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JOURNAL OF BACTERIOLOGY, Apr. 2009, p. 2512–2520 Vol. 191, No. 8 0021-9193/09/$08.00 0 doi:10.1128/JB.01596-08

Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Nanoscale Structural and Mechanical Properties of Nontypeable Haemophilus influenzae Biofilms

Fernando Ter ́an Arce,1* Ross Carlson,2 James Monds,3 Richard Veeh,2 Fen Z. Hu,4,5 Philip S. Stewart,2 Ratnesh Lal,1 Garth D. Ehrlich,4,5 and Recep Avci3

Center for Nanomedicine, Department of Medicine, University of Chicago, Chicago, Illinois 606371; Center for Biofilm Engineering, Montana State University, Bozeman, Montana 597172; Department of Physics, Montana State University, Bozeman, Montana 597173; Center for Genomic Sciences, Allegheny Singer Research Institute/Allegheny General Hospital, Pittsburgh, Pennsylvania 152124; and Departments of Microbiology and Immunology and Otolaryngology/Head and

Neck Surgery, Drexel University College of Medicine, Allegheny Campus, Pittsburgh, Pennsylvania 152125

Received 11 November 2008/Accepted 4 February 2009

Nontypeable Haemophilus influenzae (NTHI) bacteria are commensals in the human nasopharynx, as well as pathogens associated with a spectrum of acute and chronic infections. Two important factors that influence NTHI pathogenicity are their ability to adhere to human tissue and their ability to form biofilms. Extracellular polymeric substances (EPS) and bacterial appendages such as pili critically influence cell adhesion and intercellular cohesion during biofilm formation. Structural components in the outer cell membrane, such as lipopolysaccharides, also play a fundamental role in infection of the host organism. In spite of their impor- tance, these pathogenic factors are not yet well characterized at the nanoscale. Here, atomic force microscopy (AFM) was used in aqueous environments to visualize structural details, including probable Hif-type pili, of live NTHI bacteria at the early stages of biofilm formation. Using single-molecule AFM-based spectroscopy, the molecular elasticities of lipooligosaccharides present on NTHI cell surfaces were analyzed and compared between two strains (PittEE and PittGG) with very different pathogenicity profiles. Furthermore, the stiffness of single cells of both strains was measured and subsequently their turgor pressure was estimated.

Haemophilus influenzae is a gram-negative bacterium and a common commensal of the human nasopharynx; however, it can also be responsible for a number of serious infections (17, 28, 64, 65). H. influenzae strains are divided into two groups, according to the presence or absence of six antigenically dis- tinct extracellular polysaccharide capsules (serotypes a to f) (33, 54). In particular, organisms possessing the type b capsule are highly virulent and may cause bacteremia and invasive infections such as meningitis and pneumonia (28, 33, 65). Strains that do not possess one of six antigenically distinct capsules are classified as nontypeable H. influenzae (NTHI) and are associated with colonization in the great majority of healthy individuals (33). The NTHI strains are also associated with acute and chronic infections of the respiratory tract, such as acute otitis media (OM), chronic OM with effusion, otor- rhea, sinusitis, bronchitis and chronic obstructive pulmonary disease (28, 42, 68, 81). They are also increasingly linked to invasive diseases such as meningitis and sepsis (66, 67, 74, 75).

NTHI disease occurs when bacteria adhere to and colonize/ invade epithelial cells in the respiratory tract or invade into surrounding tissues. The initial interaction between NTHI bac- teria and the host is the adherence to epithelial cells (72, 81). Fibrillar appendages, called fimbriae and pili, promote and enhance adherence to epithelial cells and nasal tissue by using adhesins to bind to specific receptors on the host cell surface

* Corresponding author. Mailing address: Center for Nanomedi- cine, Department of Medicine (Pulmonary and Critical Care Section), University of Chicago, 5841 S Maryland Ave., I-505, Chicago, IL 60637. Phone: (773) 702-0654. Fax: (773) 702-4941. E-mail: ftarce @uchicago.edu.

(28). Pili and fimbriae are present in many gram-negative bac- teria, and besides adhesion they also perform other functions such as aiding genetic transfer between bacteria (sex pili) and generation of movement on surfaces via twitching motility (28). Many of the genes encoding these structures appear to be associated with mobile genetic elements and pathogenicity is- lands (51). Subsequent steps associated with chronic infection might include formation of microcolonies and ultimately a biofilm (11, 26, 35).

H. influenzae lipooligosaccharides (LOS) are important for colonization, bacterial persistence, and survival in the respira- tory system. The interaction between bacteria and host cells is influenced by LOS structure, which varies among strains and also among bacterial cells within a strain. In H. influenzae, LOS is comprised of an oligosaccharide, composed mainly of neu- tral hexose and heptose sugars, linked via a single 2-keto-3- deoxyoctulosonic acid to the membrane-anchoring lipid A moiety (39). To the best of our knowledge, detailed informa- tion about the LOS length distribution is not available for NTHI bacteria. H. influenzae bacteria do not produce a true poison or toxin (28). Disease results from the host cell’s re- sponse to bacterial factors, particularly endotoxin (LOS) (28).

Recent evidence suggests that H. influenzae is capable of forming mucosal biofilms in animals and human patients with middle ear infections (26, 35). Biofilms are surface-attached microbial communities with phenotypic and biochemical prop- erties distinct from those of their free-swimming, planktonic counterparts. Significantly, certain biofilms can develop anti- biotic resistance up to 1,000-fold greater than planktonic cells (21, 47).

Published ahead of print on 13 February 2009.

Fimbriae of different strains of H. influenzae bacteria have

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