Biofilm formation in Haemophilus parasuis: relationship with antibiotic resistance, serotype and genetic typing

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Abstract

Biofilms are surface-associated microbial communities, which are encased in self-synthesized extracellular environment. Biofilm formation may trigger drug resistance and inflammation, resulting in persistent infections. Haemophilus parasuis is the etiological agent of a systemic disease, Glässer's disease, characterized by fibrinous polyserositis, arthritis and meningitis in pigs. The purpose of this study was to examine the correlation between biofilm and antibiotic resistance among the clinical isolates of H. parasuis. In the present study, we tested biofilm-forming ability of 110 H. parasuis isolates from various farms using polystyrene microtiter plate assays. Seventy-three isolates of H. parasuis (66.4%) showed biofilm formation and most of them performed weak biofilm-forming ability (38/73). All isolates were tested for antimicrobial susceptibility to 18 antimicrobial agents by the broth microdilution method. H. parasuis isolates showed very high resistance (>90%) to sulfanilamide, nalidixic acid, and trimethoprim. Resistance to eight antibiotics such as penicillin (41.1% vs 8.1%), ampicillin (31.5% vs 8.1%), amoxicillin (28.8% vs 5.4%), gentamicin (46.6% vs 24.3%), cefazolin (19.2% vs 2.7%), doxycycline (19.2% vs 8.1%), cefotaxime (11% vs 2.7%), and cefaclor (13.7% vs 5.4%) was comparatively higher among biofilm producers than non-biofilm producers. Pulsed-field gel electrophoresis (PFGE) analyses could distinguish various isolates. Our data indicated that H. parasuis field isolates were able to form biofilms in vitro. In addition, biofilm positive strains had positive correlation with resistance to β-lactams antibiotics. Thus, biofilm formation may play important roles during H. parasuis infections.

Introduction

Haemophilus parasuis (H. parasuis) is the etiological agent of the Glasser's disease in pigs, a disease that affects swine worldwide and is characterized by fibrinous polyserositis, polyarthritis or meningitis (Biberstein, White, 1969, Little, 1970). So far 15 serovars have been defined (Kielstein and Rapp-Gabrielson, 1992); however, a high percentage of strains are non-typeable by serotyping (Kielstein, Rapp-Gabrielson, 1992, Turni, Blackall, 2005). Serovar diversity and the large amount of non-typeable isolates have negatively affected the development of effective cross-protective vaccines (Oliveira and Pijoan, 2004), leading to difficulties to prevent and control the clinical infections.

Biofilms are defined as communities of microorganisms that adhere to each other and to solid surfaces and are enclosed in an extracellular matrix composed of materials from both the microorganisms and the environment. The formation of biofilm by microorganisms is a mechanism that allows them to become persistent colonies, resist clearance by the host immune system, enhance resistance to antibiotics and exchange genetic materials (Donlan and Costerton, 2002). Although studies investigating in vitro biofilm formation have been performed on bacterial pathogens of veterinary importance, very few studies involving the formation of biofilms in vivo have been performed. Further research is required to investigate the factors conferring pathogenicity and to develop effective strategies to treat and prevent biofilm-associated diseases in animals (Clutterbuck et al., 2007). Jin et al. (2006) reported that most H. parasuis serovars were able to form biofilm in vitro. However, the role of biofilm formation in H. parasuis infections remained elusive. So it is necessary to study the relationship with antimicrobials, virulence, serovar and the genetic relatedness of the biofilm forms of the H. parasuis.

In this study, the ability of 110 H. parasuis field isolates in South China to form biofilms was investigated. The correlations between H. parasuis antibiotic resistance, serovar and genotype were also analyzed.

Section snippets

Bacterial strains

A total of 110 H. parasuis isolates from five provinces in South China were isolated from diseased swine suffering polyserositis, pneumonia or meningitis between August 2008 and July 2010. All field isolates in the present study were isolated from lung, brain, heart blood or thoracic, pericardial sac, peritoneum or joint fluids of euthanized swine. All samples were cultured on tryptic soy agar containing 10 mg/ml NAD (nicotinamide adenine dinucleotide) and 5% bovine serum for isolation of H.

Prevalence of biofilm formation among field isolates of H. parasuis

Different capabilities of biofilm formation for H. parasuis isolates were observed depending on their clinical origin. A total of 110 H. parasuis strains isolated from various farms were divided into four groups based upon the OD570 values of bacterial biofilm: non-adherent group, weak group, moderate group, and strongly adherent group (Table 1). Seventy-three out of 110 strains of H. parasuis (66.4%) showed biofilm formation and most of them performed weak biofilm-forming ability, counting for

Discussion

Biofilm formation is critical, not only for environmental survival but also for successful infection by numerous pathogenic bacteria. Many veterinary pathogens are capable to form biofilm, which are essential to cause persistent infections (Costerton et al., 1999). However, the significant role of biofilm formation in veterinary bacterial pathogens has been paid little attention. H. parasuis is an important swine pathogen that causes Glässer's disease. Factors involved in pathogenicity of H.

Acknowledgements

We would like to thank Professor Huanchun Chen (College of Veterinary Medicine, Huazhong Agricultural University) for providing 15 H. parasuis reference strains. This work was supported by grants from the Program for New Century Excellent Talents in University (No. NCET-06-0752), and the Guangdong Technology Planning Committee (Nos. 2006B0152, 2009A0201006 and 2009B03083050).

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    These authors contributed equally to this study and are regarded as joint first authors.

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