parasuis in piglets is a combination of transferable antibiotic resistance genes and multiple target gene mutations. Biofilm formation is essential for persistent infections (Costerton, Stewart & Greenberg, 1999). These structures can withstand host immune pressures and biofilm-producing strains are less susceptible to antimicrobials than biofilm-negative strains (Anderson & O'Toole, 2008;Hall-Stoodley & Stoodley, 2009;Zhang et al., 2014). H. parasuis forms biofilms in vitro and suggests a causal relationship between colonizers and biofilm growth (Jin et al., 2006). Individual H. parasuis isolates differed in their ability to form biofilms as has been reported previously (Zhang et al., 2014). Different serovars possessed different abilities to form biofilms and we confirmed a positive correlation between biofilm presence and antibiotic resistance especially with b-lactam antimicrobials (Moleres et al., 2015;Zhang et al., 2014).
In general, the 15 different serovars of H. parasuis could be divided into those that were highly virulent, moderately virulent and non-virulent although there may be individual variations (Oliveira & Pijoan, 2004). However, the high-biofilm production phenotype is not always being linked to virulence (Bello-Orti et al., 2014). It is worth noting that a recent study on the human pathogen Streptococcus pneumoniae showed that biofilm formation in vivo was associated with reduced invasiveness and a dampened cytokine response (Blanchette-Cain et al., 2013). In the present study, we detected significantly higher resistance levels among biofilm producers compared with non-biofilm producers. We also confirmed a positive correlation between biofilm presence and antibiotic resistance except for oxacillin, spectinomycin and trimethoprim/ sulfamethoxazole. Biofilm-forming H. parasuis isolates have significantly higher resistance to b-lactams than the non-producers (Zhang et al., 2014). The persistence of antibiotic resistance within biofilms is another aspect that should not be neglected because it has a potential impact on both animal and public health.
In the current study, we also utilized ERIC-PCR and found it possessed discriminatory power for H. parasuis. ERIC-PCR has been used to successfully subtype H. parasuis strains isolated from different regions, confirming the high heterogeneity and high genetic variability of H. parasuis (Macedo et al., 2011;Olvera, Calsamiglia & Aragon, 2006;Rafiee et al., 2000). We obtained 87 genotypes among the 143 H. parasuis strains and 68 genotypes in the 99 biofilm producers similar to previous results (Zhang et al., 2011). This analysis demonstrated that there were no dominant clones. Comparisons of the ERIC-PCR results of the biofilm positive strains yielded no defined correlations between serovar and ERIC-PCR profile. No correlations between biofilm formation and serovar were found in this study.

CONCLUSION
In conclusion, this study showed H. parasuis strains had high-MIC values to common antimicrobial agents in China. These showed a marked geographic variability indicating that the prudent use of antimicrobials is very important. These H. parasuis isolates were also diverse serologically and genetically and possessed with different levels of biofilmforming ability in vitro. Biofilm formation correlated with their antimicrobial susceptibility. This study suggests the need for a continuous surveillance of clinical isolates of H. parasuis.