Diseases of the Oral Cavity
The most common oral infection, affecting 60 to 90% of school-aged children and nearly all of the adult population, is dental caries (
164). Dental caries, or tooth decay, can be defined as the breakdown of the hard tooth surfaces, including the enamel, dentin, and cementum (
92). The tooth surface becomes coated with a conditioning layer of salivary and host proteins, termed the acquired pellicle, which provides a substratum for the attachment and coaggregation of microbial species to develop into matrix-producing polymicrobial biofilm communities, commonly referred to as dental plaque () (
159). Tooth surface destruction is facilitated by the fermentation of carbohydrates, mainly sucrose and fructose, into lactic acid by several species of organisms, such as
Streptococcus mutans,
Lactobacillus acidophilus,
Actinomyces viscosus,
Nocardia spp., and
Candida albicans (
106,
138). The tooth surface is constantly undergoing demineralization and remineralization, a process that can only occur homeostatically at a pH of >5.5. Microbially derived lactate drives down the microenvironmental tooth surface pH and significantly slows the remineralization process, leading to demineralized surfaces that become weakened and subsequently decayed (
128). Saini et al. examined bacterial isolates from healthy individuals and those with deep-seated dental caries and found that 100% of all cariogenic samples harbored a polymicrobial infection; it was also noted that the microbial populations of healthy teeth shifted from being dominated by Gram-positive anaerobic cocci to being dominated by Gram-positive anaerobic bacilli (
183). Furthermore, the ability of
C. albicans to cocolonize with streptococci and to grow and survive at low pH (<4.5) suggests that
C. albicans may colonize sites of active caries formation (
33,
101). In fact, evidence shows that there is a higher incidence of
Candida spp. in patient populations with higher levels of susceptibility to caries (
48). As described above, the mechanisms of coaggregation of streptococci and
C. albicans may help exacerbate caries development.
Several recent studies using culture-independent techniques have more comprehensively uncovered the polymicrobial nature of dental caries. Li et al. used denaturing gradient gel electrophoresis (DGGE) to assess microbial population shifts specific to the cariogenic state and identified increased
S. mutans colonization to be associated with active dental caries compared to healthy controls (
119). There were also several DGGE bands that were of higher abundance during the caries-free states. However, these bands were not sequenced for further analysis. Additional DGGE profiling work by Li et al. did show differences in genera isolated from caries in early childhood and from caries-free children, suggesting cariogenic roles for
Fusobacterium and
Neisseria and protective roles for
Bacteroidetes,
Treponema,
Prevotella, and
Corynebacterium (
118). 16S rRNA sequencing analysis of the oral microbiota from teeth of cariogenic and healthy children attributed a role in caries formation to several bacterial species:
S. sanguinis was correlated with health, while
Actinomyces gerencseriae,
Bifidobacterium,
S. mutans,
Veillonella,
Streptococcus salivarius,
Streptococcus constellatus,
Streptococcus parasanguinis, and
Lactobacillus fermentum (listed in order of decreasing community numbers) were associated with caries formation (
18). Interestingly,
Veillonella was originally thought to be associated with health due to its ability to readily degrade lactate and succinate, thereby negating the effects of
S. mutans caries-associated lactate production (
99). However, Arif et al. demonstrated that only certain
Veillonella species exhibited this protective effect (
6). Surprisingly, 16S rRNA sequencing of clone libraries generated from cariogenic and healthy tooth root plaques of older individuals differed still (
169). Healthy individuals showed higher numbers of
F. nucleatum,
Leptotrichia,
Selenomonas noxia,
Streptococcus cristatus, and
Kingella oralis bacteria, while cariogenic samples demonstrated increased numbers of
Actinomyces, lactobacillus,
S. mutans,
Enterococcus faecalis,
Atopobium,
Pseudoramibacter, and
Propionibacterium bacteria. While culture-dependent techniques accurately predicted a role for
S. mutans in caries formation, culture-independent methods have elucidated a much greater complexity of microbial compositions promoting cariogenesis. Combined, these data suggest that interactions between microbes and species composition may significantly affect the predisposition to dental caries or disease outcomes.
Denture stomatitis is another well-studied example of a polymicrobial biofilm-mediated oral disease. Denture stomatitis refers to erythema and edema of the soft palate and tissues of the oral cavity that are in close contact with the denture surface, as prosthetic placement restricts the lubrication achieved by normal salivary flow; redness, swelling, and burning sensations on the upper palate are most commonly caused by infections with the fungus
C. albicans (
219). A recent 16S ribosomal DNA (rDNA) survey and amplification of the fungal 18S-28S internal transcribed spacer region of biofilm material recovered from the palates of 10 denture stomatitis subjects as well as 10 healthy controls demonstrated the presence of
Candida spp. in all biofilms (
28). However, that study also identified 82 species of bacteria in both subject groups: 27 that were common to both groups, 29 that were specific to denture stomatitis, and 26 that were specific to healthy controls. Therefore, specific bacterial populations may exacerbate
Candida-induced denture stomatitis. Further studies by Baena-Monroy et al. examined saliva and culture swabs of denture surfaces from 105 subjects fitted with dentures (
12). Using culturing techniques, it was found that
C. albicans and
S. aureus could be recovered from the oral mucosa and denture surfaces of both denture stomatitis patients and healthy controls. However, more
C. albicans cells were recovered on the denture surface, while
S. aureus was found in the oral mucosa of denture stomatitis cases, suggesting that
C. albicans may facilitate colonization by
S. aureus, enabling the staphylococci to mediate the inflammation of host tissue and bacterial superinfection.
Indeed, unique associations between
C. albicans and
S. aureus in several different
in vivo and
in vitro models have been described. A series of experiments by Carlson demonstrated the ability of
C. albicans to decrease the 50% lethal dose (LD
50) of
S. aureus 200- to 70,000-fold when coinoculated into the peritoneal cavity of immunocompetent mice (
29). More strikingly, the coinfection of mice with sublethal monomicrobial infectious doses resulted in 100% mortality (
30). Quantitative CFU counts from organ homogenates demonstrated the systemic spread of staphylococci to various organs, including a polymicrobial-induced increase in staphylococcal loads in kidney, liver, pancreas, and spleen. Histological analysis revealed that staphylococci could always be found associated within the fungal growth rather than at the periphery, and it was hypothesized that this coassociation somehow protected the bacteria. In addition, when these pathogens were injected at proximal but nonoverlapping sites, the bacteria always associated at the fungal interface and not vice versa. Decades later, work by Harriott and Noverr showed that
C. albicans and
S. aureus could form polymicrobial biofilms
in vitro and that
S. aureus could be protected from vancomycin treatment, presumably through encapsulation by the
C. albicans biofilm matrix (
82). Peters et al. demonstrated that
S. aureus preferentially binds to the hyphal form of
C. albicans during polymicrobial biofilm growth and that this association is nonantagonistic (). By use of two-dimensional in-gel electrophoresis, it was also shown that metabolic, stress, and virulence factor protein expressions could be significantly altered due specifically to polymicrobial growth conditions (
163). Therefore, based on findings with these disparate model systems, the colonization of the denture surface with
C. albicans may promote the deposition of other bacterial species (or vice versa) and enable specific bacterial populations to uniquely manipulate the microenvironment via direct or cooperative efforts, leading to exacerbated disease.
Periodontitis may be the most classically defined polymicrobial biofilm-mediated disease of the oral cavity and results from a chronic inflammation of the gums that leads to the damage of structural tooth support, resorption of bone, and eventual tooth loss (
189). Symptoms of the disease include inflamed gum tissue, bleeding from the gums, gingival recession, deep-pocket formation between the gum and tooth surface, and loose teeth. Aside from dental caries, periodontitis is considered to be the second most common cause of worldwide infectious diseases, affecting nearly half of the U.S. population alone (
165). Culture-dependent analyses of biofilm plaques found on inflamed gum surfaces demonstrated that periodontal patients could be categorized into two unique disease factions, “adult periodontitis” (patients >35 years old) and a rapidly progressing form of “early-onset periodontitis” (patients <35 years old) (
68). Organisms thought to be involved in adult periodontitis included
Porphyromonas gingivalis,
Treponema denticola, and
Tannerella forsythia, while early-onset periodontitis more commonly involved
Aggregatibacter actinomycetemcomitans, which was often coisolated with
P. gingivalis,
Prevotella intermedia, and
Capnocytophaga sputigena in the periodontal plaque. However, the increased use of culture-independent techniques has led to the finding that the microbial compositions identified for each periodontal disease type were not as clearly defined as previously thought (
8). It was determined that healthy individuals often harbored these periodontal “pathogens” in low numbers as part of the normal flora, without any signs of overt disease; differences in virulence factor expressions of individual strains may partly explain this discrepancy (
85,
112). Moreover, many cases of periodontitis were mediated in the absence of the major contributing species formerly believed to facilitate disease onset (
139). Thus, the nonvalidated age-dependent terms “early-onset” and “adult” peritonitis were more broadly defined as “localized aggressive” and “chronic” peritonitis, respectively (
8). One interesting observation resulting from culture-independent analyses of the subgingival microbiota revealed the presence of
Archaea in subgingival pockets (
115). Archaea are prokaryotes that physically resemble bacteria but are more genetically similar in their 16S rRNA sequences to eukaryotes. Indeed, as a function of progressing disease, increasing numbers of
Archaea (including
Methanobrevibacter oralis) can be isolated from subgingival pockets. Despite the lack of any direct cause-effect
in vivo studies assessing the role of
Archaea in periodontitis, strikingly, these organisms have never been found in the subgingival microflora of healthy individuals or at healthy sites in patients with periodontitis (
115,
116). The difference between the onsets of chronic and localized aggressive forms of periodontitis is unclear at this time but is thought to result from a host genetic predisposition to colonization, a hyperaggressive immune response to oral bacteria involving increased levels of polymorphonuclear infiltrate, differences mediated by the microbial composition itself, or a combination of these effectors (
44,
58,
84).
While not a comprehensive analysis of the microbial interactions governing periodontal infections
in vivo, several
in vitro studies have revealed interesting associations, with possible pathogenic ramifications.
F. nucleatum, a filamentous Gram-negative anaerobic bacterium, supports
P. gingivalis during polymicrobial biofilm growth, leading to synergistic increases in biomass (
186). Furthermore,
F. nucleatum was shown to increase the penetration of
P. gingivalis during coinfection of human gingival epithelial cells
in vitro via an undefined mechanism (
185). While it is unclear at this time, invasive
P. gingivalis-mediated human periodontitis may be enhanced by interactions with
F. nucleatum. Additional studies have examined the fungal colonization of the periodontal pocket and have found that only
C. albicans, a filamentous fungal species, is harbored in both the chronic and localized aggressive forms of periodontitis (
209). It is interesting to hypothesize whether
P. gingivalis virulence is enhanced in the presence of invasive microbial species. However, comprehensive culturing and limited sequencing analyses of microbiotas obtained from periodontitis patients and healthy subjects have not yet defined these clinical polymicrobial associations.
Taken together, these diseases represent the diversity and complexity of oral polymicrobial biofilm communities and highlight the unique associations that lead to complex disease phenotypes. Although the oral cavity has been the most extensively researched polymicrobial environment, significant advancements in the mechanistic explanations of clinically relevant coassociations and the development of in vivo model systems to test these hypotheses are critical for developing therapeutic strategies against oral polymicrobial biofilm infections.
Otitis Media
Otitis media (OM), a common childhood disease, is an infection of the middle ear, often involving the Eustachian tube between the tympanic membrane and the inner ear (
191). While rarely associated with mortality, OM symptoms often include ear pain, fever, and middle ear effusion. In severe cases, a perforation of the tympanic membrane can result in purulent discharge from active chronic infection. Most cases of OM spontaneously resolve within a few weeks. However, chronic infections can lead to partial or total hearing loss (
105). The microbial species responsible for the vast majority of OM are the normally commensal bacterial species
Streptococcus pneumoniae, nontypeable
Haemophilus influenzae (NTHi), and
Moraxella catarrhalis along with upper respiratory viruses, including influenza A virus, respiratory syncytial virus (RSV), adenoviruses, and human rhinovirus (
142). Not only can these pathogens cause significant morbidity during monomicrobial infection, they also are often coassociated as polymicrobial biofilm complexes, where combinations of pathogens enhance disease or predispose the host to colonization by coinfecting microbes. In fact, the increased use of culture-independent techniques suggests a very diverse microbial etiology of OM pathogenesis (
114). It is important to note that during commensal colonization, none of the aforementioned bacterial species cause inflammation or breach epithelial barriers; it is only during times of epithelial rupture or immune dysfunction that they cause infection.
Interestingly, it appears that upper respiratory viral infections predispose the host to and enhance OM pathogenesis (
81). It has been well noted that viral infection of certain cell types enhances the bacterial colonization of the cellular surface () (
80,
102,
187). Preinfection with various viruses changes the physiological properties of infected airways, alters susceptibility to antibiotics, and modulates the innate and adaptive immune responses resulting in proinflammatory signaling events (
2,
32,
38,
217). In fact, experimental human challenge with influenza A virus leads to increases in the isolation of
S. pneumoniae and
S. aureus from the nasopharynges. Also, viral preinfection has been correlated with OM in children (
160,
214).
Giebink et al. used an animal model of OM in which chinchillas were infected with influenza A virus and subsequent pressure and inflammation of the middle ear were monitored (
69). Viral infection alone led to increases in negative pressure, inflammation, and epithelial damage in the Eustachian tube proximal to the nasopharynx; these symptoms closely mimic those of human infections and demonstrate the ability of viral infection to enhance OM pathology. In order to more closely mimic the polymicrobial nature of human disease, Krishnamurthy et al. utilized a mouse nasal colonization model of infection and pretreated animals with Sendai virus prior to inoculation with bacterial species (
110). Viral pretreatment led to significant increases in bacterial burdens regardless of the coinfecting bacterial strain. When
S. pneumoniae and
M. catarrhalis were used together as infecting species,
S. pneumoniae was the most abundant bacterial species present during experimental OM. However,
M. catarrhalis significantly increased the pneumococcal OM bacterial load, incidence, and time to infection. NTHi had a similar effect on
S. pneumoniae during coinfection but to a lesser degree. In all cases of polymicrobial disease, inflammatory responses peaked at day 1 postinfection, but in monomicrobial infections, inflammatory markers were not maximally upregulated until day 3. Recently, a chinchilla model of coinfection using
M. catarrhalis and NTHi showed that polymicrobial infection resulted in increased microbial resistance to antibiotic treatment and clearance from the host () (
7).
In vitro imaging revealed a physical coassociation of these two pathogens and a synergistic increase in the biofilm mass during polymicrobial growth. Interestingly, an NTHi
luxS mutant deficient in the production of the quorum-sensing molecule autoinducer-2 could no longer induce immunoavoidance or antimicrobial resistance mechanisms in
M. catarrhalis. These data provide evidence for interspecies microbial cross talk via quorum-sensing communication to enhance infection during polymicrobial biofilm-mediated OM.
Viral infection of the upper respiratory tract can lead to immunological changes that further enhance OM pathogenesis. Antimicrobial peptides, including defensins, lysozyme, lactoferrins, and surfactants A and D, are secreted in the middle ear and have been shown to be effective at clearing OM pathogens at micromolar concentrations (
120). McGillivary et al. demonstrated that infection of chinchilla respiratory epithelial cells
in vitro with RSV led to significant decreases in the levels of transcription of the innate antimicrobial peptide chinchilla beta-defensin 1 (cBD1), of which human beta-defensin 3 (hBD3) is an ortholog (
133). Furthermore,
in vivo RSV infection of the chinchilla nasopharynx led to nearly a 40% decrease in cBD1 transcript levels when matched to those of mock-infected controls. Consequently,
in vivo coinfections with NTHi and RSV resulted in a 10- to 100-fold increase in the level of NTHi recovered from nasopharyngeal lavage fluids of infected chinchillas compared to those infected with NTHi alone. Therefore, the modulation of innate immune effectors, such as defensins, by viral respiratory pathogens may predispose the host to more severe infection by OM pathogens.
In summary, these studies, although limited by culture-dependent techniques, identified the major microbes involved in the pathogenesis of OM and demonstrated the importance of polymicrobial interactions and cooperative viral and bacterial effects during disease predisposition and progression. Further work on a more comprehensive disease model utilizing S. pneumoniae, M. catarrhalis, NTHi, and an upper respiratory virus in concert would be extremely useful in mimicking in vivo infection and determining the potential roles that each microbe assumes during polymicrobial biofilm growth. Moreover, an increased surveillance of potential pathogenic microbial associations, by use of culture-independent techniques, will further enhance our understanding of OM pathogenesis.
Diabetic Foot Wound Infections
Diabetes mellitus, commonly referred to as diabetes, is a disease involving defects in the bodily response to insulin and affects nearly 6.4% of the population worldwide; this number is expected to double by the year 2030 and has prompted the Centers for Disease Control and Prevention (CDC) to classify diabetes as a current epidemic (
61). Diabetes can manifest itself in several forms. However, two of the most common forms are type 1 and type 2 (
3). Type 1 diabetes is caused by a lack of insulin production and is thought to be caused by the autoimmune-mediated destruction of pancreatic beta-cells, which secrete insulin. Type 2 diabetes refers to a group of metabolic disorders characterized by various levels of insulin resistance suspected to be caused by defects in or the expression of the insulin receptor (
107). In addition, impaired insulin secretion and increased hepatic glucose production are also implicated. Type 2 diabetes is thought to be mediated by genetic and environmental triggers, including obesity, diet, low activity level, and high blood pressure. The effects of low levels of insulin production or insensitivity to insulin result in high glucose blood levels, osmotic imbalance, dehydration of body tissues, and, if not treated properly, eventual organ damage (
3).
Another damaging effect of high glucose levels mediated by an insulin deficiency or resistance is the development of peripheral neuropathy and poor blood circulation, especially in extremities such as the hands and feet. Combined, these symptoms predispose diabetic individuals to an increased risk of infection, and if not identified or treated early, these infections may fulminate into chronic ulcerating polymicrobial biofilm-mediated wounds that often do not resolve with standard therapies and result in eventual limb amputation (
222). The development of such ulcers is often the synergy of two contributing factors, e.g., decreased neurosensory perception and stepping on a sharp object; it is this critical breach of the epithelial surface, coupled with neurological abnormalities, cardiovascular aberrations, and immune dysfunction, which facilitates polymicrobial colonization and subsequent pathogenesis. Due to the inability of subjects with this disease to feel cuts and irritations on visually obscured areas of the feet, these infections often go unnoticed and progress to more serious illness (
21). While not only extremely uncomfortable and cumbersome for the patient, diabetic foot wound infections also contribute to significant yearly costs of medical care, as these infections often require several rounds of therapeutic treatment and surgical debridement. In 1997, average inpatient costs for lower limb complications were $16,580 for foot ulcers, $25,241 for toe or toe and other distal amputations, and $31,436.33 for major amputations; the total estimated yearly costs are in the billions (
172).
As stated above, diabetic foot wounds are often mediated by a mixture of several species of microbes coexisting as complex biofilm communities. A large multicenter analysis of 454 individual diabetic foot wound infection swabs and aspirates resulted in the identification of over 1,600 organisms by aerobic and anaerobic culturing techniques (
40). Interestingly, of the specimens tested, 48.9% were infected with aerobic bacteria only, 1.3% were infected with anaerobic bacteria only, and 43.9% contained a mixture of aerobic and anaerobic bacteria. Bacterial growth was not identified in 5.9% of the samples. Of the positive cultures identified, 16.2% harbored one bacterial isolate, 20.4% contained two bacterial isolates, 19.7% had three bacterial isolates, 13.3% demonstrated four bacterial isolates, and 30.4% supported the growth of five or more bacterial isolates. Of these, the most abundant aerobic isolates recovered were
Corynebacterium spp.,
Enterococcus spp.,
Escherichia coli,
Staphylococcus epidermidis, and
S. aureus; among the most commonly isolated anaerobic bacteria were
Fusobacterium spp.,
Porphyromonas spp.,
Prevotella spp.,
Bacteroides spp., and
Clostridium spp. A smaller-scale study using DGGE, 16S rRNA gene sequencing techniques, and microscopy to examine debrided tissues from diabetic foot wounds resulted in the identification of highly polymicrobial communities and the detection of several species unidentifiable by standard culturing techniques (
98). Notably, DGGE analysis demonstrated the presence of several unique bands (corresponding to unique species) for each sample tested, and banding patterns differed between individual samples. While no distinct relationships between coisolated organisms can be derived from these analyses, these studies demonstrate the tremendous diversity in microbial composition and the true polymicrobial nature of diabetic foot wound diseases. As of now, it is unclear whether diabetic foot wounds arise from specific combinations of pathogens or if a simple increase in the microbial loads of any opportunistic microbes can sustain infection (
67).
Another potential mechanism for the chronic infection of diabetic foot wounds is the inability of wound healing to proceed properly. Diabetic patients have been shown to have several defects in wound healing, including decreases or impairments in angiogenic factors, growth factors, epidermal barrier function, fibroblast migration, and macrophage function (
59,
65,
66,
130). Specifically, at the edge of diabetic foot wound infections, keratinocytes demonstrate an absence of migration and the inability to differentiate completely, while unaffected cells from adjacent sites appear normal but are physiologically impaired. However, these “normal” cells can become functional by the addition of specific growth factors (
201). A recent study by Schierle et al. showed for the first time that murine cutaneous wounds cannot be resolved by the host with infection by biofilm-forming
S. aureus strains (
190). Remarkably, when a
traP S. aureus mutant defective in biofilm formation or biofilm-inhibiting peptides was used, the wound healed normally. This finding suggests that biofilm formation may specifically inhibit wound-healing mechanisms during infection in the host. Because
S. aureus is one of the bacterial strains most commonly isolated from diabetic foot wound infections, its ability to modulate wound healing and avert localized immunity may enable other microbes to colonize the wound and exacerbate disease (
22).
While there has not been an extensive development of
in vitro or
in vivo modeling systems to study the polymicrobial environment of diabetic foot wound infections, there has been some recent progress. Sun et al. devised an
in vitro system, dubbed the Lubbock chronic wound biofilm model, consisting of a series of glass tubes filled with appropriate media (replete with damaged tissue, red blood cells, and plasma) to support multispecies growth and abiotic plastic surfaces to enhance biofilm formation (
203). Both microscopically and macroscopically, the resulting polymicrobial growth strikingly resembles chronic diabetic foot wound biopsy specimens (). This model has extreme usefulness for the rapid screening of compounds for antimicrobial efficacy against diabetic foot wound infections during the growth of mixed microbial populations. Perhaps one of the most important studies demonstrating the effects of polymicrobial infection in a mouse model of type 2 diabetes was performed by Mastropaolo et al. Leptin receptor-deficient mice (BKS.Cg-
m+/+ Leprdb/J) were injected into the inner thigh with either
E. coli alone,
Bacteroides fragilis alone,
Clostridium perfringens alone, all combinations of two of the aforementioned pathogens, or all pathogens simultaneously. Infected abscesses were removed at several time points and assayed for microbiological enumeration (
131). It was found that injection with all three bacteria simultaneously led to the highest rate of mortality. When combinations of two pathogens were used, certain microbial pairings resulted in vast differences:
E. coli bestowed strong synergy to
B. fragilis but not to
C. perfringens during early infection (day 1),
B. fragilis and
C. perfringens provided moderate synergy but only in infections of young mice, and
B. fragilis reacted antagonistically during polymicrobial growth with
E. coli at later time points during infection (day 22). Most importantly, when age-matched nondiabetic C57BLKS/J mice were infected with these pathogens, they harbored anywhere from 5- to 35-fold-fewer bacterial CFU than their diabetic counterparts, demonstrating the contribution of the diabetic state to the severity of the polymicrobial infection. While only speculative at this time, the progression of human diabetic foot wound infections may be partially dependent on the species initially colonizing the wound. Furthermore, archived clinical data should be analyzed, or new studies aimed at determining polymicrobial compositions that predict disease severity or worsen disease outcome should be undertaken.
Infection of the Cystic Fibrosis Lung
Cystic fibrosis (CF) is an autosomal recessive genetic disorder, most common among Caucasians, and is caused by an inherited mutation in a specific chloride ion channel named the cystic fibrosis transmembrane conductance regulator (CFTR) (
47). Mutations in phenylalanine residue 508 of the CFTR are responsible for the majority of cystic fibrosis cases; this precise mutation leads to improper protein folding and subsequent cellular degradation. However, there are nearly 1,900 documented mutations associated with the disease (
http://genet.sickkids.on.ca/app). Regardless of the source of genetic polymorphism, the lack of functional CFTR molecules on the surface of mucosal tissues severely affects the production of sweat, components of the digestive juices, and mucous composition, as imbalances in chloride ion secretion outside the cell lead to cationic influx, osmotic imbalance, and eventual dehydration (
135). Early symptoms of cystic fibrosis are increased susceptibility to lung infections, persistent coughing, and heightened sputum production. As infecting microbes fail to be cleared, uncontrolled inflammation begins to cause permanent damage to the lung architecture, resulting in bronchiectasis, pulmonary hypertension, and hypoxia (
19). End-stage cystic fibrosis requires the use of positive-pressure air masks or ventilators to mechanically assist breathing. Besides destructive effects on the airway, cystic fibrosis can also lead to nutrient loss by the progressive scarring of the pancreas, which becomes dehydrated in a fashion similar to that of the lungs (
54).
One of the most notable features of CF is the loss of normal mucociliary clearance, resulting in extensive mucous buildup and excessive lung inflammation. Not only is the mucous unable to be cleared efficiently, dehydration results in thick and adhesive secretions; these conditions are ideal for supporting robust bacterial growth (
140). Individuals with functional CFTR receptors inhale potentially pathogenic microbes on a routine basis, but they are efficiently trapped in the respiratory tract and eliminated by the mucociliary escalator. However, individuals suffering from cystic fibrosis are unable to clear such organisms, and these organisms often develop into polymicrobial biofilm-mediated infections in the lower airways (
194). Bacterial lung infection still remains the primary cause of morbidity and mortality in CF patients. Some of the most common isolates from human bronchiolar lavage samples of CF patients are
Pseudomonas aeruginosa,
S. aureus,
Streptococcus milleri group (SMG) pathogens,
Burkholderia cepacia,
Stenotrophomonas maltophilia,
H. influenzae, and
C. albicans. However, this list ignores members of the normal flora that are not regularly screened in the clinical laboratory (
143,
179,
182,
194). Several interesting findings and trends from coinfections with several of these pathogens will be discussed.
Due to undergoing repeated rounds of antimicrobial therapy and relatively high levels of colonization in the nasopharynx,
C. albicans is often found in the lower airways of individuals with cystic fibrosis (
39).
C. albicans, a dimorphic yeast, can switch from a usually commensally associated round yeast form to an elongated hyphal form, a transition central to its pathogenesis. These hyphae are important for the progression of biofilm formation and are also needed to puncture the epithelial and endothelial layers to gain access to deep tissue (
100,
175). A series of eloquent experiments demonstrated that
P. aeruginosa, one of the organisms most often cultured from the lower airways of infected CF patients, is able to inhibit candidal germination by the secretion of the molecule 3-oxo-C
12 homoserine lactone (
89). Furthermore,
P. aeruginosa can attach to localized areas of the hyphal surface and induce cell lysis, thereby effectively killing the hyphal cell, but are unable to attach to or kill the round yeast form of
C. albicans (); mutants lacking the production of this quorum-sensing molecule were unable to propagate hyphal lysis (
88). Although the molecules that enable pseudomonal binding to hyphal surfaces were not entirely elucidated, work by Brand et al. demonstrated that binding requires both contact dependence and soluble factors (
24).
P. aeruginosa culture supernatants were capable of inducing lysis independent of cellular contact, but cocultured cells displayed only localized points of
C. albicans cell wall weakening. It was also noted that
C. albicans mutants with severely truncated O-linked, but not N-linked, cell wall glycans were hypersusceptible to killing by
P. aeruginosa. The truncation of fungal O-linked glycans may cause cell wall rearrangements that are more amenable to
P. aeruginosa binding or result in the exposure of high-affinity binding sites.
However, this association is not unilaterally antagonistic.
C. albicans produces the quorum-sensing molecule farnesol, a sesquiterpene alcohol, which inhibits the transition of yeast to hyphae. As discovered by Cugini et al., farnesol is toxic to
P. aeruginosa by significantly downregulating the transcription of the
pqsA gene, an important mediator of the expression of the pseudomonal iron-scavenging virulence factor pyocyanin (
43). Interestingly, pyocyanin production is upregulated when
P. aeruginosa is cultivated in sputum from CF patients compared to that of growth in standard culture (
156). Pyocyanin is also toxic to
C. albicans. Therefore, the secretion of farnesol may be a protective mechanism to reduce pyocyanin levels during the intimate cocolonization of the CF lung (
103). Farnesol secretion has also been shown to inhibit swarming motility in
P. aeruginosa; this may slow the initial deposition of pseudomonads onto the hyphal surface and result in less lytic activity (
132).
Another clinically relevant polymicrobial association in the CF lung is that which exists between
S. aureus and
P. aeruginosa. Liou et al. analyzed data obtained from 5,820 randomly selected patients to develop a multivariate 5-year survivorship model and to partially identify predictors of disease outcomes (
122). While the model accurately predicted several known risk factors for CF disease, such as decreased forced expiry volume, increased age, and decreased weight, one interesting result was uncovered: infection with
S. aureus resulted in increased survivorship over a 5-year period, independent of age, while patients infected with
B. cepacia were predicted to have the worst outcome. In fact, it has been shown that CF patients often initially become colonized early with
S. aureus or
H. influenzae, followed by a lengthy colonization with
P. aeruginosa, and are then terminally colonized with
B. cepacia (
83). The reason for the increased 5-year survivorship associated with
S. aureus infection is unknown, but it was hypothesized that chronic low-level lung inflammation induced by
S. aureus may be partially protective against
P. aeruginosa colonization and progressive disease. It could also be due to the staphylococcal production of an unidentified pseudomonicidal compound.
While potentially helpful for the CF patient, infection with
S. aureus assumes both antagonistic and beneficial roles when cocultured with
P. aeruginosa. During
in vitro polymicrobial growth in CF sputum,
S. aureus growth is repressed by
P. aeruginosa within 5 h (
156). This growth inhibition could be due to the production of staphylolytic secreted compounds or 2-heptyl-4-hydroxyquinoline
N-oxide (HQNO) (
125). Hoffman et al. demonstrated that the coculturing of
S. aureus and
P. aeruginosa protected staphylococci from killing by the antibiotic tobramycin via the temporary inhibition of staphylococcal respiration, thereby impeding rapid growth (
87). Incubation and the prolonged exposure of
S. aureus to purified HQNO led to the development and recovery of small-colony-variant (SCV) phenotypes. SCVs are often morphologically and phenotypically distinct from their parental generations, with the most common change being defects in the electron transport chain. Therefore, exposure to respiratory inhibitors, such as HQNO, naturally selects for clones that are inherently resistant to such compounds. Because SCV phenotypes do not respond to commonly used antibiotics, they can serve as sources of chronic infection. Indeed, HQNO was found to be present in the airways of the CF lung but not in those of matched healthy controls, which may partially explain the relatively high incidence of SCV identification in CF patients. Additionally, Duan et al. used a rat lung infection model to demonstrate that the coinfection of
P. aeruginosa with both
Staphylococcus spp. and
Streptococcus spp. resulted in increased lung histopathology. In addition, a luciferase reporter promoter library screen showed the modulation of
P. aeruginosa virulence and metabolic genes during coculture (
56). Although not fully elucidated, clearly,
S. aureus and
P. aeruginosa have the potential to serve both antagonistic and synergistic roles during polymicrobial infection of the CF lung.
Burkholderia cepacia complex infection, consisting of several phenotypically indistinguishable genomovars, is often associated with high mortality rates in CF patients (
49). The coinfection of
B. cepacia with
P. aeruginosa was reported to result in a more rapid decline in pulmonary function and worse clinical outcomes (
97). Preliminary
in vitro experiments have shown that coculture with
P. aeruginosa upregulates putative burkholderial virulence factors and increases epithelial adhesion (
134). Using the Calgary biofilm device (CBD), Tomlin et al. demonstrated the ability of
B. cepacia and CF
P. aeruginosa isolates to form mixed polymicrobial biofilm communities. However, when pseudomonal laboratory strain PAO1 was used,
B. cepacia was not incorporated into the developing biofilm (
205). The detection of copious pyocyanin production in strain PAO1 was found to inhibit
B. cepacia growth. By utilizing green fluorescent protein (GFP)-based sensor plasmids capable of recognizing specific
N-acylhomoserine-lactone quorum-sensing molecules, Riedel et al. determined that
B. cepacia could respond to quorum-sensing signals of
P. aeruginosa both
in vitro and during
in vivo infection of mouse lungs, but this chemically mediated exchange was not reciprocated by
P. aeruginosa (
177). Unfortunately, the target effectors of this quorum-sensing cross talk have not yet been identified, requiring the need for further studies of this interesting and clinically relevant polymicrobial interaction.
Members of the SMG, composed of
Streptococcus constellatus,
Streptococcus intermedius, and
Streptococcus anginosus, common members of the airway microflora, have recently been identified as clinically relevant causes of pulmonary exacerbations in CF patients (
193). The coinfection of rat lungs (agar-bead model of CF) with streptococci and
P. aeruginosa led to increased microbial burden and percent lung damage compared to infection with either microbe alone. Importantly,
in vitro studies using a promoter-reporter plasmid harboring the
luxABCDE cassette revealed that several
P. aeruginosa genes, including virulence factors and drug efflux pumps, could be differentially expressed during coculture with streptococcal flora (
56). Furthermore, it was demonstrated that some of these same gene modulations could be mediated by the quorum-sensing molecule AI-2, which can accumulate to high levels in the sputum of CF patients, suggesting that intermicrobial communication between microbes of the “normal” flora may exacerbate CF disease caused by classical pathogens. A culture-independent analysis of a small cohort of CF patients revealed that SMG organisms not only were found during episodes of CF airway exacerbations but also were the numerically dominant species present (
195). While the virulence mechanisms of SMG are unclear at this time, future studies should determine whether the increased infectious burden, the quorum-sensing-enhanced virulence of other species present, or a combination of these effects results in enhanced CF disease.
Tunney and colleagues recently reported stark differences between the composition of anaerobic species found in CF patients and that of anaerobic species found in healthy controls by using anaerobic culturing techniques (
206). Sputum samples from adult CF patients contained increased numbers and various compositions of anaerobic organisms, including
Prevotella,
Veillonella,
Propionibacterium, and
Actinomyces. A similar composition of microbial isolates was obtained from the bronchoalveolar lavage fluid of pediatric patients. Interestingly, the identification of
P. aeruginosa positively predicted increased numbers of anaerobic bacteria in the sputum. Later studies using culture-independent techniques confirmed previous findings but revealed an even more diverse composition of anaerobic flora, with significant patient-to-patient variation (
207). Despite the microbial community composition remaining relatively stable, it was shown that antimicrobial therapy resulted in a decreased overall microbial burden that was more pronounced in aerobic than in anaerobic flora. If anaerobic flora do indeed play a critical role in mediating the severity of disease, current antimicrobial therapies may be ineffective at targeting the reduction of this crucial group of organisms.
While not classically identified as confounding causes of CF chronic infection, the presence of a variety of viruses infecting the upper respiratory tract results in an exacerbation of CF symptoms. Mice coinfected intranasally with RSV and
P. aeruginosa developed more severe disease than mice infected with
P. aeruginosa alone (
51). Even more drastic was the nearly 2,000-fold increase in CFU counts from lung homogenates after coinfection compared to monomicrobial infection. Differences in inflammatory mediators were negligible between the two groups, except that coinfection slightly induced the expression of MIG (monokine induced by gamma interferon), a T-cell chemoattractant. Van Ewijk et al. showed that preinfection and coinfection of epithelial cell monolayers with RSV strongly increased the adherence of
P. aeruginosa to the epithelial surface; a direct binding of
P. aeruginosa and RSV virus particles was also observed (
210). Therefore, viral particles may serve as a bridge between contact points on the bacterial and host surfaces. Similarly, using PCR detection with nasal swabs, Wat et al. showed that children afflicted with CF at the time of temporary exacerbated pathology were colonized with an upper respiratory virus 46% of the time, compared to unexacerbated controls (16.9%); rhinovirus, influenza A virus, and influenza B virus were the most commonly isolated viruses (
218). In support of these findings, Oliver et al. showed that
in vitro infection of alveolar macrophages with rhinovirus resulted in a replication-dependent release of tumor necrosis factor alpha (TNF-α) and the neutrophil chemokine interleukin-8 (IL-8) (
152). However, the preinfection of alveolar macrophages with rhinovirus which were then challenged with either lipopolysaccharide or lipoteichoic acid, major constituents of bacterial Gram-negative and Gram-positive cell walls, respectively, failed to induce the secretion of TNF-α or IL-8. Therefore, viral coinfection with bacterial pathogens may lead to increased colonization or immunomodulatory effects that repress bacterial clearance mechanisms in the CF lung.
The overproduction of mucous and reduced ciliary clearance predispose the CF lung to infection with a variety of organisms. While not fully appreciated, the clinical significance of microbial associations and polymicrobial infection in the lower airways will lead to a better understanding of CF disease progression and selective therapies to eradicate potentially hypervirulent pathogenic microbial compositions.
Parenteral Nutrition Feeding Tubes
While the polymicrobial diseases discussed above are commonly associated with biofilm formation on human mucosal tissues or epidermal layers, an overwhelming number of human polymicrobial diseases are propagated on abiotic surfaces, such as intravenous and urinary catheters, cosmetic and cochlear implants, stents, artificial lenses, internal nondissolving stitches, tympanostomy tubes, artificial heart valves, ventilator tubes, cerebrospinal shunts, pacemakers, and orthopedic devices, including prostheses, intramedullary rods, external fixation devices, screws, and plates (
113,
146,
180). These infected implants may serve as sources of chronic infection and can potentially serve as a source of inoculation into the bloodstream, leading to sepsis. One such example of an implanted medical device-related polymicrobial biofilm infection is the biofilm contamination of parenteral nutrition feeding tubes.
Patients unable to masticate—due in most cases to neurological or pharyngeal disease—may require nutritional support for extended periods. For such individuals, enteral nutrition (EN) is preferred over the parenteral route because it is both associated with a lower risk of serious sequelae and more physiologically relevant in that it preserves the barrier, absorptive, and immunological functions of the gut (
31,
94,
117,
226). Percutaneous endoscopic gastrostomy (PEG) tube feeding involves the delivery of nutrients via a silicone tube directly into the stomach and is usually begun after patients have been received EN nasogastrically (NG). PEG feeding is preferred over NG feeding because NG tubes are uncomfortable and easily displaced by the patient (
202). Either type of EN bypasses many of the mechanisms preventing microbial colonization of the upper gut, and the feeding tube itself acts both as a conduit through which microorganisms can migrate into the stomach from the external environment and as a convenient surface upon which biofilms can accumulate. The result of these ruptures in the body's innate defenses is the development of an abnormal microflora in the stomach and duodenum and, inevitably, the formation of a polymicrobial biofilm on the surface of the PEG tube.
In immunocompetent individuals, the upper gastrointestinal (GI) tract is sparsely colonized by microorganisms. Based on culture-dependent analyses, the stomach is generally devoid of significant microbiota other than
Helicobacter pylori and some lactobacilli that are present in low numbers (ca. 10
1 to 10
3 CFU per ml contents) (
78,
174). In contrast, the duodenum contains a resident microbiota in which lactobacilli and streptococci are the main culturable species, reaching population densities of approximately 10
2 to 10
4 CFU per ml contents (
148). The microbial density in the intestinal tract increases with proximity to the rectum; colonic contents contain up to 10
12 CFU per gram, principally comprising obligately anaerobic genera such as
Bacteroides (
93).
Low gastric pH is thought to be a major factor suppressing the microbial colonization of the stomach (
220). However, some enteric bacteria possess acid resistance mechanisms that may confer protection in the GI tract (
34). Additionally, a number of innate defense mechanisms break down in patients with PEG tubes. The absence of the sensory stimuli associated with food intake inhibits saliva production and peristalsis, while reduced swallowing increases pH and reduces gastric nitrite concentrations. The net effect is a greater susceptibility to microbial overgrowth in the stomach and duodenum. The most common pathology in such patients is chronic diarrhea, although more serious complications, such as malabsorption and sepsis, also occur (
27). The formation of microbial biofilms on PEG tubes is an unavoidable consequence of bacterial overgrowth. Such microcommunities are highly recalcitrant to antibiotic therapy (
200,
216). Moreover, biofilms can harbor pathogens and/or microorganisms carrying antibiotic resistance genes and often cause problems with indwelling devices (
17,
151,
158).
Candida spp. are known to colonize PEG tubes, a phenomenon that can lead to tube deterioration (
72–
74). There is a consistent overlap of microorganisms found on PEG tube biofilm samples, whether they are obtained from pediatric or from adult patients, where enterococci, staphylococci,
Candida spp., pseudomonads, and bacilli predominate (
46,
137). This is similar to the microbiota present in the upper GI tract of critically ill patients in surgical intensive care units (
129). Such colonization has been shown to result most commonly in chronic diarrhea but also poses an increased risk of microbial translocation across the epithelial barrier, potentially leading to bacteremia—the “gut-origin-of-sepsis” hypothesis (
124). Thus, the potential clinical consequences of the long-term colonization of the upper GI tract are obvious, especially where the PEG tube surface-associated biofilm acts as a potential refuge and locus of recolonization after antibiotic treatment.
A comprehensive study of the microbiota of patients receiving EN via a PEG tube was carried out by O'May et al. An array of 11 selective solid media was used to isolate microorganisms from the luminal surface of PEG tubes and from gastric and duodenal aspirates from the same individuals (
154). The genera isolated were similar to those isolated in previous studies, comprising mainly
Candida spp.,
Enterobacteriaceae, streptococci, staphylococci, and lactobacilli. Data suggested that the gastric pH had no significant effect on the density of colonization in the stomachs and duodena of patients on EN, although it did affect the composition of the microbiota:
Bifidobacterium,
Klebsiella, and
Staphylococcus spp. were detected only in aspirates with a pH of >3.
Candida spp.,
E. coli, and streptococci were detected in aspirates with a pH of 1 to 7. PEG tube surface-associated microbiota were similar in composition to those detected in aspirates; thus, it seems likely that these communities are able to seed the lumen of the stomach, thereby allowing recolonization after antibiotic treatment. Significantly,
E. coli, staphylococci, and
Candida spp. were detected only in aspirates from patients who had received antibiotic treatment during their stay in the hospital. The same researchers developed a chemostat-based model system using a defined microbiota composed of the microorganisms isolated most commonly from patients on EN (
153).
This model system was used to investigate empirically the effect of pH on planktonic and PEG tube surface-associated microbiota. In general,
in vitro data mirrored those obtained from the above-mentioned
ex vivo studies. The lowering of the pH from 6 to 3 had no significant effect on the density of planktonic or biofilm communities and a significant (circa 10
7 CFU/ml) microbiota was detected at pH 3. It is important that because of the continuous-culture methods employed in that study, these recovery data must represent cells actively multiplying at such low pH values and not merely dormant microorganisms surviving. These data are in stark contrast to those from other studies which suggested that
E. coli is unable to multiply in environments with pH values of <4.4 (
121). However, the lowering of the pH altered markedly the composition of the recovered microbiota:
Candida spp. and lactobacilli were aciduric, while the numbers of
E. coli and
Klebsiella pneumoniae bacteria detected decreased steadily with decreasing pH. Visualization by fluorescent
in situ hybridization of PEG tube surface-associated biofilms revealed microcolonies surrounded by significant areas of sparsely colonized interstitial space. Microcolonies were comprised of both live and dead cells; in many cases, yeast pseudohyphae were found to be invading the interior of the microcolonies. Where this occurred, any bacterial cells surrounding the pseudohyphae were invariably dead, as determined by BacLight Live/Dead staining (). The mechanism behind this killing remains unknown and requires further research but could be due to the bactericidal
Candida quorum-sensing compound farnesol, as described above.
The increasing use of EN in the nosocomial setting renders an understanding of the mechanisms behind and consequences of microbial colonization in such patients increasingly important. Biofilm formation is inevitable when the upper GI tract becomes overgrown and when the stable nonshedding surface of the tube luminal surface is present for long periods. Data suggesting that the use of antibiotics in such patients may actually increase the probability of colonization by potentially pathogenic microorganisms, such as S. aureus and C. albicans, make the search for alternative therapies ever more urgent.
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