Mustafa Fazli

Distribution, Organization, and Ecology of Bacteria in Chronic Wounds

ABSTRACT Between 1 and 2% of the population in the developed world experiences a nonhealing or chronic wound characterized by an apparent arrest in a stage dominated by inflammatory processes. Lately, research groups have proposed that bacteria might be involved in and contribute to the lack of healing of these wounds. To investigate this, we collected and examined samples from chronic wounds obtained from 22 different patients, all selected because of suspicion of Pseudomonas aeruginosa colonization. These wound samples were investigated by standard culturing methods and peptide nucleic acid-based fluorescence in situ hybridization (PNA FISH) for direct identification of bacteria. By means of the culturing methods, Staphylococcus aureus was detected in the majority of the wounds, whereas P. aeruginosa was observed less frequently. In contrast, using PNA FISH, we found that a large fraction of the wounds contained P. aeruginosa. Furthermore, PNA FISH revealed the structural organization of bacteria in the samples. It appeared that P. aeruginosa aggregated as microcolonies imbedded in the matrix component alginate, which is a characteristic hallmark of the biofilm mode of growth. The present investigation suggests that bacteria present within these wounds tend to be aggregated in microcolonies imbedded in a self-produced matrix, characteristic of the biofilm mode of growth. Additionally, we must conclude that there exists no good correlation between bacteria detected by standard culturing methods and those detected by direct detection methods such as PNA FISH. This strongly supports the development of new diagnostic and treatment strategies for chronic wounds.

Nonrandom Distribution of Pseudomonas aeruginosa and Staphylococcus aureus in Chronic Wounds

ABSTRACT The spatial organization of Pseudomonas aeruginosa and Staphylococcus aureus in chronic wounds was investigated in the present study. Wound biopsy specimens were obtained from patients diagnosed as having chronic venous leg ulcers, and bacterial aggregates in these wounds were detected and located by the use of peptide nucleic acid-based fluorescence in situ hybridization and confocal laser scanning microscopy (CLSM). We acquired CLSM images of multiple regions in multiple sections cut from five wounds containing P. aeruginosa and five wounds containing S. aureus and measured the distance of the bacterial aggregates to the wound surface. The distance of the P. aeruginosa aggregates to the wound surface was significantly greater than that of the S. aureus aggregates, suggesting that the distribution of the bacteria in the chronic wounds was nonrandom. The results are discussed in relation to our recent finding that swab culturing techniques may underestimate the presence of P. aeruginosa in chronic wounds and in relation to the hypothesis that P. aeruginosa bacteria located in the deeper regions of chronic wounds may play an important role in keeping the wounds arrested in a stage dominated by inflammatory processes.

Biofilms in chronic infections – a matter of opportunity – monospecies biofilms in multispecies infections

It has become evident that aggregation or biofilm formation is an important survival mechanism for bacteria in almost any environment. In this review, we summarize recent visualizations of bacterial aggregates in several chronic infections (chronic otitis media, cystic fibrosis, infection due to permanent tissue fillers and chronic wounds) both as to distribution (such as where in the wound bed) and organization (monospecies or multispecies microcolonies). We correlate these biofilm observations to observations of commensal biofilms (dental and intestine) and biofilms in natural ecosystems (soil). The observations of the chronic biofilm infections point toward a trend of low bacterial diversity and sovereign monospecies biofilm aggregates even though the infection in which they reside are multispecies. In contrast to this, commensal and natural biofilm aggregates contain multiple species that are believed to coexist, interact and form biofilms with high bacterial and niche diversity. We discuss these differences from both the diagnostic and the scientific point of view.

The CRP/FNR family protein Bcam1349 is a c-di-GMP effector that regulates biofilm formation in the respiratory pathogen Burkholderia cenocepacia

Burkholderia cenocepacia is an opportunistic respiratory pathogen that can cause severe infections in immune‐compromised individuals and is associated with poor prognosis for patients suffering from cystic fibrosis. The second messenger cyclic diguanosine monophosphate (c‐di‐GMP) has been shown to control a wide range of functions in bacteria, but little is known about these regulatory mechanisms in B. cenocepacia. Here we investigated the role that c‐di‐GMP plays in the regulation of biofilm formation and virulence in B. cenocepacia. Elevated intracellular levels of c‐di‐GMP promoted wrinkly colony, pellicle and biofilm formation in B. cenocepacia. A screen for transposon mutants unable to respond to elevated levels of c‐di‐GMP led to the identification of the mutant bcam1349 that did not display increased biofilm and pellicle formation with excessive c‐di‐GMP levels, and displayed a biofilm defect with physiological c‐di‐GMP levels. The bcam1349 gene is predicted to encode a transcriptional regulator of the CRP/FNR superfamily. Analyses of purified Bcam1349 protein and truncations demonstrated that it binds c‐di‐GMP in vitro. The Bcam1349 protein was shown to regulate the production of a number of components, including cellulose and fimbriae. It was demonstrated that the Bcam1349 protein binds to the promoter region of the cellulose synthase genes, and that this binding is enhanced by the presence of c‐di‐GMP. The bcam1349 mutant showed reduced virulence in a Galleria mellonella wax moth larvae infection model. Taken together, these findings suggest that the Bcam1349 protein is a transcriptional regulator that binds c‐di‐GMP and regulates biofilm formation and virulence in B. cenocepacia in response to the level of c‐di‐GMP.

Quantitative analysis of the cellular inflammatory response against biofilm bacteria in chronic wounds.

Chronic wounds are an important problem worldwide. These wounds are characterized by a persistent inflammatory stage associated with excessive accumulation and elevated cell activity of neutrophils, suggesting that there must be a persistent stimulus that attracts and recruits neutrophils to the wound. One such stimulus might be the presence of bacterial biofilms in chronic wounds. In the present study, biopsy specimens from chronic venous leg ulcers were investigated for the detection of bacteria using peptide nucleic acid‐based fluorescence in situ hybridization (PNA‐FISH) and confocal laser scanning microscopy. The bacteria in the wounds were often situated in large aggregates. To obtain a measure of the cellular inflammatory response against the bacteria in the chronic wounds, the amount of neutrophils accumulated at the site of infection was evaluated through differential neutrophil counting on the tissue sections from wounds containing either Pseudomonas aeruginosa or Staphylococcus aureus. The P. aeruginosa‐containing wounds had significantly higher numbers of neutrophils accumulated compared with the S. aureus‐containing wounds. These results are discussed in relation to the hypothesis that the presence of P. aeruginosa biofilms in chronic wounds may be one of the main factors leading to a persistent inflammatory response and impaired wound healing.

Influence of putative exopolysaccharide genes on Pseudomonas putida KT2440 biofilm stability

We report a study of the role of putative exopolysaccharide gene clusters in the formation and stability of Pseudomonas putida KT2440 biofilm. Two novel putative exopolysaccharide gene clusters, pea and peb, were identified, and evidence is provided that they encode products that stabilize P. putida KT2440 biofilm. The gene clusters alg and bcs, which code for proteins mediating alginate and cellulose biosynthesis, were found to play minor roles in P. putida KT2440 biofilm formation and stability under the conditions tested. A P. putida KT2440 derivative devoid of any identifiable exopolysaccharide genes was found to form biofilm with a structure similar to wild-type biofilm, but with a stability lower than that of wild-type biofilm. Based on our data, we suggest that the formation of structured P. putida KT2440 biofilm can occur in the absence of exopolysaccharides; however, exopolysaccharides play a role as structural stabilizers.

The exopolysaccharide gene cluster Bcam1330–Bcam1341 is involved in Burkholderia cenocepacia biofilm formation, and its expression is regulated by c-di-GMP and Bcam1349

In Burkholderia cenocepacia, the second messenger cyclic diguanosine monophosphate (c‐di‐GMP) has previously been shown to positively regulate biofilm formation and the expression of cellulose and type‐I fimbriae genes through binding to the transcriptional regulator Bcam1349. Here, we provide evidence that cellulose and type‐I fimbriae are not involved in B. cenocepacia biofilm formation in flow chambers, and we identify a novel Bcam1349/c‐di‐GMP‐regulated exopolysaccharide gene cluster which is essential for B. cenocepacia biofilm formation. Overproduction of Bcam1349 in trans promotes wrinkly colony morphology, pellicle, and biofilm formation in B. cenocepacia. A screen for transposon mutants unable to respond to the overproduction of Bcam1349 led to the identification of a 12‐gene cluster, Bcam1330–Bcam1341, the products of which appear to be involved in the production of a putative biofilm matrix exopolysaccharide and to be essential for flow‐chamber biofilm formation. We demonstrate that Bcam1349 binds to the promoter region of genes in the Bcam1330–Bcam1341 cluster and that this binding is enhanced by the presence of c‐di‐GMP. Furthermore, we demonstrate that overproduction of both c‐di‐GMP and Bcam1349 leads to increased transcription of these genes, indicating that c‐di‐GMP and Bcam1349 functions together in regulating exopolysaccharide production from the Bcam1330–Bcam1341 gene cluster. Our results suggest that the product encoded by the Bcam1330–Bcam1341 gene cluster is a major exopolysaccharide that provides structural stability to the biofilms formed by B. cenocepacia, and that its production is regulated by c‐di‐GMP through binding to and promotion of the activity of the transcriptional regulator Bcam1349.

PNA-based fluorescence in situ hybridization for identification of bacteria in clinical samples.

Fluorescence in situ hybridization with PNA probes (PNA-FISH) that target specific bacterial ribosomal RNA sequences is a powerful and rapid tool for identification of bacteria in clinical samples. PNA can diffuse readily through the bacterial cell wall due to its uncharged backbone, and PNA-FISH can be performed with high specificity due to the extraordinary thermal stability of RNA-PNA hybrid complexes. We describe a PNA-FISH procedure and provide examples of the application of PNA-FISH for the identification of bacteria in chronic wounds, cystic fibrosis lungs, and soft tissue fillers. In all these cases, bacteria can be identified in biofilm aggregates, which may explain their recalcitrance to antibiotic treatment.

Colony morphology and transcriptome profiling of Pseudomonas putida KT2440 and its mutants deficient in alginate or all EPS synthesis under controlled matric potentials.

Pseudomonas putida is a versatile bacterial species adapted to soil and its fluctuations. Like many other species living in soil, P. putida often faces water limitation. Alginate, an exopolysaccharide (EPS) produced by P. putida, is known to create hydrated environments and alleviate the effect of water limitation. In addition to alginate, P. putida is capable of producing cellulose (bcs), putida exopolysaccharide a (pea), and putida exopolysaccharide b (peb). However, unlike alginate, not much is known about their roles under water limitation. Hence, in this study we examined the role of different EPS components under mild water limitation. To create environmentally realistic water limited conditions as observed in soil, we used the Pressurized Porous Surface Model. Our main hypothesis was that under water limitation and in the absence of alginate other exopolysaccharides would be more active to maintain homeostasis. To test our hypothesis, we investigated colony morphologies and whole genome transcriptomes of P. putida KT2440 wild type and its mutants deficient in synthesis of either alginate or all known EPS. Overall our results support that alginate is an important exopolysaccharide under water limitation and in the absence of alginate other tolerance mechanisms are activated.

In-Frame and Unmarked Gene Deletions in Burkholderia cenocepacia via an Allelic Exchange System Compatible with Gateway Technology

ABSTRACT Burkholderia cenocepacia is an emerging opportunistic pathogen causing life-threatening infections in immunocompromised individuals and in patients with cystic fibrosis, which are often difficult, if not impossible, to treat. Understanding the genetic basis of virulence in this emerging pathogen is important for the development of novel treatment regimes. Generation of deletion mutations in genes predicted to encode virulence determinants is fundamental to investigating the mechanisms of pathogenesis. However, there is a lack of appropriate selectable and counterselectable markers for use in B. cenocepacia, making its genetic manipulation problematic. Here we describe a Gateway-compatible allelic exchange system based on the counterselectable pheS gene and the I-SceI homing endonuclease. This system provides efficiency in cloning homology regions of target genes and allows the generation of precise and unmarked gene deletions in B. cenocepacia. As a proof of concept, we demonstrate its utility by deleting the Bcam1349 gene, encoding a cyclic di-GMP (c-di-GMP)-responsive regulator protein important for biofilm formation.