Article

The Exopolysaccharide Alginate Protects Pseudomonas aeruginosa Biofilm Bacteria from IFN- -Mediated Macrophage Killing

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Abstract

The ability of Pseudomonas aeruginosa to form biofilms and cause chronic infections in the lungs of cystic fibrosis patients is well documented. Numerous studies have revealed that P. aeruginosa biofilms are highly refractory to antibiotics. However, dramatically fewer studies have addressed P. aeruginosa biofilm resistance to the host's immune system. In planktonic, unattached (nonbiofilm) P. aeruginosa, the exopolysaccharide alginate provides protection against a variety of host factors yet the role of alginate in protection of biofilm bacteria is unclear. To address this issue, we tested wild-type strains PAO1, PA14, the mucoid cystic fibrosis isolate, FRD1 (mucA22+), and the respective isogenic mutants which lacked the ability to produce alginate, for their susceptibility to human leukocytes in the presence and absence of IFN-gamma. Human leukocytes, in the presence of recombinant human IFN-gamma, killed biofilm bacteria lacking alginate after a 4-h challenge at 37 degrees C. Bacterial killing was dependent on the presence of IFN-gamma. Killing of the alginate-negative biofilm bacteria was mediated through mononuclear cell phagocytosis since treatment with cytochalasin B, which prevents actin polymerization, inhibited leukocyte-specific bacterial killing. By direct microscopic observation, phagocytosis of alginate-negative biofilm bacteria was significantly increased in the presence of IFN-gamma vs all other treatments. Addition of exogenous, purified alginate to the alginate-negative biofilms restored resistance to human leukocyte killing. Our results suggest that although alginate may not play a significant role in bacterial attachment, biofilm development, and formation, it may play an important role in protecting mucoid P. aeruginosa biofilm bacteria from the human immune system.

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... 22 Alginate plays an essential role in protecting biofilm forming P. aeruginosa from human immune response. 23 The elastase enzyme damages tissues and degrades plasma proteins such as complement factors and immunoglobulins which are vital for the immune system of host. 24 A high frequency of plcH, plcN and exoS was observed in the current study. ...
... PCR: Polymerase chain reaction DNA: Deoxyribonucleic acid. PCR amplification of plcH gene: lane L (100 bp DNA ladder); C (positive control); Lanes 1,3,4,5,6,7,8,9,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26 and 27 haveplcH (608 bp) gene positive Pseudomonas aeruginosa samples. PCR: Polymerase chain reaction DNA: Deoxyribonucleic acid. ...
... PCR: Polymerase chain reaction DNA: Deoxyribonucleic acid. PCR amplification of toxA gene: lane L (100 bp DNA ladder); C (positive control);Lanes 3,4,5,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24, 25 and 26 havetoxA(270 bp) gene positive Pseudomonas aeruginosa samples. PCR: Polymerase chain reaction DNA: Deoxyribonucleic acid. ...
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Abstract Objective: To investigate six virulence genes in clinical isolates of pseudomonas aeruginosa collected from tertiary care hospitals. Methods: The cross-sectional study was conducted from December 2014 to June 2016 at the Centre of Biotechnology and Microbiology, University of Peshawar, Pakistan and comprised pseudomonas aeruginosa isolates collected from Khyber Teaching Hospital, Peshawar, and Hayatabad Medical Complex, Peshawar, Pakistan. The isolates were recovered from pus, urine, sputum, wound, bronchial wash, cerebrospinal fluid, blood, high vaginal swab and diabetic foot. Genomic deoxyribonucleic acid was extracted from the isolates after identification and polymerase chain reaction technique was performed for the molecular detection of six virulence genes using specific primers. The six genes were: algD, lasB, toxA, plcH, plcN, and exoS Results: There were 182 pseudomonas aeruginosa isolates. The prevalence of algD was 179(98.3%), toxA 156(85.7%), lasB 179(98.3%), plcH 178(97.8%), plcN 170(93.4%) and exoS 175(96.15%). Conclusions: There was high prevalence of virulence factors among regional isolates of Pseudomonas aeruginosa. Keywords: Pseudomonas aeruginosa, Virulence factors, Exotoxin A, Exoenzyme S, Elastase B. (JPMA 68: 1787; 2018)
... The fragments were joined in a PCR SOEing reaction via their overlap ping regions around codon 456. The prc-His 6 and prc(S456A)-His 6 fragments were cloned into pHERD20T using XbaI and HindIII restriction sites incorporated in the primers. Plasmids encoding MBP-′MucA or -′MucA22 periplasmic domain fusion proteins were made by amplifying the mucA and mucA22 periplasmic domains from genomic DNA. ...
... In vitro proteolysis reactions contained approximately 2 µM Prc-His 6 or Prc-S456A-His 6 and approximately 2 µM MBP-′MucA or MBP-′MucA22. Reactions were incubated at 37°C, and aliquots were removed after 1, 3, 5, and 8 h and terminated by adding SDS-PAGE sample buffer and boiling. ...
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In Pseudomonas aeruginosa, alginate biosynthesis gene expression is inhibited by the transmembrane anti-sigma factor MucA, which sequesters the AlgU sigma factor. Cell envelope stress initiates cleavage of the MucA periplasmic domain by site-1 protease AlgW, followed by further MucA degradation to release AlgU. However, after colonizing the lungs of people with cystic fibrosis, P. aeruginosa converts to a mucoid form that produces alginate constitutively. Mucoid isolates often have mucA mutations, with the most common being mucA22, which truncates the periplasmic domain. MucA22 is degraded constitutively, and genetic studies suggested that the Prc protease is responsible. Some studies also suggested that Prc contributes to induction in strains with wild-type MucA, whereas others suggested the opposite. However, missing from all previous studies is a demonstration that Prc cleaves any protein directly, which leaves open the possibility that the effect of a prc null mutation is indirect. To address the ambiguities and shortfalls, we reevaluated the roles of AlgW and Prc as MucA and MucA22 site-1 proteases. In vivo analyses using three different assays and two different inducing conditions all suggested that AlgW is the only site-1 protease for wild-type MucA in any condition. In contrast, genetics suggested that AlgW or Prc act as MucA22 site-1 proteases in inducing conditions, whereas Prc is the only MucA22 site-1 protease in non-inducing conditions. For the first time, we also show that Prc is unable to degrade the periplasmic domain of wild-type MucA but does degrade the mutated periplasmic domain of MucA22 directly. IMPORTANCE After colonizing the lungs of individuals with cystic fibrosis, Pseudomonas aeruginosa undergoes mutagenic conversion to a mucoid form, worsening the prognosis. Most mucoid isolates have a truncated negative regulatory protein MucA, which leads to constitutive production of the extracellular polysaccharide alginate. The protease Prc has been implicated, but not shown, to degrade the most common MucA variant, MucA22, to trigger alginate production. This work provides the first demonstration that the molecular mechanism of Prc involvement is direct degradation of the MucA22 periplasmic domain and perhaps other truncated MucA variants as well. MucA truncation and degradation by Prc might be the predominant mechanism of mucoid conversion in cystic fibrosis infections, suggesting that Prc activity could be a useful therapeutic target.
... Research has shown that culture conditions can change the physiological state of bacteria and influence their production of natural products or metabolites. Here we also cultured the bacteria in solid culture to induce the formation of colony biofilms and also change the expression of genes that may induce production of antibiofilm compounds, particularly exopolysaccharides [22][23][24][25]. The aim of this research was to compare liquid and solid cultures of these eleven environmental isolates in terms of their antibiofilm inhibition and destruction activity against representative pathogenic bacteria. ...
... This strain might produce compounds inside the colony biofilm that are not normally produced when living in the planktonic stage. It has been reported that polysaccharides are elevated in biofilms [22][23][24][25], and one study suggested that Vibrio sp. can produce an exopolysaccharide capable of inhibiting or eradicating biofilms. We infer that Vibrio strain B32 might be producing polysaccharide compounds; however, further investigation and characterization remains needed. ...
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Background Biofilm-associated infections are a global threat to our economy and human health; as such, development of antibiofilm compounds is an urgent need. Our previous study identified eleven environmental isolates of endophyte bacteria, actinomycetes, and two strains of Vibrio cholerae as having strong antibiofilm activity, but only tested crude extracts from liquid culture. Here we grew the same bacteria in solid culture to induce the formation of colony biofilms and the expression of genes that may ultimately produce antibiofilm compounds. This research aimed to compare antibiofilm inhibition and destruction activities between liquid and solid cultures of these eleven environmental isolates against the biofilms of representative pathogenic bacteria. Results We measured antibiofilm activity using the static antibiofilm assay and crystal violet staining. The majority of our isolates exhibited higher inhibitory antibiofilm activity in liquid media, including all endophyte bacteria, V. cholerae V15a, and actinomycetes strains (CW01, SW03, CW17). However, for V. cholerae strain B32 and two actinomycetes bacteria (TB12 and SW12), the solid crude extracts showed higher inhibitory activity. Regarding destructive antibiofilm activity, many endophyte isolates and V. cholerae strains showed no significant difference between culture methods; the exceptions were endophyte bacteria isolate JerF4 and V. cholerae B32. The liquid extract of isolate JerF4 showed higher destructive activity relative to the corresponding solid culture extract, while for V. cholerae strain B32 the solid extract showed higher activity against some biofilms of pathogenic bacteria. Conclusions Culture conditions, namely solid or liquid culture, can influence the activity of culture extracts against biofilms of pathogenic bacteria. We compared the antibiofilm activity and presented the data that majority of isolates showed a higher antibiofilm activity in liquid culture. Interestingly, solid extracts from three isolates (B32, TB12, and SW12) have a better inhibition or/and destruction antibiofilm activity compared to their liquid culture. Further research is needed to characterize the activities of specific metabolites in solid and liquid culture extracts and to determine the mechanisms of their antibiofilm actions.
... At least 80% of the PJI-causing bacteria are believed to exist in the biofilm form; therefore, the planktonic structure used this study may not correctly reflect the PJI status in patients (Springer and Parvizi 2013). It has been reported that the immune system can clear planktonic microorganisms faster than those in the biofilms (Cerca et al. 2006;Leid et al. 2005); however, planktonic bacteria are commonly used to avoid problems associated with the standardization of the inoculum with the biofilm structure. Another limitation of this study is the exclusive use of ATCC strains, which, while ensuring standardization, may not fully capture the variability of clinical isolates. ...
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Purpose The most frequently used surgical procedures for periprosthetic joint infections (PJIs) are debridement, antibiotics, and implant retention (DAIR), as well as single- or two-stage revision arthroplasty. The choice of surgery is made depending on the full maturation of the biofilm layer. The purpose of this study was to evaluate the biofilm formation and microbial growth using common PJI-causing agents and compare its development on the implant surface. Methods The in vivo study was performed using 40 Sprague–Dawley rats divided into five groups (n = 8/group): Staphylococcus aureus, Staphylococcus epidermidis, Pseudomonas aeruginosa, Candida albicans, and control. Six standard titanium alloy discs were placed into the subcutaneous air pouches of the interscapular areas of the rats. After the inoculation of microorganisms, disc and soft tissue cultures were collected at 2-week intervals for 6 weeks, and the microbial load and the microscopic appearance of the biofilm were compared. Results The disc samples from the S. aureus group had the highest infection load at all time points; however, in soft tissue samples, this was only observed at week 4 and 6. Electron microscopic images showed no distinctive differences in the biofilm structures between the groups. Conclusion S. aureus microbial burden was significantly higher in implant cultures at week 2 compared to other PJI-causing agents examined. These results may explain the higher failure rate seen if the DAIR procedure was performed at < 3–4 weeks after the PJI symptom onset and support the observation that DAIR may not be effective against PJIs caused by S. aureus.
... 25 Some may give a creamy consistency resembling alginate while others are hard to turn into a gel and their uses are based on technical aspects since color difference is irrelevant. 26 Carrageenan furthermore is classied as linear sulfated polysaccharide biopolymers that are extracted from red edible seaweeds. The most common uses are in food industries because of their gelling and thickening as well as stabilizing abilities. ...
Article
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Natural polysaccharides play a crucial role across diverse fields such as medicine, food, and cosmetics, for their various physiochemical and biological properties. In this study, we developed a new crosslinked biopolymer using sodium alginate (AG) and carrageenan (CAR) polysaccharides. Various metal complexes involving different metal salts such as CoCl2·6H2O and CrCl3·6H2O were synthesized using the crosslinked biopolymer formed above. The two polymeric complexes were characterized using Fourier-transform infrared spectroscopy (FT-IR), elemental analysis, ultraviolet-visible spectroscopy (UV-Vis), magnetic susceptibility, molar conductivity techniques, and thermogravimetric analysis. The Co(ii) polymeric complex exhibits a tetrahedral X-ray crystal structure and belongs to the monoclinic crystal system. Cr(iii) complex is octahedral and crystal data are in compliance with the cubic crystal system. The antimicrobial study showed a significant activity improvement for all the developed complexes against both Gram-positive as well as Gram-negative bacterial pathogens – Staphylococcus aureus, Microscus luteum, Escherichia coli and Salmonella typhimurium. Similarly, the different polymeric complexes showed an efficient activity against Candida albicans as anti-fungal effect. Moreover, higher antioxidant values of the two complexes were obtained with DPPH scavenging activity ranging between 73% and 94%. In addition, both the polymeric complexes were subjected to biocompatibility cell viability assays along with in vitro anticancer evaluation. The alginate/carrageenan crosslinked coordination complexes revealed excellent cytocompatibility with normal human breast epithelial cells (MCF10A) and a high anticancer potential with human breast cancer cells (MCF-7) which increase significantly in a dose-dependent manner.
... The water binding capacity of the polymer increases with O-acetylation, which in turn influences its solubility, viscosity, and gelling properties. One study found that alginate protects P. aeruginosa biofilms from IFN-γmediated macrophage killing (Leid et al. 2005). Another study showed that O-acetylation of alginate helps P. aeruginosa evade the host immune system by preventing opsonization (Pier et al. 2001). ...
Chapter
This chapter examines the composition of exopolysaccharides within the bacterial biofilm matrix, unraveling their intricate roles in biofilm formation and infection pathogenesis. Serving as key structural components of the biofilm matrix, exopolysaccharides form part of the complex network of polymers secreted by biofilm bacteria. Here, we explore biosynthesis pathways, regulatory mechanisms, and multifaceted functions of exopolysaccharides—drawing insights from notable exopolysaccharides like alginate, Pel, Psl, cellulose, and poly-N-acetylglucosamine (PNAG). Specifically, we focus on exopolysaccharide-mediated mechanisms of adhesion, biofilm mechanics, immune evasion, and resistance to antimicrobials. Furthermore, we discuss the translational potential of antibacterial therapeutics that target exopolysaccharides. Through an exploration of the structure and functions of exopolysaccharides, this chapter contributes to a more comprehensive understanding of biofilm biology, highlighting unresolved questions and potential avenues for future research in this critical area.
... 15 On the other hand, Psl is involved in biofilm formation in both nonmucoid and mucoid strains of P. aeruginosa. 30,31 Divyashree et al. 17 found that PslB was the most dominant gene, and that most of the isolates were resistant to several drugs due to moderate and high biofilm formation. ...
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Background The ability of Pseudomonas aeruginosa to produce biofilm has established it as one of the most significant pathogens. The purpose of this study was to evaluate antimicrobial resistance and conduct a molecular investigation of the virulence genes PslB and PelD in Pseudomonas aeruginosa species isolated from patients. Methodology One hundred clinical isolates were collected from patients of different age groups who were hospitalized in Kermanshah and Sonqor hospitals. The isolates were obtained through culture on specific media, biochemical confirmatory tests, and gram staining for confirmation. Biofilm production was assessed using an indirect quantification method with crystal violet. Additionally, antibiotic resistance was determined through the disc various method following Clinical and Laboratory Standards Institute guidelines. Finally, the presence of genes related to PlsB and PelD in resistant strains was examined using The polymerase chain reaction (PCR). Results The results indicate that the highest resistance and lowest sensitivity were related to nitrofurantoin 100 μg, while the lowest resistance and highest sensitivity were related to cefepime 30 mg. Biofilm phenotypes were categorized as weak in 7% (n = 7) of isolates, medium in 13% (n = 13), and high in 80% (n = 80). The PslB and PelD genes were identified in 86% (n = 86) and 38% (n = 38) of isolates, respectively, while 4% (n = 4) did not possess either of these two genes. Additionally, a majority of the isolates exhibited multidrug-resistance (87%) due to their moderate-to-high biofilm formation. Conclusion All isolates were capable of producing biofilm. A significant association were between strains with the high biofilm and multidrug-resistance species (p < 0.05). Multidrug-resistance (78%) isolates included 28% (n = 28) of isolates that were PslB+ PelD+, 45% (n = 45) of isolates that were only PslB+, and 5 (n = 5) isolates that were only PelD+. A significant relationship was found between the presence of the PslB gene multidrug-resistance and high producer (p < 0.05).
... Additionally, exoproducts from P. aeruginosa have been shown to promote the production of staphyloxanthin, further enhancing the virulence of S. aureus 67 . Other studies have shown that S. aureus supports colonisation and pathogenicity of P. aeruginosa by inhibiting its phagocytosis by eukaryotic cells 8 . It has been proposed that, during the course of chronic infection, P. aeruginosa may find evolutionarily favourable to maintain a population of S. aureus to help counteract the host's immune response. ...
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Staphylococcus aureus and Pseudomonas aeruginosa are the most common bacteria co-isolated from chronic infected wounds. Their interactions remain unclear but this coexistence is beneficial for both bacteria and may lead to resistance to antimicrobial treatments. Besides, developing an in vitro model where this coexistence is recreated remains challenging, making difficult their study. The aim of this work was to develop a reliable polymicrobial in vitro model of both species to further understand their interrelationships and the effects of different antimicrobials in coculture. In this work, bioluminescent and fluorescent bacteria were used to evaluate the activity of two antiseptics (chlorhexidine and thymol) against these bacteria planktonically grown, or when forming single and mixed biofilms. At the doses tested (0.4-1,000 mg/L), thymol showed selective antimicrobial action against S. aureus in planktonic and biofilm states, in contrast with chlorhexidine which exerted antimicrobial effects against both bacteria. Furthermore, the initial conditions for both bacteria in the co-culture determined the antimicrobial outcome, showing that P. aeruginosa impaired the proliferation and metabolism of S. aureus. Moreover, S. aureus showed an increased tolerance against antiseptic treatments when co-cultured, attributed to the formation of a thicker mixed biofilm compared to those obtained when monocultured, and also, by the reduction of S. aureus metabolic activity induced by diffusible molecules produced by P. aeruginosa. This work underlines the relevance of polymicrobial populations and their crosstalk and microenvironment in the search of disruptive and effective treatments for polymicrobial biofilms.
... Similarly, the genes for elastase (lasB), alkaline protease (apr), alginate (algD), and epoxide hydrolase (cif) were detected in all strains associated with bacteremia (24/24) from patients with hospital infections (n = 67), as well as in respiratory infections (10/10) and catheter-associated infections (10/10) from patients with community-acquired infections (n = 57), highlighting the virulence capacity of the strains to causing severe and fatal infections in patients, especially in immunocompromised patients, because LasB activity has been described to disrupt epithelial tight junctions and degrade surfactant proteins, as well as host cytokines and immunoglobulins [44,45]. The Apr protease interferes with endothelial fibronectin and laminin, degrading cytokines and complement proteins [46], while alginate polysaccharide (AlgD) evades pulmonary phagocytosis and promotes bacterial resistance to antibiotics [47,48], and the Cif protein promotes the severity of infections in patients with cystic fibrosis and pneumonia [49]. Overall, the high percentages of the virulence genotype of more than 90% for pilA, oprL, oprI, algD, apr, lasA, lasB, cif, and groEL found in our strains of P. aeruginosa isolated from patients with hospital-and community-acquired infections were higher than those described in strains from patients with different infections in other countries; pilA (approximately 30%), oprL (29.6%), oprI (40.7%), algD (64.6% to 70.1%), apr (63%), lasA (approximately 4%), lasB (13% to 95.4%), and cif (94%) [11,31,50,51], where the frequency of ndvB (94.3%) and groEL (100%) detected in our strains was very similar (ndvB; 95.7% and groEL; 100%) to that described in strains causing chronic otitis [10]. ...
Article
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The increase in the number of hospital strains of hypervirulent and multidrug resistant (MDR) Pseudomonas aeruginosa is a major health problem that reduces medical treatment options and increases mortality. The molecular profiles of virulence and multidrug resistance of P. aeruginosa-associated hospital and community infections in Mexico have been poorly studied. In this study, we analyzed the different molecular profiles associated with the virulence genotypes related to multidrug resistance and the genotypes of multidrug efflux pumps (mex) in P. aeruginosa causing clinically critical infections isolated from Mexican patients with community- and hospital-acquired infections. Susceptibility to 12 antibiotics was determined using the Kirby–Bauer method. The identification of P. aeruginosa and the detection of virulence and efflux pump system genes were performed using conventional PCR. All strains isolated from patients with hospital-acquired (n = 67) and community-acquired infections (n = 57) were multidrug resistant, mainly to beta-lactams (ampicillin [96.7%], carbenicillin [98.3%], cefalotin [97.5%], and cefotaxime [87%]), quinolones (norfloxacin [78.2%]), phenicols (chloramphenicol [91.9%]), nitrofurans (nitrofurantoin [70.9%]), aminoglycosides (gentamicin [75%]), and sulfonamide/trimethoprim (96.7%). Most strains (95.5%) isolated from patients with hospital- and community-acquired infections carried the adhesion (pilA) and biofilm formation (ndvB) genes. Outer membrane proteins (oprI and oprL) were present in 100% of cases, elastases (lasA and lasB) in 100% and 98.3%, respectively, alkaline protease (apr) and alginate (algD) in 99.1% and 97.5%, respectively, and chaperone (groEL) and epoxide hydrolase (cif) in 100% and 97.5%, respectively. Overall, 99.1% of the strains isolated from patients with hospital- and community-acquired infections carried the efflux pump system genes mexB and mexY, while 98.3% of the strains carried mexF and mexZ. These findings show a wide distribution of the virulome related to the genotypic and phenotypic profiles of antibiotic resistance and the origin of the strains isolated from patients with hospital- and community-acquired infections, demonstrating that these molecular mechanisms may play an important role in high-pathogenicity infections caused by P. aeruginosa.
... The innate immune response to P. aeruginosa biofilm involves the activation of NK cells, macrophages, neutrophils, dendritic cells, and the complement system. The most convincing evidence of the function of innate immune responses in bacterial biofilm has been obtained by founding human neutrophils and macrophages to P. aeruginosa biofilms that lack planktonic bacteria [43][44][45][46]. Similarly, research on mouse lungs exposed to biofilms has demonstrated that the innate immune response involves a significant accumulation of activated neutrophils in the airways. ...
Article
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“An impregnable stronghold where one or more warrior clans can evade enemy attacks" may serve as a description of bacterial biofilm on a smaller level than human conflicts. Consider this hypothetical conflict: who would emerge victorious? The occupants of secure trenches or those carrying out relentless assault? Either faction has the potential for triumph; the defenders will prevail if they can fortify the trench with unwavering resolve, while the assailants will succeed if they can devise innovative means to breach the trench. Hence, bacterial biofilms pose a significant challenge and are formidable adversaries for medical professionals, often leading to the failure of antibiotic treatments in numerous hospital infections. Phage engineering has become the foundation for the targeted enhancement of various phage properties, facilitating the eradication of biofilms. Researchers across the globe have studied the impact of engineered phages and phage-derived enzymes on biofilms formed by difficult-to-treat bacteria. These novel biological agents have shown promising results in addressing biofilm-related challenges. The compilation of research findings highlights the impressive capabilities of engineered phages in combating antibiotic-resistant bacteria, superbugs, and challenging infections. Specifically, these engineered phages exhibit enhanced biofilm destruction, penetration, and prevention capabilities compared to their natural counterparts. Additionally, the engineered enzymes derived from phages demonstrate improved effectiveness in addressing bacterial biofilms. As a result, these novel solutions, which demonstrate high penetration, destruction, and inhibition of biofilms, can be regarded as a viable option for addressing infectious biofilms in the near future.
... Редкие и генетически детерминированные заболевания легких путей микроколонии, которые образуют вокруг себя биопленку (мукоид), охраняющую их от действия защитных факторов макроорганизма и противосинегнойных препаратов. В процессе размножения микроколонии продуцируют вирулентные факторы, которые повреждают клетки макроорганизма, стимулируют выработку медиаторов воспаления, повышают проницаемость капилляров, вызывают лейкоцитарную инфильтрацию [53]. ...
Chapter
Второй том издания посвящен клиническим аспектам респираторной медицины. Представлена информация о наиболее распространенных хронических воспалительных заболеваниях органов дыхания: хронической обструктивной болезни легких и бронхиальной астме, подробно изложены современные представления об их гено-, эндо- и фенотипах, приведены обновленные классификации, существенно дополнены главы по лечению с учетом последних рекомендаций GOLD и GINA, включая генно-инженерную биологическую терапию. Значительно расширен раздел по инфекционным заболеваниям органов дыхания. Представлена информация о COVID-19 и постковидном синдроме, в частности его бронхолегочных проявлениях. Обновлены рекомендации по лечению пневмонии, туберкулеза и других инфекционных заболеваний органов дыхания. Специальный раздел посвящен их специфической иммунопрофилактике и иммунотерапии. Впервые в особом разделе объединены главы о редких и генетически детерминированных заболеваниях легких – муковисцидозе, первичной цилиарной дискинезии, лимфангиолейомиоматозе и др., описаны современные подходы к их диагностике и терапии. В настоящем издании, составленном с учетом актуальных клинических рекомендаций, рассматриваются вопросы клиники, диагностики и лечения основных заболеваний респираторной системы. Руководство предназначено для повышения квалификации широкого круга врачей: терапевтов, пульмонологов, фтизиатров, аллергологов и иммунологов, онкологов, хирургов, оториноларингологов, педиатров, инфекционистов, патологоанатомов и представителей смежных специальностей. Также издание адресовано студентам старших курсов медицинских вузов, ординаторам, аспирантам и может быть использовано в качестве учебника для подготовки к практическим занятиям и итоговой государственной аттестации.
... Another study reported that the modification in LPS core affects adherence and biofilm formation in P. aeruginosa PAO1 (Lau et al., 2009). The exopolysaccharide alginate, a major substance in biofilm, protects P. aeruginosa from IFN-γ mediated macrophage killing, according to an in vivo study showing that the P. aeruginosa biofilm reduced the intensity of local neutrophil response in murine wounds (Leid et al., 2005;Trøstrup et al., 2013). ...
... ASTM International standardized methods for biofilm research and other proposed biofilm research protocols are notably repeatable, fast, and reliant on accessible supplies like coupons and conical tubes or a combination [14]. Moreover, there are four ASTM International-approved standardized devices for studying biofilms: the CDC biofilm reactor [15,16], a drip flow reactor [17], a rotating disk reactor [16], and the MBEC Assay® or similar peg lid microtiter plate assays all with limited in situ microscopy access [15,18,19]. The MBEC Assay® is a 96-well plate lid with pegs that sit in the wells [20]. ...
Article
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Bacteria biofilm responses to disinfectants and antibiotics are quantified and observed using multiple methods, though microscopy, particularly confocal laser scanning microscopy (CLSM) is preferred due to speed, a reduction in user error, and in situ analysis. CLSM can resolve biological and spatial heterogeneity of biofilms in 3D with limited throughput. The microplate peg-lid-based assay, described in ASTM E2799-22, is a medium-throughput method for testing biofilms but does not permit in situ imaging. Breaking off the peg, as recommended by the manufacturer, risks sample damage, and is limited to easily accessible pegs. Here we report modifications to the peg optimized for in situ visualization and visualization of all pegs. We report similar antibiotic challenge recovery via colony formation following the ASTM E2799-22 protocol and in situ imaging. We report novel quantifiable effects of antibiotics on biofilm morphologies, specifically biofilm streamers. The new design bridges the MBEC® assays design that selects for biofilm phenotypes with in situ imaging needs.
... These results emphasize the membrane role but do not exclude the intervention of certain virulence factors in the antibiotic tolerance of Pseudomonas. In this context, the study of Liang et al. (2023) confirmed the role of alginate, one of the most studied virulence factors of planktonic and biofilm growth P.aeruginosa (Leid et al. 2005), in their tolerance toward tobramycin and meropenem, but not ciprofloxacin (Liang et al.2023). Moreover, the role of the flagellar hook was also confirmed in the antibiotic tolerance of P. aeruginosa biofilms, in the study of Valentin et al. (2022). ...
Article
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The protective role of superoxide dismutase (Sod) against oxidative stress, resulting from the common antibiotic pathway of action, has been studied in the wild type and mutant strains of swarmer Pseudomonas aeruginosa, lacking Cytosolic Mn-Sod (sodM), Fe-Sod (sodB) or both Sods (sodMB). Our results showed that inactivation of sodB genes leads to significant motility defects and tolerance to meropenem. This resistance is correlated with a greater membrane unsaturation as well as an effective intervention of Mn-Sod isoform, in antibiotic tolerance. Moreover, loss of Mn-Sod in sodM mutant, leads to polymixin intolerance and is correlated with membrane unsaturation. Effectivelty, sodM mutant showed an enhanced swarming motility and a conserved rhamnolipid production. Whereas, in the double mutant sodMB, ciprofloxacin tolerance would be linked to an increase in the percentage of saturated fatty acids in the membrane, even in the absence of superoxide dismutase activity. The overall results showed that Mn-Sod has a protective role in the tolerance to antibiotics, in swarmer P.aeruginosa strain. It has been further shown that Sod intervention in antibiotic tolerance is through change in membrane fatty acid composition.
... In vitro studies suggested that microorganisms in biofilms are less recognised by the immune system [63][64][65] and thus resistant to phagocytosis and killing by neutrophils [66,67]. For example, the P. aeruginosa exopolysaccharide Psl reduces opsonophagocytosis by inhibiting complement molecules deposition at the surface of the bacteria and promotes intracellular bacterial survival, whereas alginate protects against phagocyte activation, uptake, and killing [68,69]. Similarly, exopolysaccharide GAGs mask b-glucans from recognition by Dectin-1, a receptor of innate immunity, and protect hyphae from neutrophil extracellular traps [35,70]. ...
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... Alginic acid (AA) is a naturally occurring edible polysaccharide [32,33]. It has many carboxyl and hydroxyl groups, and can easily combine with metal ions (such as sodium and calcium) to form the corresponding salt, called alginate. ...
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... In response to tissue-located biofilms, antibodies bind to virulence factors and other bacterial antigens, forming immune complexes. The tolerance of the biofilm to PMNs is mediated by (i) the increased biofilm size which prevents phagocytosis, (ii) the protection provided by the EPS, and (iii) the inactivation of the complement system, opsonization avoidance, and the evasion of immune recognition [20,21]. If the host response reduces the pathogenic biofilm size and causes the dispersion of the biofilm cells, low-grade focal inflammation may occur, and a relapse of infection may follow due to the regrowth of persister cells. ...
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... Rhamnolipids produced by P. aeruginosa can also cause necrotic cell death in neutrophils [107]. Additionally, alginate, Psl and Pel polysaccharides present in the P. aeruginosa biofilm prevent opsonization by the host complement system and phagocytosis by host macrophages [117][118][119]. Thus, P. aeruginosa utilizes several signals from the host to transition between the biofilm state or the rhamnolipid-producing state. ...
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... The extra-cellular polymeric substances (EPS) matrix surrounding cells in the mature biofilm also limits penetration of antibiotic compounds and diminishes the effectiveness of the immune system [21]. The EPS protects bacterial cells from leukocyte-based killing of bacteria and reduces immune-cell phagocytosis [22][23][24][25][26]. It also reduces complement activation, and affects polymorphonuclear leukocyte bactericidal ability [27]. ...
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... Different strains of P. aeruginosa produce three exopolysaccharides (EPS): Pel, Psl, and alginate. These proteins aid in cell adhesion and protect P. aeruginosa from the human immune cells [105,106]. Alginate is an overproduced EPS by mucoid strains of P. aeruginosa, which is a common characteristic of isolates from chronic lung infections [107,108]. LPS is a major part of the outer membrane of Gram-negative bacteria. ...
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... Flagella mutant strains ΔflgK and pili mutant strain ΔPilA showed significant decrease (7.3% and 14% respectively) in the growth of planktonic cells. ΔcupA1 which is deficient in fimbrial ushar protein required for fimbriae production[12,13] and ΔpslC which is mutant for exopolysaccharide production[14] ...
... Alginate plays many important roles in biofilms. For example, alginate retains water and nutrients and provides antibiotic resistance and immune evasion [32]. ...
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P. aeruginosaisan opportunistic microorganism that impose a versatile modifications in its genome to sustain an infection that can range from otitis media to a life threating disease. Moreover, antibiotic resistance endows P. aeruginosa characteristics to be one of the most resistant microorganisms to be treated. P. aeruginosa possess both cell associated and extracellular virulence factors. Cell associated virulence factors include flagellum, pili, adhesins, alginate, and lipopolysaccharide. While, extracellular virulence factors include hemolysins, proteases, lipases, exotoxin A, and cytotoxins.
... Alginate reduces the penetration of the leukocyte antigens, and production of AMPs in the biofilm 2), biofilm matrix or bacterial components can suppress leukocyte-specific processes. 3) the QS signaling and genetic switching can increase resistance to the immune system [97]. ...
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The infection caused by P. aeruginosa still is dangerous throughout the world. This is partly due to its immune escape mechanisms considerably increasing the bacterial survival in the host. By escape from recognition by TLRs, interference with complement system activation, phagocytosis inhibition, production of ROS, inhibition of NET production, interference with the generation of cytokines, inflammasome inhibition, reduced antigen presentation, interference with cellular and humoral immunity, and induction of apoptotic cell death and MDSc, P. aeruginosa breaks down the barriers of the immune system and causes lethal infections in the host. Recognition of other immune escape mechanisms of P. aeruginosa may provide a basis for the future treatment of the infection. This manuscript may provide new insights and information for the development of new strategies to combat P. aeruginosa infection. In the present manuscript, the escape mechanisms of P. aeruginosa against immune response would be reviewed.
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Chronic leg wounds represent a major burden of disease worldwide, costing health care systems billions of dollars each year. Aside from the financial implications, they also impose a significant physical and psychosocial burden on the patient, their relatives and/or carers, and the community. Whilst measures such as maintenance of wound hygiene, debridement, dressings and compression are the current standard of care, complete healing is not always achievable and ulcer recurrence is common. Thus, there is still a gap to breach in terms of understanding the intricate pathophysiology of chronic wounds and the role this plays on treatment and management. Pseudomonas aeruginosa has been linked to poor wound healing, with the pathogen being frequently isolated from chronic leg ulcers. Characterized by its multi-drug resistance, targeting P. aeruginosa requires the development of novel therapeutic options. Thus, the aim of this literature review is to describe the pathophysiology of P. aeruginosa in chronic leg ulcers and discuss novel treatment strategies. Here, we describe the key molecular mechanisms driving the observed clinical effect of P. aeruginosa on wounds and discuss novel strategies of molecular targeting of this common bacteria, establishing new approaches that could benefit patients with chronic hard to heal wounds.
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Periprosthetic joint infection is a major complication of total joint arthroplasty. Even with current treatments, failure rates are unacceptably high with a 5‐year mortality rate of 26%. Majority of the literature in the field has focused on development of better biomarkers for diagnostics and treatment strategies including innovate antibiotic delivery systems, anti‐biofilm agents, and bacteriophages. Nevertheless, the role of the immune system, our first line of defense during PJI, is not well understood. Evidence of infection in PJI patients is found within circulation, synovial fluid, and tissue and include numerous cytokines, metabolites, antimicrobial peptides, and soluble receptors that are part of the PJI diagnosis workup. Macrophages, neutrophils, and myeloid derived suppressor cells (MDSCs) are initially recruited into the joint by chemokines and cytokines produced by immune cells and bacteria and are activated by pathogen‐associated molecular patterns (PAMPs). While these cells are efficient killers of planktonic bacteria by phagocytosis, opsonization, degranulation, and recruitment of adaptive immune cells, biofilm‐associated bacteria are troublesome. Biofilm is not only a physical barrier for the immune system but also elicit effector functions. Additionally, bacteria have developed mechanisms to evade the immune system by inactivating effector molecules, promoting killing or anti‐inflammatory effector cell phenotypes, and intracellular persistence and dissemination. Understanding these shortcomings and the mechanisms by which bacteria can subvert the immune system may open new approaches to better prepare our own immune system to combat PJI. Furthermore, preoperative immune system assessment and screening for dysregulation may aid in developing preventative interventions to decrease PJI incidence. This article is protected by copyright. All rights reserved.
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Bacterial biofilms can be found in most environments on our planet, and the human body is no exception. Consisting of microbial cells encased in a matrix of extracellular polymers, biofilms enable bacteria to sequester themselves in favorable niches, while also increasing their ability to resist numerous stresses and survive under hostile circumstances. In recent decades, biofilms have increasingly been recognized as a major contributor to the pathogenesis of chronic infections. However, biofilms also occur in or on certain tissues in healthy individuals, and their constituent species are not restricted to canonical pathogens. In this review, we discuss the evidence for where, when, and what types of biofilms occur in the human body, as well as the diverse ways in which they can impact host health under homeostatic and dysbiotic states.
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Upon undergoing mucoid conversion within the lungs of cystic fibrosis patients, the pathogenic bacterium Pseudomonas aeruginosa synthesises copious quantities of the virulence factor and exopolysaccharide alginate. The enzyme guanosine diphosphate mannose dehydrogenase (GMD) catalyses the rate-limiting step and irreversible formation of the alginate sugar nucleotide building block, guanosine diphosphate mannuronic acid. Since there is no corresponding enzyme in humans, strategies that could prevent its mechanism of action could open a pathway for new and selective inhibitors to disrupt bacterial alginate production. Using virtual screening, a library of 1447 compounds within the Known Drug Space parameters were evaluated against the GMD active site using the Glide, FRED and GOLD algorithms. Compound hit evaluation with recombinant GMD refined the panel of 40 potential hits to 6 compounds which reduced NADH production in a time-dependent manner; of which, an usnic acid derivative demonstrated inhibition six-fold stronger than a previously established sugar nucleotide inhibitor, with an IC50 value of 17 μM. Further analysis by covalent docking and mass spectrometry confirm a single site of GMD alkylation.
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This review addresses the topic of biofilms, including their development and the interaction between different counterparts. There is evidence that various diseases, such as cystic fibrosis, otitis media, diabetic foot wound infections, and certain cancers, are promoted and aggravated by the presence of polymicrobial biofilms. Biofilms are composed by heterogeneous communities of microorganisms protected by a matrix of polysaccharides. The different types of interactions between microorganisms gives rise to an increased resistance to antimicrobials and to the host's defense mechanisms, with the consequent worsening of disease symptoms. Therefore, infections caused by polymicrobial biofilms affecting different human organs and systems will be discussed, as well as the role of the interactions between the gram-negative bacteria Pseudomonas aeruginosa, which is at the base of major polymicrobial infections, and other bacteria, fungi, and viruses in the establishment of human infections and diseases. Considering that polymicrobial biofilms are key to bacterial pathogenicity, it is fundamental to evaluate which microbes are involved in a certain disease to convey an appropriate and efficacious antimicrobial therapy.
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Antimicrobial resistance is one of the greatest threats to human health. Gram-positive methicillin resistant Staphylococcus aureus (MRSA), in both its planktonic and biofilm form, is of particular concern. Herein we identify the hydrogelation properties for a series of intrinsically fluorescent, structurally related supramolecular self-associating amphiphiles and determine their efficacy against both planktonic and biofilm forms of MRSA. To further explore the potential translation of this hydrogel technology for real-world applications, the toxicity of the amphiphiles was determined against the eukaryotic multicellular model organism, Caenorhabditis elegans. Due to the intrinsic fluorescent nature of these supramolecular amphiphiles, material characterisation of their molecular self-associating properties included; comparative optical density plate reader assays, rheometry and widefield fluorescence microscopy. This enabled determination of amphiphile structure and hydrogel sol dependence on resultant fibre formation.
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Introduction Pseudomonas aeruginosa is known for its ability to form biofilms, which are dependent on the production of exopolysaccharides. During chronic colonization of the airway and biofilm formation, P. aeruginosa converts to a mucoid phenotype, indicating production of the exopolysaccharide alginate. The mucoid phenotype promotes resistance to phagocytic killing, but the mechanism has not been established. Methods and Results To better understand the mechanism of phagocytic evasion conferred by alginate production, Human (THP-1) and murine (MH-S) macrophage cell lines were used to determine the effects of alginate production on macrophage binding, signaling and phagocytosis. Phagocytosis assays using mucoid clinical isolate FRD1 and its non-mucoid algD mutant showed that alginate production inhibited opsonic and non-opsonic phagocytosis, but exogenous alginate was not protective. Alginate caused a decrease in binding to murine macrophages. Blocking antibodies to CD11b and CD14 showed that these receptors were important for phagocytosis and were blocked by alginate. Furthermore, alginate production decreased the activation of signaling pathways required for phagocytosis. Mucoid and non-mucoid bacteria induced similar levels of MIP-2 from murine macrophages. Discussion This study demonstrated for the first time that alginate on the bacterial surface inhibits receptor-ligand interactions important for phagocytosis. Our data suggest that there is a selection for alginate conversion that blocks the earliest steps in phagocytosis, leading to persistence during chronic pulmonary infections.
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Pseudomonas aeruginosa (P. aeruginosa) is commonly implicated in hospital-acquired infections where its capacity to form biofilms on a variety of surfaces and the resulting enhanced antibiotic resistance seriously limit treatment choices. Because surface attachment sensitizes P. aeruginosa to quorum sensing (QS) and induces virulence through both chemical and mechanical cues, we investigate the effect of surface properties through spatially patterned mucin, combined with sub-inhibitory concentrations of tobramycin on QS and virulence factors in both mucoid and non-mucoid P. aeruginosa strains using multi-modal chemical imaging combining confocal Raman microscopy and matrix-assisted laser desorption/ionization-mass spectrometry. Samples comprise surface-adherent static biofilms at a solid-water interface, supernatant liquid, and pellicle biofilms at an air-water interface at various time points. Although the presence of a sub-inhibitory concentration of tobramycin in the supernatant retards growth and development of static biofilms independent of strain and surface mucin patterning, we observe clear differences in the behavior of mucoid and non-mucoid strains. Quinolone signals in a non-mucoid strain are induced earlier and are influenced by mucin surface patterning to a degree not exhibited in the mucoid strain. Additionally, phenazine virulence factors, such as pyocyanin, are observed in the pellicle biofilms of both mucoid and non-mucoid strains but are not detected in the static biofilms from either strain, highlighting the differences in stress response between pellicle and static biofilms. Differences between mucoid and non-mucoid strains are consistent with their strain-specific phenology, in which the mucoid strain develops highly protected biofilms.
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aeruginosa to undergo the typical adaptation to the intractable mu- coid form in the CF lung. These findings indicate that gene activation in bacteria by toxic oxygen radicals, similar to that found in plants and mammalian cells, may serve as a defence mechanism for the bacteria. This suggests that mucoid conversion is a response to oxygen radical exposure and that this response is a mechanism of defence by the bacteria. This is the first report to show that PMNs and their oxygen radicals can cause this phenotypic and genotypic change which is so typical of the intractable form of P. aeruginosa in the CF lung. These findings may provide a basis for the development of anti-oxidant and anti-inflammatory therapy for the early stages of infection in CF patients.
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Alginate, a viscous polysaccharide from mucoid Pseudomonas aeruginosa, may interfere with the host defenses in patients with cystic fibrosis and chronic P. aeruginosa lung infection. The alginate concentration in the sol phase of expectorated sputum was quantitated by a biochemical method and a newly developed enzyme-linked immunosorbent assay. There was a high degree of correlation between the methods, and the concentration of alginate ranged from 4 to 101 micrograms/ml with a median of 35.5 micrograms/ml when measured by enzyme-linked immunosorbent assay. Alginate could not be detected in the bronchial secretions from patients without P. aeruginosa infection. In vitro investigation of alginate did not show any activation of the alternative pathway of complement, as determined by a hemolytic kinetic assay and by testing for neutrophil chemotaxis. At a high concentration, P. aeruginosa alginate caused a slight activation of the classical pathway of complement. Alginate did not cause neutrophil chemotaxis by itself but was able to reduce the neutrophil chemotactic response to N-formylmethionylleucylphenylalanine and for zymosan-activated serum. P. aeruginosa and seaweed alginates were able to prime neutrophils for increased N-formylmethionylleucylphenylalanine-induced neutrophil oxidative burst, as determined by chemiluminescence. Because of its ability to prevent attraction of neutrophils to the site of infection, lack of complement activation, and ability to enhance neutrophil oxidative burst, alginate from P. aeruginosa may contribute to the persistence and pathogenesis of chronic P. aeruginosa infection in cystic fibrosis.
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Alginic acid was purified from a mucoid clinical isolate of Pseudomonas aeruginosa. Luminol-dependent chemiluminescence of phorbol myristate acetate-stimulated neutrophils was inhibited by this alginate, but oxygen consumption was unaffected. Further studies indicated that this effect was due to the ability of the pseudomonal alginate to scavenge hypochlorite. A seaweed alginate was less effective and dextran T500 was ineffective in hypochlorite scavenging. It appears that the uronic acid core and the O-acetyl groups of pseudomonal alginate are involved in its hypochlorite-scavenging ability. The relevance of this phenomenon was demonstrated by the greater resistance to killing by hypochlorite of mucoid P. aeruginosa compared with a nonmucoid revertant, and the addition of purified alginate to the nonmucoid revertant protected the organism from hypochlorite. Thus, this extracellular polysaccharide may enhance the virulence of P. aeruginosa by scavenging the phagocyte-generated oxidant HOCl. This enhanced virulence may be involved in disease processes in which mucoid organisms predominate, such as cystic fibrosis.
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A unique, recently described rat alveolar macrophage cell line (NR8383) was used to study the interaction of the pulmonary immune system with a mucoid cystic fibrosis isolate of Pseudomonas aeruginosa (SRM-3), its nonmucoid revertant (SRM-3R), and a non-cystic fibrosis isolate (PAO-1). Strain SRM-3 was cultivated in a chemostat system to allow maintenance of an entirely mucoid population. The alveolar macrophage response to the mucoid and nonmucoid strains of P. aeruginosa was determined by visually quantitating phagocytosis in acridine orange-stained monolayers and measuring the induction of an oxidative burst as indicated by chemiluminescence and H2O2 production. In all experiments, fewer than 2% of the NR8383 cells engulfed the mucoid SRM-3 isolate, while SRM-3R and PAO-1 were phagocytized by 15 and 41%, respectively. Opsonization by normal serum (complement) provided minimal phagocytic enhancement of these strains, whereas specific anti-P. aeruginosa antibody slightly elevated phagocytic responses to strains with nonmucoid phenotypes while providing a sevenfold increase in uptake of SRM-3. Chemiluminescent and H2O2 responses were comparable with the levels of phagocytosis observed, with very little or no response to the mucoid strain SRM-3. The data indicate that the strains with mucoid phenotypes are refractile to ingestion and that studies which describe ingestion of mucoid strains were likely measuring ingestion of revertants. Alginic acid (2 mg/ml) was found to inhibit stimulation of macrophage response to the opsonized and unopsonized nonmucoid strain PAO-1.
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Direct electron microscopic examination of postmortem lung material from cystic fibrosis patients infected with Pseudomonas aeruginosa has shown that these bacterial cells form distinct fiber-enclosed microcolonies in the infected alveoli. Similar examination of bronchoscopy material from infected cystic fibrosis patients showed that the fibres of the enveloping matrix are definitely associated with the bacterial cells. The fibers of the extracellular matrix stain with ruthenium red and are therefore presumed to be polyanionic. When mucoid strains of P. aeruginosa were recovered from cystic fibrosis patients and grown in a suitable liquid medium, they were found to produce large microcolonies whose component cells were embedded in a very extensive matrix of polyanionic fibers that could be stabilized by reaction with antibodies to prevent collapse during the dehydration steps of preparation for electron microscopy. When these mucoid strains of P. aeruginosa were used to produce pulmonary infections of rats by the agar bead method, the infected alveoli contained large fiber-enclosed bacterial microcolonies. We conclude that the cells of P. aeruginosa that infect cystic fibrosis patients form microcolonies that are enveloped in a fibrous anionic matrix and that these microcolonies can be duplicated in in vitro cultures and in animal model systems.
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Mucoid Pseudomonas aeruginosa strain FRD, a sputum isolate from a cystic fibrosis patient, was used to develop a genetic system. The mucoid appearance is due to the biosynthesis of the exopolysaccharide alginate and is a potential virulence factor of the organism. The sex factor plasmid FP2 was used for uninterrupted genetic exchange to investigate the nature of spontaneous mutations which produce frequent alginate-negative (Alg-) derivatives. Crosses between Alg+ donors and Alg- recipients demonstrated linkage between alginate genes and chromosomal markers. Crosses between an Alg- donor and Alg- recipients produced Alg+ recombinants at frequencies that varied, depending on the recipient strains used. This indicated that more than one genetic locus was associated with spontaneous mutation leading to loss of the mucoid character. Three classes of Alg- mutants were identified. Genetic exchange experiments showed that the loci of the alginate (alg) mutations of the three mutant classes are in the same region of the chromosome. The sex factor plasmid R68.45 was used for nonpolarized chromosome transfer and demonstrated close linkage between chromosomal markers (his-1, met-1) and alg markers. This was consistent with the data obtained in FP2-mediated crosses. Thus, the evidence obtained indicated that the alg genes which undergo frequent mutation are chromosomal, that several loci are involved, and that these alg loci are apparently clustered on the chromosome.
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Mucoid Pseudomonas aeruginosa strains isolated from cystic fibrosis patients are very heterogeneous and include a class which is hypersusceptible to carbenicillin (minimum inhibitory concentration, less than or equal to 1 microgram/ml). Hypersusceptible mucoid P. aeruginosa isolates were found in 12 of 22 cystic fibrosis patients examined. In cystic fibrosis patients having both resistant and hypersusceptible mucoid strains, 24 of 54 mucoid colonies obtained from a sputum sample were found to belong to the hypersusceptible class. In most instances, hypersusceptible and resistant strains isolated from the same sputum sample were indistinguishable, aside from their antibiotic susceptibilities, by classical methods. A particular pair of mucoid isolates (one hypersusceptible and one resistant) was chosen for further study. The hypersusceptibility was not limited to carbenicillin but was found to extend to other penicillins, tetracycline, and trimethoprim but not to the aminoglycosides gentamicin and tobramycin. The hypersusceptibility of the mucoid strain was found to be unrelated to amount or ability to synthesize alginate. The hypersusceptible strain was found to have two additional outer membrane proteins (32,000 and 25,000 daltons) as compared with the resistant strain. The 32,000-dalton protein, termed protein N1, was found to be correlated to the hypersusceptibility phenotype, as all spontaneous mutants of the hypersusceptible mucoid strain which were capable of growing in the presence of 50 microgram of carbenicillin per ml had lost the 32,000-dalton outer membrane protein. The possible origins of the hypersusceptibility phenotype and the implications of the heterogeneity of mucoid P. aeruginosa in the pathogenesis of P. aeruginosa are discussed.
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A Pseudomonas aeruginosa strain (UCBPP-PA14) is infectious both in an Arabidopsis thaliana leaf infiltration model and in a mouse full-thickness skin burn model. UCBPP-PA14 exhibits ecotype specificity for Arabidopsis, causing a range of symptoms from none to severe in four different ecotypes. In the mouse model, UCBPP-PA14 is as lethal as other well-studied P. aeruginosa strains. Mutations in the UCBPP-PA14 toxA, plcS, and gacA genes resulted in a significant reduction in pathogenicity in both hosts, indicating that these genes encode virulence factors required for the full expression of pathogenicity in both plants and animals.
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Conversion to mucoidy, caused by the overproduction of the exopolysaccharide alginate in laboratory and cystic fibrosis strains of Pseudomonas aeruginosa, can occur via frameshift or nonsense mutations in the second gene of the algU mucA mucB cluster. The first gene of the cluster, algU, encodes a putative alternative sigma factor required for algD transcription. The algD gene encodes a critical alginate biosynthetic enzyme and is invariably activated in mucoid P. aeruginosa cells. To investigate the function of the genes controlling conversion to mucoidy, the wild-type algU mucA mucB cluster from the standard genetic strain PAO1 was used to reconstitute algD transcription in Escherichia coli. Transcription of an algD-lacZ chromosomal fusion in E. coli was detected upon introduction of plasmid-borne algU mucA mucB. Moreover, insertional inactivation of either mucA or mucB resulted in further stimulation of transcriptional activity from the algD promoter. This activation was dependent on algU, since a double algU mucA mutation abrogated transcription of algD. These experiments suggest that the phenotypic manifestations of muc mutations, i.e., increased algD expression and mucoid phenotype, depend on the presence of an active algU gene and that this regulator and the factors encoded by the downstream genes interact. Further support for these conclusions came from the investigations of the mechanism of reversion to nonmucoidy in P. aeruginosa, a phenomenon frequently referred to as the instability of mucoid phenotype. Spontaneous nonmucoid derivatives of the mucoid strain PAO578 carrying the mucA22 mutation were examined for the presence of alterations within the algU mucA mucB locus. Point mutations which inactivated algU were detected in some, but not all, nonmucoid revertants. No reversion of the original mucA22 mutation (a deletion of one C) was observed in any of the investigated strains. This observation suggests that the process of conversion to nonmucoidy ban be explained, at least partially, by second-site suppressor mutations and that a fraction of such mutations occurs in algU.
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Respiratory infections with Pseudomonas aeruginosa and Burkholderia cepacia play a major role in the pathogenesis of cystic fibrosis (CF). This review summarizes the latest advances in understanding host-pathogen interactions in CF with an emphasis on the role and control of conversion to mucoidy in P. aeruginosa, a phenomenon epitomizing the adaptation of this opportunistic pathogen to the chronic chourse of infection in CF, and on the innate resistance to antibiotics of B. cepacia, person-to-person spread, and sometimes rapidly fatal disease caused by this organism. While understanding the mechanism of conversion to mucoidy in P. aeruginosa has progressed to the point where this phenomenon has evolved into a model system for studying bacterial stress response in microbial pathogenesis, the more recent challenge with B. cepacia, which has emerged as a potent bona fide CF pathogen, is discussed in the context of clinical issues, taxonomy, transmission, and potential modes of pathogenicity.
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Biofilms are considered to be highly resistant to antimicrobial agents. Strictly speaking, this is not the case-biofilms do not grow in the presence of antimicrobials any better than do planktonic cells. Biofilms are indeed highly resistant to killing by bactericidal antimicrobials, compared to logarithmic-phase planktonic cells, and therefore exhibit tolerance. It is assumed that biofilms are also significantly more tolerant than stationary-phase planktonic cells. A detailed comparative examination of tolerance of biofilms versus stationary- and logarithmic-phase planktonic cells with four different antimicrobial agents was performed in this study. Carbenicillin appeared to be completely ineffective against both stationary-phase cells and biofilms. Killing by this beta-lactam antibiotic depends on rapid growth, and this result confirms the notion of slow-growing biofilms resembling the stationary state. Ofloxacin is a fluoroquinolone antibiotic that kills nongrowing cells, and biofilms and stationary-phase cells were comparably tolerant to this antibiotic. The majority of cells in both populations were eradicated at low levels of ofloxacin, leaving a fraction of essentially invulnerable persisters. The bulk of the population in both biofilm and stationary-phase cultures was tolerant to tobramycin. At very high tobramycin concentrations, a fraction of persister cells became apparent in stationary-phase culture. Stationary-phase cells were more tolerant to the biocide peracetic acid than were biofilms. In general, stationary-phase cells were somewhat more tolerant than biofilms in all of the cases examined. We concluded that, at least for Pseudomonas aeruginosa, one of the model organisms for biofilm studies, the notion that biofilms have greater resistance than do planktonic cells is unwarranted. We further suggest that tolerance to antibiotics in stationary-phase or biofilm cultures is largely dependent on the presence of persister cells.
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While originally characterized as a collection of related syndromes, cystic fibrosis (CF) is now recognized as a single disease whose diverse symptoms stem from the wide tissue distribution of the gene product that is defective in CF, the ion channel and regulator, cystic fibrosis transmembrane conductance regulator (CFTR). Defective CFTR protein impacts the function of the pancreas and alters the consistency of mucosal secretions. The latter of these effects probably plays an important role in the defective resistance of CF patients to many pathogens. As the modalities of CF research have changed over the decades from empirical histological studies to include biophysical measurements of CFTR function, the clinical management of this disease has similarly evolved to effectively address the ever-changing spectrum of CF-related infectious diseases. These factors have led to the successful management of many CF-related infections with the notable exception of chronic lung infection with the gram-negative bacterium Pseudomonas aeruginosa. The virulence of P. aeruginosa stems from multiple bacterial attributes, including antibiotic resistance, the ability to utilize quorum-sensing signals to form biofilms, the destructive potential of a multitude of its microbial toxins, and the ability to acquire a mucoid phenotype, which renders this microbe resistant to both the innate and acquired immunologic defenses of the host.
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Staphylococcus aureus is a common pathogen responsible for nosocomial and community infections. It readily colonizes indwelling catheters, forming microbiotic communities termed biofilms. S. aureus bacteria in biofilms are protected from killing by antibiotics and the body's immune system. For years, one mechanism behind biofilm resistance to attack from the immune system's sentinel leukocytes has been conceptualized as a deficiency in the ability of the leukocytes to penetrate the biofilm. We demonstrate here that under conditions mimicking physiological shear, leukocytes attach, penetrate, and produce cytokines in response to maturing and fully matured S. aureus biofilm.
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The bacterium Pseudomonas aeruginosa causes chronic respiratory infections in cystic fibrosis (CF) patients. Such infections are extremely difficult to control because the bacteria exhibit a biofilm-mode of growth, rendering P. aeruginosa resistant to antibiotics and phagocytic cells. During the course of infection, P. aeruginosa usually undergoes a phenotypic switch to a mucoid colony, which is characterized by the overproduction of the exopolysaccharide alginate. Alginate overproduction has been implicated in protecting P. aeruginosa from the harsh environment present in the CF lung, as well as facilitating its persistence as a biofilm by providing an extracellular matrix that promotes adherence. Because of its association with biofilms in CF patients, it has been assumed that alginate is also the primary exopolysaccharide expressed in biofilms of environmental nonmucoid P. aeruginosa. In this study, we examined the chemical nature of the biofilm matrix produced by wild-type and isogenic alginate biosynthetic mutants of P. aeruginosa. The results clearly indicate that alginate biosynthetic genes are not expressed and that alginate is not required during the formation of nonmucoid biofilms in two P. aeruginosa strains, PAO1 and PA14, that have traditionally been used to study biofilms. Because nonmucoid P. aeruginosa strains are the predominant environmental phenotype and are also involved in the initial colonization in CF patients, these studies have implications in understanding the early events of the infectious process in the CF airway.
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Pseudomonas aeruginosa is an opportunistic respiratory pathogen that accounts for most of the morbidity and mortality in cystic fibrosis (CF) patients. In CF-affected lungs, the bacteria undergo conversion from a non-mucoid to a non-tractable mucoid phenotype, due to overproduction of alginate. The effect of alginate production on pathogenicity was investigated by using an acute lung infection mouse model that compared a non-mucoid P. aeruginosa strain, PAO1, to its constitutive alginate-overproducing derivative, Alg(+) PAOmucA22, and an alginate-defective strain, Alg(-) PAOalgD. Bacterial suspensions were instilled into the left bronchus and examined 24 and 48 h post-infection. The highest bacterial loads and the most severe lung pathology were observed with strain Alg(-) PAOalgD at 24 h post-infection, which may have been due to an increase in expression of bacterial elastase by the mutant. Significantly lower lung and spleen bacterial loads were found in the two non-mucoid (PAO1 and Alg(-) PAOalgD) groups, compared to the mucoid Alg(+) PAOmucA22 group, between 24 and 48 h post-infection. The positive correlation between lung bacteriology and lung macroscopic pathology in the Alg(+) PAOmucA22 group suggests that alginate production not only impedes pulmonary clearing, but also results in severe lung damage. Positive correlations between IL12 levels and lung macroscopic pathology, and between IL12 and IFN-gamma levels in the Alg(+) PAOmucA22 group, suggested a possible contribution of these pro-inflammatory cytokines to tissue damage. No significant differences were found between the three groups in lung cytokine responses at 24 or 48 h post-infection. However, on comparison within each group at 24 and 48 h post-infection, a significant increase in the pro-inflammatory cytokine IFN-gamma was observed. Higher ratios of IFN-gamma/IL4 and IFN-gamma/IL10, but lower IL10 levels, were also found in all three groups. These results indicate a Th1-predominated immune response in these animals. Such cytokine responses could have aided the clearance of non-mucoid P. aeruginosa, but were not sufficient to alleviate infection by the mucoid variants. Alginate production may promote survival and persistence of this pathogenic micro-organism in the lung.
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Respiratory infections with Pseudomonas aeruginosa and Burkholderia cepacia play a major role in the pathogenesis of cystic fibrosis (CF). This review summarizes the latest advances in understanding host-pathogen interactions in CF with an emphasis on the role and control of conversion to mucoidy in P. aeruginosa, a phenomenon epitomizing the adaptation of this opportunistic pathogen to the chronic chourse of infection in CF, and on the innate resistance to antibiotics of B. cepacia, person-to-person spread, and sometimes rapidly fatal disease caused by this organism. While understanding the mechanism of conversion to mucoidy in P. aeruginosa has progressed to the point where this phenomenon has evolved into a model system for studying bacterial stress response in microbial pathogenesis, the more recent challenge with B. cepacia, which has emerged as a potent bona fide CF pathogen, is discussed in the context of clinical issues, taxonomy, transmission, and potential modes of pathogenicity.
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Bacteria that adhere to implanted medical devices or damaged tissue can encase themselves in a hydrated matrix of polysaccharide and protein, and form a slimy layer known as a biofilm. Antibiotic resistance of bacteria in the biofilm mode of growth contributes to the chronicity of infections such as those associated with implanted medical devices. The mechanisms of resistance in biofilms are different from the now familiar plasmids, transposons, and mutations that confer innate resistance to individual bacterial cells. In biofilms, resistance seems to depend on multicellular strategies. We summarise the features of biofilm infections, review emerging mechanisms of resistance, and discuss potential therapies.
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Bacteria that attach to surfaces aggregate in a hydrated polymeric matrix of their own synthesis to form biofilms. Formation of these sessile communities and their inherent resistance to antimicrobial agents are at the root of many persistent and chronic bacterial infections. Studies of biofilms have revealed differentiated, structured groups of cells with community properties. Recent advances in our understanding of the genetic and molecular basis of bacterial community behavior point to therapeutic targets that may provide a means for the control of biofilm infections.
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Bacteria that attach to a surface and grow as a biofilm are protected from killing by antibiotics. Reduced antibiotic susceptibility contributes to the persistence of biofilm infections such as those associated with implanted devices. The protective mechanisms at work in biofilms appear to be distinct from those that are responsible for conventional antibiotic resistance. In biofilms, poor antibiotic penetration, nutrient limitation and slow growth, adaptive stress responses, and formation of persister cells are hypothesized to constitute a multi-layered defense. The genetic and biochemical details of these biofilm defenses are only now beginning to emerge. Each gene and gene product contributing to this resistance may be a target for the development of new chemotherapeutic agents. Disabling biofilm resistance may enhance the ability of existing antibiotics to clear infections involving biofilms that are refractory to current treatments.
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Microbial biofilm has become inexorably linked with man's failure to control them by antibiotic and biocide regimes that are effective against suspended bacteria. This failure relates to a localized concentration of biofilm bacteria, and their extracellular products (exopolymers and extracellular enzymes), that moderates the access of the treatment agent and starves the more deeply placed cells. Biofilms, therefore, typically present gradients of physiology and concentration for the imposed treatment agent, which enables the less susceptible clones to survive. Such clones might include efflux mutants in addition to genotypes with modifications in single gene products. Clonal expansion following subeffective treatment would, in the case of many antibiotics, lead to the emergence of a resistant population. This tends not to occur for biocidal treatments where the active agent exhibits multiple pharmacological activity towards a number of specific cellular targets. Whilst resistance development towards biocidal agents is highly unlikely, subeffective exposure will lead to the selection of less susceptible clones, modified either in efflux or in their most susceptible target. The latter might also confer resistance to antibiotics where the target is shared. Thus, recent reports have demonstrated that sublethal concentrations of the antibacterial and antifungal agent triclosan can select for resistant mutants in Escherichia coli and that this agent specifically targets the enzyme enoyl reductase that is involved in lipid biosynthesis. Triclosan may, therefore, select for mutants in a target that is shared with the anti-E. coli diazaborine compounds and the antituberculosis drug isoniazid. Although triclosan may be a uniquely specific biocide, sublethal concentrations of less specific antimicrobial agents may also select for mutations within their most sensitive targets, some of which might be common to therapeutic agents. Sublethal treatment with chemical antimicrobial agents has also been demonstrated to induce the expression of multidrug efflux pumps and efflux mutants. Whilst efflux does not confer protection against use concentrations of biocidal products it is sufficient to confer protection against therapeutic doses of many antibiotics. It has, therefore, been widely speculated that biocide misuse may have an insidious effect, contributing to the evolution and persistence of drug resistance within microbial communities. Whilst such notions are supported by laboratory studies that utilize pure cultures, recent evidence has strongly refuted such linkage within the general environment where complex, multispecies biofilms predominate and where biocidal products are routinely deployed. In such situations the competition, for nutrients and space, between community members of disparate sensitivities far outweighs any potential benefits bestowed by the changes in an individual's antimicrobial susceptibility.
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Mucoid strains of P. aeruginosa isolated from the sputa of patients with cystic fibrosis or in vitro through selection with phage were more resistant to carbenicillin, flucloxacillin and tobramycin and more sensitive to tetracycline than related non-mucoid strains. The observations led to the development of a technique for the isolation of mucoid variants in vitro based on enhanced resistance to carbenicillin. Mucoid variants were isolated from various strains of P. aeruginosa and the exopolysaccharide found to be similar to that obtained from mucoid P. aeruginosa isolated from patients with cystic fibrosis.
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Mucoid Pseudomonas aeruginosa is an important respiratory pathogen in patients with cystic fibrosis, and once acquired is virtually impossible to eradicate. Although mucoid P. aeruginosa is generally believed to be resistant to phagocytosis, the mechanism is not understood fully. We studied the nonopsonic phagocytosis by human neutrophils or macrophages of eight mucoid/nonmucoid P. aeruginosa pairs (three isogenic and five "wild-type"). Mucoid strains were relatively resistant to nonopsonic phagocytosis but the nonmucoid types were phagocytosis-susceptible as assessed by visual inspection and chemiluminescence assays. The mucoid and nonmucoid variants had equal numbers of pili but different surface characteristics as determined by biphasic partitioning in polyethylene glycol and dextran. The mucoid exopolysaccharide of mucoid strains appears to alter the surface characteristics of P. aeruginosa thereby rendering them resistant to nonopsonic phagocytosis. The resistance of mucoid variants of P. aeruginosa to nonopsonic phagocytosis may provide a survival advantage to these bacteria early in the course of pulmonary infection before opsonic antibody and complement are present in respiratory secretions.
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We developed a solid-phase radioimmunoassay with a reference standard pseudomonas antigen and used this with 125I-labeled anti-human immunoglobulin to evaluate specific antibodies to Pseudomonas aeruginosa, qualitatively and quantitatively, in sera from children with cystic fibrosis (CF) whose lungs were colonized by this bacterium. The results of this IgG assay correlated with the number of precipitin antibodies to the standard reference antigen determined by cross-immunoelectrophoresis in the same sera. Forced expiratory volume (FEV1; percentage predicted), determined as an indicator of lung injury in CF, was evaluated as an immunologic response to pseudomonas, against a profile derived from combined serial data on both the circulating immune complexes (CIC) and the Ps. aeruginosa antibodies (N = 25 CF patients; 108 sera). This revealed that in CF patients who had no specific IgG antibodies to Ps. aeruginosa and no IgG-CIC had the best pulmonary function (FEV1 = 115 +/- 14.52%) and those with high levels of antibodies to this organism and high IgG-CIC levels had the poorest lung function (FEV1 = 69.75 +/- 10.99%) (P less than 0.05). We believe that this indicates an immunologic basis for lung injury in cystic fibrosis.
Article
The adherence of coagulase-negative staphylococci to smooth surfaces was assayed by measuring the optical densities of stained bacterial films adherent to the floors of plastic tissue culture plates. The optical densities correlated with the weight of the adherent bacterial film (r = 0.906; P less than 0.01). The measurements also agreed with visual assessments of bacterial adherence to culture tubes, microtiter plates, and tissue culture plates. Selected clinical strains were passed through a mouse model for foreign body infections and a rat model for catheter-induced endocarditis. The adherence measurements of animal passed strains remained the same as those of the laboratory-maintained parent strain. Spectrophotometric classification of coagulase-negative staphylococci into nonadherent and adherent categories according to these measurements had a sensitivity, specificity, and accuracy of 90.6, 80.8, and 88.4%, respectively. We examined a previously described collection of 127 strains of coagulase-negative staphylococci isolated from an outbreak of intravascular catheter-associated sepsis; strains associated with sepsis were more adherent than blood culture contaminants and cutaneous strains (P less than 0.001). We also examined a collection of 84 strains isolated from pediatric patients with cerebrospinal fluid (CSF) shunts; once again, pathogenic strains were more adherent than were CSF contaminants (P less than 0.01). Finally, we measured the adherence of seven endocarditis strains. As opposed to strains associated with intravascular catheters and CSF shunts, endocarditis strains were less adherent than were saprophytic strains of coagulase-negative staphylococci. The optical densities of bacterial films adherent to plastic tissue culture plates serve as a quantitative model for the study of the adherence of coagulase-negative staphylococci to medical devices, a process which may be important in the pathogenesis of foreign body infections.
Article
Alginate obtained from a mucoid strain of Pseudomonas aeruginosa was shown to inhibit the phagocytosis of an isogenic non-mucoid revertant by rat alveolar macrophages. Phagocytosis of Staphylococcus albus, binding of sensitized sheep erythrocytes to Fc receptors and uptake of latex particles were also inhibited. These results suggest that the alginate acts as a barrier, surrounding the macrophage preventing the attachment of bacteria or other particles to the plasma membrane. This conclusion was supported by showing that alginic acid, a polysaccharide from seaweed structurally similar to alginate also inhibited the phagocytosis of non-mucoid Ps. aeruginosa. The alginate also inhibited opsonisation of the non-mucoid revertant by a non-agglutinating hyperimmune serum. It is proposed that alginate confers a selective advantage on mucoid producing forms of Ps. aeruginosa by impairing the host immune response by its action on alveolar macrophages and opsonization of bacteria.
Article
Cystic fibrosis is the most common fatal inherited disease of Caucasians. At present, cystic fibrosis accounts for most cases of chronic progressive pulmonary disease and for many other clinical features in the first three decades of life. Thus, it is a challenge to both pediatricians and internists, particularly chest physicians. The diagnosis is based on the triad of chronic obstructive pulmonary disease, pancreatic insufficiency, and increased levels of electrolytes in the sweat. The cardinal test for confirmation of the diagnosis is the "sweat test," which is an excellent discriminant for cystic fibrosis, even in adults. Ancillary features of cystic fibrosis may be of diagnostic assistance (eg, nasal polyposis, Pseudomonas aeruginosa in sputum, azoospermia, and others). Treatment of the pulmonary disease must be emphasized. Choice of antibiotics should be based on the results of sputum culture, but P aeruginosa is the most common pathogen. Removal of secretions by regular postural drainage and percussion is an integral part of the program. Pneumothorax, massive hemoptysis, cor pulmonale, and other complications may be encountered. Sinusitis is almost universal, and nasal polyposis is frequently present. Pancreatic insufficiency occurs in over 80 percent of the patients with cystic fibrosis and may result in intestinal malabsorption. Massive salt loss through the sweat in hot weather, a distinctive type of biliary cirrhosis without jaundice, gallbladder abnormalities, cholelithiasis, and diabetes mellitus also may be found. Of special importance are intestinal obstructive complications (meconium ileus in newborn infants with cystic fibrosis and intestinal obstruction due to fecal accumulation or intussusception in adults). Azoospermia is present in 95 percent of men and there is reduced fertility in women; however, pregnancy does occur in cystic fibrosis. This chronic and ultimately fatal disease produces a predictable set of psychosocial complications.
Article
In order to evaluate the role of mucoid coating of clinical isolates of Pseudomonas aeruginosa as a virulence factor, opsonophagocytosis of mucoid and nonmucoid strains of P. aeruginosa were studied by three different methods: uptake of [3H]adenine-labeled bacteria, oxygen consumption, and chemiluminescence production by phagocytosing polymorphonuclear leukocytes. All three methods showed a strong correlation with regard to phagocytosis of the bacterial isolates. As a group, the mucoid strains of P. aeruginosa demonstrated significantly reduced phagocytic uptake by leukocytes when compared to nonmucoid strains, although occasionally mucoid strains did exhibit normal uptake. In vitro growth of mucoid strains under nonstationary conditions was associated with reduced mucoid coating and with a corresponding increase in susceptibility to leukocyte phagocytosis. Although the overall bactericidal activity of human polymorphonuclear leukocytes for mucoid strains was diminished, this appears to be a function of reduced ingestion since the rate of intracellular killing was similar for mucoid and nonmucoid strains. The enhanced susceptibility of mucoid strains to spontaneous bactericidal activity of pooled human serum was confirmed. Mucoid-producing strains produced less protease in vitro; however, the role of this phenomenon in influencing phagocytosis is unknown.
Article
The interaction between Pseudomonas aeruginosa strains (6 non-mucoid and 4 mucoid strains), serum factors and phagocytic cells was investigated. Strains were incubated in different concentrations of normal serum, chelated serum (with only the alternative complement pathway intact), IgG, Cls, C2 and C3 deficient serum and immune serum. After incubation complement consumption, C3 fixation and phagocytosis by polymorphonuclear leukocytes (PMN) were measured. In contrast to normal serum, immune serum raised against a mucoid and a non-mucoid strain exhibited heat-stable opsonic activity. All ten Pseudomonas aeruginosa strains were able to activate complement in 20% normal serum, leading to deposition of the activated form of the third complement component on the bacterial cell wall and to subsequent recognition and phagocytosis. One mucoid and four non-mucoid strains activated the alternative complement pathway and were effectively opsonized in chelated or in Cls, C2 or IgG deficient serum. Although mucoid strains were less able to activate complement via the alternative route, no differences were observed in opsonic requirements and phagocytosis between mucoid and non-mucoid strains.
Article
Serum opsonophagocytic-killing titers often indicate the level of immune resistance to bacterial pathogens, yet in almost all cystic fibrosis (CF) patients that have chronic lung infections with mucoid Pseudomonas aeruginosa, high titers of opsonic-killing Abs can be measured and the infectious pathology still progresses through pulmonary failure and death. This anomalous finding may be due to the use of suspended cells of P. aeruginosa to evaluate phagocytic killing, whereas in the lungs of CF patients the organisms grow in a microcolony or biofilm, encased in mucoid exopolysaccharide (MEP, also called alginate). To determine whether the microcolony mode of growth contributes to bacterial resistance to host defenses, we evaluated opsonophagocytic killing of mucoid P. aeruginosa growing in a biofilm. Abs from infected CF patients were poorly able to mediate opsonic killing of biofilm, but not suspended, mucoid P. aeruginosa cells. Bacterial resistance to killing could be overcome by disruption of the biofilm layer with an enzyme that degrades MEP. Chronically infected CF patients also fail to produce opsonic-killing Abs specific to MEP, and when these Abs were evaluated in sera of older, noninfected CF patients and humans vaccinated with MEP, comparable killing of P. aeruginosa in biofilms and suspensions was obtained. In this case, C3 was deposited onto the MEP layer and could be visualized by fluorescence microscopy deposited throughout the biofilm. We conclude that opsonic Abs made by CF patients in response to chronic infection are ineffective at mediating phagocytic killing and elimination of bacterial cells growing as microcolonies in their lungs.
Article
Pseudomonas aeruginosa produced alginate and elevated algD (encoding GDPmannose 6-dehydrogenase) transcription under strict anaerobic conditions, especially when using nitrate as a terminal electron acceptor. Purified alginate added to bacterial suspensions caused a decrease in growth, suggesting that alginate contributes to oxygen limitation for the organism and likely for patients afflicted with the inherited autosomal disease cystic fibrosis.
Article
Populations of surface-attached microorganisms comprising either single or multiple species are commonly referred to as biofilms. Using a simple assay for the initiation of biofilm formation (e.g. attachment to an abiotic surface) by Pseudomonas fluorescens strain WCS365, we have shown that: (i) P. fluorescens can form biofilms on an abiotic surface when grown on a range of nutrients; (ii) protein synthesis is required for the early events of biofilm formation; (iii) one (or more) extracytoplasmic protein plays a role in interactions with an abiotic surface; (iv) the osmolarity of the medium affects the ability of the cell to form biofilms. We have isolated transposon mutants defective for the initiation of biofilm formation, which we term surface attachment defective (sad). Molecular analysis of the sad mutants revealed that the ClpP protein (a component of the cytoplasmic Clp protease) participates in biofilm formation in this organism. Our genetic analyses suggest that biofilm formation can proceed via multiple, convergent signalling pathways, which are regulated by various environmental signals. Finally, of the 24 sad mutants analysed in this study, only three had defects in genes of known function. This result suggests that our screen is uncovering novel aspects of bacterial physiology.
Article
Bacteria that attach to surfaces aggregate in a hydrated polymeric matrix of their own synthesis to form biofilms. Formation of these sessile communities and their inherent resistance to antimicrobial agents are at the root of many persistent and chronic bacterial infections. Studies of biofilms have revealed differentiated, structured groups of cells with community properties. Recent advances in our understanding of the genetic and molecular basis of bacterial community behavior point to therapeutic targets that may provide a means for the control of biofilm infections.
Article
This review discusses some diagnostic aspects of cystic fibrosis (CF) including direct mutational analysis. Treatment of major disease manifestations is discussed in more detail with an emphasis on lung disease, in particular chronic infection with Pseudomonas aeruginosa which is responsible for the majority of excess morbidity and mortality. Centralised care and aggressive antimicrobial treatment have led to increased life expectancy and this may be even further increased by the demonstration that chronic P. aeruginosa infection may be prevented, or at least postponed for many years in a majority of patients. Adjunct treatment such as the use of local and systemic anti-inflammatory agents and inhalation of human recombinant DNase are also briefly touched upon. It is emphasised that important questions concerning the link(s) between the mutated gene and lung disease are still missing but that current research raises hope of a more causal treatment in the near future.
Article
Most cystic fibrosis (CF) patients with chronic Pseudomonas aeruginosa lung infection have a persistent acute type lung inflammation dominated by polymorphonuclear neutrophils (PMN) and a pronounced antibody response against P. aeruginosa. We speculated whether this immune response in CF is of the Th2 type and whether a change to a Th1 type immune response could improve the prognosis. Therefore, we studied 14 CF patients with (CF +P) and 14 CF patients without (CF -P) chronic P. aeruginosa lung infection. The specific production of interferon-gamma (IFN-gamma) and interleukin-4 (IL-4) by peripheral blood mononuclear cells was determined. Cells from CF +P patients had lower IFN-gamma (p<0.05) and higher IL-4 (p<0.005) production as compared to cells from CF -P patients. Furthermore, a positive correlation between IFN-gamma production and lung function was found (FVC: Rho = 0.637; p<0.03; FEV1: Rho=0.524; p<0.07). We conclude that a Th2 type immune response is most frequent in CF patients with chronic P. aeruginosa lung infection, and the patients with a Th1-dominated immune response had the best lung function. The clinical implication is that a change to a Th1 type immune response might improve the prognosis in these patients.
Article
Biofilms can be defined as communities of microorganisms attached to a surface. It is clear that microorganisms undergo profound changes during their transition from planktonic (free-swimming) organisms to cells that are part of a complex, surface-attached community. These changes are reflected in the new phenotypic characteristics developed by biofilm bacteria and occur in response to a variety of environmental signals. Recent genetic and molecular approaches used to study bacterial and fungal biofilms have identified genes and regulatory circuits important for initial cell-surface interactions, biofilm maturation, and the return of biofilm microorganisms to a planktonic mode of growth. Studies to date suggest that the planktonic-biofilm transition is a complex and highly regulated process. The results reviewed in this article indicate that the formation of biofilms serves as a new model system for the study of microbial development.
Article
The bacterium Pseudomonas aeruginosa permanently colonizes cystic fibrosis lungs despite aggressive antibiotic treatment. This suggests that P. aeruginosa might exist as biofilms--structured communities of bacteria encased in a self-produced polymeric matrix--in the cystic fibrosis lung. Consistent with this hypothesis, microscopy of cystic fibrosis sputum shows that P. aeruginosa are in biofilm-like structures. P. aeruginosa uses extracellular quorum-sensing signals (extracellular chemical signals that cue cell-density-dependent gene expression) to coordinate biofilm formation. Here we found that cystic fibrosis sputum produces the two principal P. aeruginosa quorum-sensing signals; however, the relative abundance of these signals was opposite to that of the standard P. aeruginosa strain PAO1 in laboratory broth culture. When P. aeruginosa sputum isolates were grown in broth, some showed quorum-sensing signal ratios like those of the laboratory strain. When we grew these isolates and PAO1 in a laboratory biofilm model, the signal ratios were like those in cystic fibrosis sputum. Our data support the hypothesis that P. aeruginosa are in a biofilm in cystic fibrosis sputum. Moreover, quorum-sensing signal profiling of specific P. aeruginosa strains may serve as a biomarker in screens to identify agents that interfere with biofilm development.
Article
Biofilms are formed by a spectrum of microorganisms, including pathogens, and provide a means for these organisms to protect themselves against antimicrobial agents. Several mechanisms have been proposed to explain this phenomenon of resistance within biofilms, including delayed penetration of the antimicrobial into the biofilm extracellular matrix, slowing of growth rate of organisms within the biofilm, or other physiologic changes brought about by interaction of the organisms with a surface. The practical implications of biofilm formation are that alternative control strategies must be devised both for testing the susceptibility of the organisms within the biofilm and treating the established biofilm to alter its structure. A number of testing protocols have been developed. Effective treatment strategies will incorporate antimicrobials or other agents that have been demonstrated to penetrate and kill biofilm organisms, or treatments that disrupt or target specific components of the biofilm matrix. A better understanding of the role of biofilms in infection and how in vivo biofilms respond to selected treatments requires more study.
Article
Biofilms are communities of microorganisms attached to a surface. It has become clear that biofilm-grown cells express properties distinct from planktonic cells, one of which is an increased resistance to antimicrobial agents. Recent work has indicated that slow growth and/or induction of an rpoS-mediated stress response could contribute to biocide resistance. The physical and/or chemical structure of exopolysaccharides or other aspects of biofilm architecture could also confer resistance by exclusion of biocides from the bacterial community. Finally, biofilm-grown bacteria might develop a biofilm-specific biocide-resistant phenotype. Owing to the heterogeneous nature of the biofilm, it is likely that there are multiple resistance mechanisms at work within a single community. Recent research has begun to shed light on how and why surface-attached microbial communities develop resistance to antimicrobial agents.