Nonmucoid Isolates Research Articles

Identification of hypermucoviscous Klebsiella pneumoniae K1, K2, K54 and K57 capsular serotypes by Raman spectroscopy

Antimicrobial resistance poses a significant challenge in modern medicine, affecting public health. Klebsiella pneumoniae infections compound this issue due to their broad range of infections and the emergence of multiple antibiotic resistance mechanisms. Efficient detection of its capsular serotypes is crucial for immediate patient treatment, epidemiological tracking and outbreak containment. Current methods have limitations that can delay interventions and increase the risk of morbidity and mortality. Raman spectroscopy is a promising alternative to identify capsular serotypes in hypermucoviscous K. pneumoniae isolates. It provides rapid and in situ measurements with minimal sample preparation. Moreover, its combination with machine learning tools demonstrates high accuracy and reproducibility. This study analyzed the viability of combining Raman spectroscopy with one-dimensional convolutional neural networks (1-D CNN) to classify four capsular serotypes of hypermucoviscous K. pneumoniae: K1, K2, K54 and K57. Our approach involved identifying the most relevant Raman features for classification to prevent overfitting in the training models. Simplifying the dataset to essential information maintains accuracy and reduces computational costs and training time. Capsular serotypes were classified with 96 % accuracy using less than 30 Raman features out of 2400 contained in each spectrum. To validate our methodology, we expanded the dataset to include both hypermucoviscous and non-mucoid isolates and distinguished between them. This resulted in an accuracy rate of 94 %. The results obtained have significant potential for practical healthcare applications, especially for enabling the prompt prescription of the appropriate antibiotic treatment against infections.

Non-starved mucoid Pseudomonas aeruginosa isolates cause a high death rate in strains of mice.

Pseudomonas aeruginosa is an opportunistic pathogen that affects many patients in a hospital setting as well as immunocompromised patients. Pseudomonas aeruginosa is resistant to many antibiotics because of the formation of biofilms and pigmentations serving as virulence factors. Pseudomonas aeruginosa isolates are either mucoid or alginate-producing and non-alginate-producing (non-mucoid) isolates. The main objective of this experiment was to analyze the pathogenicity of mucoid and non-mucoid isolates in P. aeruginosa in a mouse model. We hypothesized that mucoid isolates are more pathogenic compared to non-pathogenic ones. Pseudomonas aeruginosa isolates PAO1 (non-mucoid) and 2192 (mucoid) were used intranasally to infect and monitor mortality rates. Isolates starved in water for at least three months were tested for infectivity. The mucoid isolate 2192 showed a 67% death rate compared to 33% of PAO1, suggesting the mucoid is more pathogenic to BALB/c mice than the non-mucoid PAO1. The death rate of mice infected with strain DBA susceptible to P. aeruginosa infection was higher than that of the BALB/c strain. The results also suggest that non-starved bacteria are more pathogenic to mice than starved bacteria. We extend our study by infecting mouse strains under hindlimb-unloading (HU) conditions with P. aeruginosa mucoid and non-mucoid isolates. Our results show that HU conditions enhance infection, suggesting the effect of stress and physiological changes could lead to susceptibility to infection. Our data indicate that the mucoid isolate is more likely to kill all mouse strains.

Strain-specific predation of Bdellovibrio bacteriovorus on Pseudomonas aeruginosa with a higher range for cystic fibrosis than for bacteremia isolates

This work aimed to evaluate the predatory activity of Bdellovibrio bacteriovorus 109J on clinical isolates of Pseudomonas aeruginosa selected from well-characterized collections of cystic fibrosis (CF) lung colonization (n = 30) and bloodstream infections (BSI) (n = 48) including strains selected by genetic lineage (frequent and rare sequence types), antibiotic resistance phenotype (susceptible and multidrug-resistant isolates), and colony phenotype (mucoid and non-mucoid isolates). The intraspecies predation range (I-PR) was defined as the proportion of susceptible strains within the entire collection. In contrast, the predation efficiency (PE) is the ratio of viable prey cells remaining after predation compared to the initial inoculum. I-PR was significantly higher for CF (67%) than for BSI P. aeruginosa isolates (35%) probably related to an environmental origin of CF strains whereas invasive strains are more adapted to humans. I-PR correlation with bacterial features such as mucoid morphotype, genetic background, or antibiotic susceptibility profile was not detected. To test the possibility of increasing I-PR of BSI isolates, a polyhydroxyalkanoate depolymerase deficient B. bacteriovorus bd2637 mutant was used. Global median I-PR and PE values remained constant for both predators, but 31.2% of 109J-resistant isolates were susceptible to the mutant, and 22.9% of 109J-susceptible isolates showed resistance to predation by the mutant, pointing to a predator–prey specificity process. The potential use of predators in the clinical setting should be based on the determination of the I-PR for each species, and the PE of each particular target strain.

Characterization of group A beta-haemolytic streptococcus with mucoid phenotype isolated in a tertiary hospital

IntroductionThe objective of this study is to characterize Streptococcus pyogenes isolates with a mucoid phenotype and to compare them with non-mucoid isolates obtained between April and August 2016. Material and methodsIdentification and antimicrobial susceptibility were performed in all isolates. The emm type and exotoxin genes speA, speB, speC, speF, speG, speH, speJ, speZ and ssa were analyzed. Clinical and demographic data were collected. ResultsFrom 96 isolates analyzed, 47% had a mucoid phenotype and 95.5% of them presented speA-speB-speF-speG-ssa genes and emm3 genotype. The main clinical manifestation was pharyngotonsillitis (77.1%) evolving to scarlet fever in 67.5% of the cases. ConclusionThis study describes the circulation of a mucoid phenotype strain with a speA-speB-speF-speG-ssa toxin profile and emm3.1 genotype considered one of the most frequent and virulent of SGA.

Bacterin Vaccination Provides Insufficient Protection Against Streptococcus equi Subspecies zooepidemicus Infection in Pigs.

Streptococcus equi subspecies zooepidemicus (SEZ) is a zoonotic pathogen capable of causing severe disease in many mammalian species. Historically, SEZ has not been a common cause of disease in pigs in North America; however, in 2019, SEZ caused mortality events leading to severe illness and 30–50% mortality in exposed animal groups. Because of the rapid progression of disease, it is important to investigate intervention strategies to prevent disease development. In this study, pigs were divided into four groups: (1) vaccinated with an inactivated SEZ vaccine generated from a highly mucoid 2019 mortality event isolate; (2) vaccinated with an inactivated SEZ vaccine generated from a genetically similar, non-mucoid isolate from a guinea pig; (3) and (4) sham vaccinated. Following boost vaccination, groups 1–3 were challenged with a 2019 mortality event isolate and group 4 were non-challenged controls. Antibody titers were higher for SEZ vaccinated animals than sham vaccinated animals; however, no anamnestic response was observed, and titers were lower than typically seen following the use of inactivated vaccines. Vaccination did not provide protection from disease development or mortality following challenge, which could be associated with the comparatively low antibody titers generated by vaccination. Surviving pigs also remained colonized and transmitted SEZ to naïve contact pigs 3 weeks following challenge, indicating that healthy animals can act as a source of SEZ exposure. Future investigation should evaluate different vaccine formulations, such as increased antigen load or an alternative adjuvant, that could induce a more robust adaptive immune response.

Pseudomonas aeruginosa Affects Airway Epithelial Response and Barrier Function During Rhinovirus Infection

Cystic fibrosis (CF) lung disease is aggravated by recurrent and ultimately chronic bacterial infections. One of the key pathogens in adult CF lung disease is P. aeruginosa (PA). In addition to bacteria, respiratory viral infections are suggested to trigger pulmonary exacerbations in CF. To date, little is known on how chronic infections with PA influence susceptibility and response to viral infection. We investigated the interactions between PA, human rhinovirus (HRV) and the airway epithelium in a model of chronic PA infection using differentiated primary bronchial epithelial cells (pBECs) and clinical PA isolates obtained from the respiratory sample of a CF patient. Cells were repeatedly infected with either a mucoid or a non-mucoid PA isolate for 16 days to simulate chronic infection, and subsequently co-infected with HRV. Key cytokines and viral RNA were quantified by cytometric bead array, ELISA and qPCR. Proteolytic degradation of IL-6 was analyzed by Western Blots. Barrier function was assessed by permeability tests and transepithelial electric resistance measurements. Virus infection stimulated the production of inflammatory and antiviral mediators, including interleukin (IL)-6, CXCL-8, tumor necrosis factor (TNF)-α, and type I/III interferons. Co-infection with a non-mucoid PA isolate increased IL-1β protein concentrations (28.88 pg/ml vs. 6.10 pg/ml), but in contrast drastically diminished levels of IL-6 protein (53.17 pg/ml vs. 2301.33 pg/ml) compared to virus infection alone. Conditioned medium obtained from co-infections with a non-mucoid PA isolate and HRV was able to rapidly degrade recombinant IL-6 in a serine protease-dependent manner, whereas medium from individual infections or co-infections with a mucoid isolate had no such effect. After co-infection with HRV and the non-mucoid PA isolate, we detected lower mRNA levels of Forkhead box J1 (FOXJ1) and Cilia Apical Structure Protein (SNTN), markers of epithelial cell differentiation to ciliated cells. Moreover, epithelial permeability was increased and barrier function compromised compared to single infections. These data show that PA infection can influence the response of bronchial epithelial cells to viral infection. Altered innate immune responses and compromised epithelial barrier function may contribute to an aggravated course of viral infection in PA-infected airways.

Caracterización de estreptococos beta-hemolíticos del grupo A con fenotipo mucoide aislados en un hospital de tercer nivel

IntroductionThe objective of this study is to characterize Streptococcus pyogenes isolates with a mucoid phenotype and to compare them with non-mucoid isolates obtained between April and August 2016. Material and methodsIdentification and antimicrobial susceptibility were performed in all isolates. The emm type and exotoxin genes speA, speB, speC, speF, speG, speH, speJ, speZ and ssa were analyzed. Clinical and demographic data were collected. ResultsFrom 96 isolates analyzed, 47% had a mucoid phenotype and 95.5% of them presented speA-speB-speF-speG-ssa genes and emm3 genotype. The main clinical manifestation was pharyngotonsillitis (77.1%) evolving to scarlet fever in 67.5% of the cases. ConclusionThis study describes the circulation of a mucoid phenotype strain with a speA-speB-speF-speG-ssa toxin profile and emm3.1 genotype considered one of the most frequent and virulent of SGA.

Acquired mucoid phenotype of Acinetobacter baumannii: Impact for the molecular characteristics and virulence

Mucoid phenotype is an important adaptive defense response for Acinetobacter baumannii (A. baumannii). The aim of this study was to analyze the impact of mucoid phenotype for the molecular characteristics and virulence of A. baumannii. We observed that the colonies of mucoid A. baumannii were moist, with an elevated surface, and the wire drawing result was positive. Transmission electron microscopy data showed that the outer wall of the mucoid colonies was not smooth, had protruding pseudopodia, and was surrounded by a layer of unknown material. Antibiotic susceptibility testing showed that the mucoid strains were multidrug resistant. Notably, the mucoid phenotype and antibiotic resistance were not correlated with the amount of biofilm produced by A. baumannii. MLST data demonstrated that the mucoid A. baumannii strains belonged to type ST2. Most (82.6 %, 38/46) of the multidrug-resistant nonmucoid strains also belonged to the molecular type ST2 and to other types, including ST129, ST158, ST195, ST80 and ST3. Moreover, mucoid A. baumannii strains were more virulent than nonmucoid isolates in a mouse model. The comparative transcriptomic data indicated that 15 genes, especially IX87_RS16955 (acnA), IX87_RS10800 (XanP), IX87_RS12875 (GlmM), IX87_RS00885 and IX87_RS12395 (bfr), were possibly associated with the phenotype and virulence of mucoid A. baumannii. In conclusions, the study comprehensively describes the molecular characteristics and virulence regulatory mechanism of mucoid A. baumannii, and provides novel insights for the prevention and treatment of infections associated with these strains.

Kinetic studies on the expression of alginate and extracellular proteins by Pseudomonas aeruginosa FRD1 and PAO1.

Pseudomonas aeruginosa is characterized by its capability to produce extracellular virulence proteins and to establish biofilm-based infections that do not respond easily to conventional treatments. However, the physiological conditions that decrease the fitness of such a persistent pathogen would assist the host to defend itself and reduce the infection prevalence. Therefore, developing treatments against P. aeruginosa requires a quantitative understanding of the relationship between bacterial growth kinetics and secretion of alginate and proteins, in addition to the ecological factors that control their synthesis. For this purpose, we examined various environmental factors that affect the specific product yield coefficients (expressed as g product/OD600) of alginate and extracellular proteins using a mucoid (FRD1) and a non-mucoid (PAO1) clinical isolate of P. aeruginosa, respectively. The results suggested magnesium sulfate, trace elements and hydrogen peroxide as significant variables that positively affect alginate synthesis by the FRD1 cells. However, the production of extracellular proteins by PAO1 was negatively affected by the concentration of ferrous sulfate. For understanding the kinetics of expressing alginate and extracellular proteins by the cells, a well-controlled 5 L tank bioreactor was used. The results suggested that under the bioreactor controlled conditions, both alginate and extracellular proteins are expressed parallel to biomass increase in the cells of P. aeruginosa.

Evolution of Klebsiella pneumoniae with mucoid and non-mucoid type colonies within a single patient

We obtained nine Klebsiella pneumoniae isolates successively isolated from a single patient. Four pairs (M1–M4 and NM1–NM4) obtained simultaneously from the same site showed different colony types, mucoid and non-mucoid, while the final isolate (M5) was isolated alone from the blood and showed a mucoid phenotype. The whole genome of isolate M5 was sequenced de novo using the PacBio RSII system, while the others were sequenced with an Illumina Hiseq4000 and mapped to the genome sequences of M5. To identify insertions or deletions in the cps locus, we amplified and sequenced cps locus genes. We identified insertion sequence (IS) elements in several genes of the cps locus or one amino acid substitution in WcaJ in all non-mucoid isolates. Five additional amino acid alterations in RpsJ, LolE, Lon-2, PpsE, and a hypothetical protein were detected in some mucoid and non-mucoid isolates. Based on the genome data and cps locus sequences, the mucoid phenotype may have been lost or converted into the non-mucoid phenotype because of the insertion of IS elements or amino acid alterations at this locus. We inferred a within-host evolutionary scenario, in which non-mucoid variants emerged repeatedly from mucoid isolates, but may be short-lived because of their low fitness.

Remodeling of O Antigen in Mucoid Pseudomonas aeruginosa via Transcriptional Repression of wzz2.

Pseudomonas aeruginosa is an opportunistic pathogen that causes chronic lung infections in people with cystic fibrosis (CF). Chronic P. aeruginosa isolates generally do not express O antigen and often have a mucoid phenotype, which is characterized by the overproduction of the exopolysaccharide alginate. Therefore, O antigen expression and the mucoid phenotype may be coordinately regulated upon chronic adaption to the CF lung. Here we demonstrate that PDO300, a mucoid strain derived from the nonmucoid laboratory isolate PAO1, does not produce very long O antigen due to decreased expression of Wzz2, the very long O antigen chain length control protein, and that mucoid clinical isolates express reduced levels of Wzz2 compared to nonmucoid isolates. Further, we show that forcing the expression of very long O antigen by PDO300, by providing wzz2 in trans, does not alter alginate production, suggesting that sugar precursors are not limited between the two biosynthesis pathways. Moreover, we confirm that AmrZ, a transcription factor highly expressed in mucoid strains, is a negative regulator of wzz2 promoter activity and very long O antigen expression. These experiments identify the first transcriptional regulator of O antigen chain length in P. aeruginosa and support a model where transition to a chronic mucoid phenotype is correlated with downregulation of very long O antigen through decreased Wzz2 production.IMPORTANCE Detection of mucoid Pseudomonas aeruginosa, characterized by the overproduction of alginate, is correlated with the establishment of a chronic pulmonary infection and disease progression in people with cystic fibrosis (CF). In addition to the overproduction of alginate, loss of O antigen lipopolysaccharide production is also selected for in chronic infection isolates. In this study, we have identified the regulatory network that inversely regulates O antigen and alginate production. Understanding the regulation of these chronic phenotypes will elucidate mechanisms that are important for the establishment of a long-term P. aeruginosa lung infection and ultimately provide an opportunity for intervention. Preventing P. aeruginosa from chronically adapting to the CF lung environment could provide a better outcome for people who are infected.

Adaptation of Pseudomonas aeruginosa to the chronic phenotype by mutations in the algTmucABD operon in isolates from Brazilian cystic fibrosis patients.

Chronic lung infection by Pseudomonas aeruginosa is the leading cause of morbidity and mortality in cystic fibrosis (CF) patients. This is associated with the conversion of the non-mucoid to the mucoid phenotype. However, there is little information about the occurrence of alginate-producing P. aeruginosa in CF patients outside Europe and North America. The aim of the present study was to investigate mutations in the algTmucABD operon in mucoid and non-mucoid isolates from Brazilian CF patients. Twenty-seven mucoid and 37 non-mucoid isolates from 40 CF patients chronically infected by P. aeruginosa attending a CF reference center in Brazil were evaluated by sequence analysis. Mutations in mucA were observed in 93% of the mucoid isolates and 54% of the non-mucoid isolates. Among these non-mucoid isolates, 55% were considered revertants, since they also had mutations in algT (algU). Most isolates associated with moderate alginate production presented point mutations in mucB and/or mucD. We identified 30 mutations not previously described in the operon. In conclusion, mutations in mucA were the main mechanism of conversion to mucoidy, and most of the non-mucoid isolates were revertants, but the mechanism of revertance is not fully explained by changes in algT.

Hyperencapsulated mucoid pneumococcal isolates from patients with cystic fibrosis have increased biofilm density and persistence in vivo.

Mucoid bacteria, predominately Pseudomonas aeruginosa, are commonly associated with decline in pulmonary function in children with cystic fibrosis (CF), and are thought to persist at least in part due to a greater propensity toward forming biofilms. We isolated a higher frequency of mucoid Streptococcus pneumoniae (Sp) expressing high levels of capsular polysaccharides from sputa from children with CF, compared to those without CF. We compared biofilm formation and maturation by mucoid and non-mucoid isolates of Sp collected from children with and without CF. Non-mucoid Sp serotype 19A and 19F isolates had significantly higher levels of biofilm initiation and adherence to CF epithelial cells than did serotype 3 isolates. However, strains expressing high levels of capsule had significantly greater biofilm maturation, as evidenced by increased density and thickness in static and continuous flow assays via confocal microscopy. Finally, using a serotype 3 Sp strain, we showed that highly encapsulated mucoid phase variants predominate during late adherence and better colonize CFTR-/- as compared to wild-type mice in respiratory infection studies. These findings indicate that overexpression of capsule can enhance the development of mature pneumococcal biofilms in vitro, and may contribute to pneumococcal colonization in CF lung disease.

An Antipersister Strategy for Treatment of Chronic Pseudomonas aeruginosa Infections.

Bacterial persisters are a quasidormant subpopulation of cells that are tolerant to antibiotic treatment. The combination of the aminoglycoside tobramycin with fumarate as an antibacterial potentiator utilizes an antipersister strategy that is aimed at reducing recurrent Pseudomonas aeruginosa infections by enhancing the killing of P. aeruginosa persisters. Stationary-phase cultures of P. aeruginosa were used to generate persister cells. A range of tobramycin concentrations was tested with a range of metabolite concentrations to determine the potentiation effect of the metabolite under a variety of conditions, including a range of pH values and in the presence of azithromycin or cystic fibrosis (CF) patient sputum. In addition, 96-well dish biofilm and colony biofilm assays were performed, and the cytotoxicity of the tobramycin-fumarate combination was determined utilizing a lactate dehydrogenase (LDH) assay. Enhanced killing of up to 6 orders of magnitude of P. aeruginosa persisters over a range of CF isolates, including mucoid and nonmucoid strains, was observed for the tobramycin-fumarate combination compared to killing with tobramycin alone. Furthermore, significant fumarate-mediated potentiation was seen in the presence of azithromycin or CF patient sputum. Fumarate also reduced the cytotoxicity of tobramycin-treated P. aeruginosa to human epithelial airway cells. Finally, in mucoid and nonmucoid CF isolates, complete eradication of P. aeruginosa biofilm was observed in the colony biofilm assay due to fumarate potentiation. These data suggest that a combination of tobramycin with fumarate as an antibacterial potentiator may be an attractive therapeutic for eliminating recurrent P. aeruginosa infections in CF patients through the eradication of bacterial persisters.

A Low-Molecular-Weight Alginate Oligosaccharide Disrupts Pseudomonal Microcolony Formation and Enhances Antibiotic Effectiveness

In chronic respiratory disease, the formation of dense, 3-dimensional "microcolonies" by Pseudomonas aeruginosa within the airway plays an important role in contributing to resistance to treatment. An in vitro biofilm model of pseudomonal microcolony formation using artificial-sputum (AS) medium was established to study the effects of low-molecular-weight alginate oligomers (OligoG CF-5/20) on pseudomonal growth, microcolony formation, and the efficacy of colistin. The studies employed clinical cystic fibrosis (CF) isolates (n = 3) and reference nonmucoid and mucoid multidrug-resistant (MDR) CF isolates (n = 7). Bacterial growth and biofilm development and disruption were studied using cell viability assays and image analysis with scanning electron and confocal laser scanning microscopy. Pseudomonal growth in AS medium was associated with increased ATP production (P < 0.05) and the formation (at 48 h) of discrete (>10-μm) microcolonies. In conventional growth medium, colistin retained an ability to inhibit growth of planktonic bacteria, although the MIC was increased (0.1 to 0.4 μg/ml) in AS medium compared to Mueller-Hinton (MH) medium. In contrast, in an established-biofilm model in AS medium, the efficacy of colistin was decreased. OligoG CF-5/20 (≥2%) treatment, however, induced dose-dependent biofilm disruption (P < 0.05) and led to colistin retaining its antimicrobial activity (P < 0.05). While circular dichroism indicated that OligoG CF-5/20 did not change the orientation of the alginate carboxyl groups, mass spectrometry demonstrated that the oligomers induced dose-dependent (>0.2%; P < 0.05) reductions in pseudomonal quorum-sensing signaling. These findings reinforce the potential clinical significance of microcolony formation in the CF lung and highlight a novel approach to treat MDR pseudomonal infections.

Small Colony Variants of Pseudomonas aeruginosa Display Heterogeneity in Inhibiting Aspergillus fumigatus Biofilm.

Pseudomonas aeruginosa and Aspergillus fumigatus are major microbes in cystic fibrosis (CF). We reported non-mucoid P. aeruginosa isolates more inhibitory to A. fumigatus than mucoid ones. Another CF P. aeruginosa phenotype, small colony variants (SCVs), is an unknown factor in intermicrobial competition with A. fumigatus. Clinical SCV isolates and reference CF non-mucoid isolate (Pa10, producing normal-sized colonies) were compared. Live cells of P. aeruginosa or filtrates from P. aeruginosa planktonic or biofilm cultures were co-incubated with A. fumigatus growing under conditions allowing biofilm formation or with preformed biofilm. Metabolic activity of A. fumigatus biofilm was then measured. When necessary, assays were done after adjustment for growth differences by adding fresh medium to the planktonic culture filtrate. Pyoverdine determinations were performed spectrophotometrically on the planktonic culture filtrates. In all experimental conditions (live cells and planktonic or biofilm culture filtrates of P. aeruginosa versus A. fumigatus biofilm formation or preformed biofilm), three SCV isolates were less inhibitory than Pa10, two equal or more inhibitory. Adjusting planktonic culture filtrates for growth differences showed SCV inhibition differences variably related to growth or deficient inhibitor production. Studies suggested the principal P. aeruginosa inhibitor to be pyoverdine. SCV isolates appear heterogeneous in their capacity to inhibit A. fumigatus biofilm. SCV isolates can be important in the CF microbiome, because they are capable of intermicrobial inhibition.

Exopolysaccharide-Repressing Small Molecules with Antibiofilm and Antivirulence Activity against Pseudomonas aeruginosa.

Biofilm formation is a universal virulence strategy in which bacteria grow in dense microbial communities enmeshed within a polymeric extracellular matrix that protects them from antibiotic exposure and the immune system. Pseudomonas aeruginosa is an archetypal biofilm-forming organism that utilizes a biofilm growth strategy to cause chronic lung infections in cystic fibrosis (CF) patients. The extracellular matrix of P. aeruginosa biofilms is comprised mainly of exopolysaccharides (EPS) and DNA. Both mucoid and nonmucoid isolates of P. aeruginosa produce the Pel and Psl EPS, each of which have important roles in antibiotic resistance, biofilm formation, and immune evasion. Given the central importance of the EPS for biofilms, they are attractive targets for novel anti-infective compounds. In this study, we used a high-throughput gene expression screen to identify compounds that repress expression of the pel genes. The pel repressors demonstrated antibiofilm activity against microplate and flow chamber biofilms formed by wild-type and hyperbiofilm-forming strains. To determine the potential role of EPS in virulence, pel/psl mutants were shown to have reduced virulence in feeding behavior and slow killing virulence assays in Caenorhabditis elegans The antibiofilm molecules also reduced P. aeruginosa PAO1 virulence in the nematode slow killing model. Importantly, the combination of antibiotics and antibiofilm compounds increased killing of P. aeruginosa biofilms. These small molecules represent a novel anti-infective strategy for the possible treatment of chronic P. aeruginosa infections.

Dynamic in vivo mutations within the ica operon during persistence of Staphylococcus aureus in the airways of cystic fibrosis patients.

Cystic fibrosis (CF) is associated with chronic bacterial airway infections leading to lung insufficiency and decreased life expectancy. Staphylococcus aureus is one of the most prevalent pathogens isolated from the airways of CF patients. Mucoid colony morphology has been described for Pseudomonas aeruginosa, the most common pathogen in CF, but not for S. aureus. From the airways of 8 of 313 CF patients (2.5%) mucoid S. aureus isolates (n = 115) were cultured with a mean persistence of 29 months (range 1 month, 126 months). In contrast to non-mucoid S. aureus, mucoid isolates were strong biofilm formers. The upstream region of the ica operon, which encodes the proteins responsible for the synthesis of the polysaccharide intercellular adhesin (PIA), of mucoid isolates was sequenced. Spa-types of mucoid and non-mucoid strains were identical, but differed between patients. Mucoid isolates carried a 5 bp deletion in the intergenic region between icaR and icaA. During long-term persistence, from two patients subsequent non-mucoid isolates (n = 12) with 5 bp deletions were cultured, which did not produce biofilm. Sequencing of the entire ica operon identified compensatory mutations in various ica-genes including icaA (n = 7), icaD (n = 3) and icaC (n = 2). Six sequential isolates of each of these two patients with non-mucoid and mucoid phenotypes were subjected to whole genome sequencing revealing a very close relationship of the individual patient’s isolates. Transformation of strains with vectors expressing the respective wild-type genes restored mucoidy. In contrast to the non-mucoid phenotype, mucoid strains were protected against neutrophilic killing and survived better under starvation conditions. In conclusion, the special conditions present in CF airways seem to facilitate ongoing mutations in the ica operon during S. aureus persistence.

Mucoid and Nonmucoid Pseudomonas aeruginosa Isolated from Respiratory Tract Infections

ABSTRACT Introduction Pseudomonas aeruginosa is a common etiological agent causing various infections. The isolates can be mucoid or nonmucoid. It harbors innate resistance to a broad range of antibiotics. Objectives The aim of this study is to determine the frequency of the mucoid and nonmucoid isolates along with their antibiogram from respiratory samples. Materials and methods A total of 220 isolates of P. aeruginosa were obtained from various clinical specimens and 72 from respiratory samples. Pseudomonas aeruginosa was identified by standard colony morphology and bacteriological methods, and antibiotic sensitivity was determined by the Kirby Bauer method. Results Out of the 72 respiratory samples, 15 (20.83%) were mucoid strains and 57 (79.87%) nonmucoid strains. The mucoid colonies showed high resistance to antibiotics, such as co-trimoxazole (COT) (73.33%), ciprofloxacin (CIP) (60%), and ceftazidime (CAZ) (53.33%), whereas high resistance rate among the nonmucoid isolates were seen in (COT) (78.95%), (CIP) (71.93%), (CAZ) (54.39%), cefepime (52.63%). Conclusion The antibiotic susceptibility pattern helps in constituting the guidelines for treatment and management of the infections by P. aeruginosa. How to cite this article Mishra PP, Prakash V. Mucoid and Nonmucoid Pseudomonas aeruginosa Isolated from Respiratory Tract Infections. Int J Adv Integ Med Sci 2016;1(2):43-45.

The Effects of CFTR and Mucoid Phenotype on Susceptibility and Innate Immune Responses in a Mouse Model of Pneumococcal Lung Disease.

Recent studies have reported the isolation of highly mucoid serotype 3 Streptococcus pneumoniae (Sp) from the respiratory tracts of children with cystic fibrosis (CF). Whether these highly mucoid Sp contribute to, or are associated with, respiratory failure among patients with CF remains unknown. Other mucoid bacteria, predominately Pseudomonas aeruginosa, are associated with CF respiratory decline. We used a mouse model of CF to study pneumococcal pneumonia with highly mucoid serotype 3 and non-mucoid serotype 19A Sp isolates. We investigated susceptibility to infection, survival, and bacterial counts from bronchoaviolar lavage samples and lung homogenates, as well as associated inflammatory cytokines at the site of infection, and lung pathology. Congenic CFTR–/– mice and wild-type (WT)-mice were infected intranasally with CHB756, CHB1126, and WU2 (highly mucoid capsular serotype 3, intermediately mucoid serotype 3, and less mucoid serotype 3, respectively), or CHB1058 (non-mucoid serotype 19A). BAL, lung homogenates, and blood were collected from mice 5 days post-infection. Higher CFU recovery and shorter survival were observed following infection of CFTR–/– mice with CHB756 compared to infection with CHB1126, WU2, or CHB1058 (P≤0.001). Additionally, CFTR–/– mice infected with CHB756 and CHB1126 were more susceptible to infection than WT-mice (P≤0.05). Between CFTR–/– mice and WT-mice, no significant differences in TNF-α, CXCL1/KC concentrations, or lung histopathology were observed. Our results indicate that highly mucoid type 3 Sp causes more severe lung disease than non-mucoid Sp, and does so more readily in the lungs of CFTR–/– than WT-mice.