Mucoid Isolates Research Articles

At what cost? The impactof bacteriophage resistance on the growth kinetics and protein synthesis of Escherichia coli.

Cost of bacteriophage resistance (COR) is important in explaining processes of diversification and coexistence in microbial communities. COR can be expressed in different traits, and the lack of universally applicable methods to measure fitness trade-offs makes COR challenging to study. Due to its fundamental role in growth, we explored protein synthesis as a target for quantifying COR. In this study, the growth kinetics of three genome-sequenced strains of phage-resistant Escherichia coli, along with the phage-susceptible wild-type, were characterized over a range of glucose concentrations. Bioorthogonal non-canonical amino acid tagging (BONCAT) was used to track differences in protein synthetic activity between the wild-type and phage-resistant E. coli. Two of the resistant strains, with different levels of phage susceptibility, showed mucoid phenotypes corresponding with mutations in genes associated with the Rcs phosphorelay. These mucoid isolates, however, had reduced growth rates and potentially lower protein synthetic activity. Another resistant isolate with a different mutational profile maintained the same growth rate as the wild-type and showed increased BONCAT fluorescence, but its yield was lower. Together, these findings present different patterns of trade-offs resulting from the phage-induced mutations and demonstrate the potential applicability of BONCAT as a tool for measuring COR.

In vivo and in vitro analyses of the role of the Prc protease in inducing mucoidy in Pseudomonas aeruginosa.

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.

Clinical, phenotypic characterization and genomic analysis of the mucoid Acinetobacter baumannii from a teaching hospital

BackgroundAcinetobacter baumannii (A. baumannii) has become a significant nosocomial pathogen globally over the past decade due to the increasing prevalence of antibiotic-resistant isolates. The formation of the mucoid phenotype is a crucial adaptive defense response to external pressure, but the clinical, phenotypic and genotypic characteristics and their relationship with sequence types (ST) and K locus (KL) types remain unclear. MethodsIn this study, we screened a total of 736 A. baumannii isolates, from which we identified and characterized 13 mucoid isolates. The study explored the clinical characteristics of patients with mucoid isolates, investigated the mucoid phenotype, performed capsule observation, quantified capsule production, and assessed antimicrobial susceptibility. Subsequently, whole-genome sequencing (WGS) was used to analyze the sequence types (ST), loci for capsular polysaccharide (KL), antibiotic resistance genes, virulence genes, and core-genome single-nucleotide polymorphisms (SNPs). Additionally, the virulence of all mucoid strains was evaluated through serum resistance assay, biofilm-forming assay, and Galleria mellonella survival assay. ResultsAll mucoid A. baumannii isolates were found to be encapsulated and extremely drug-resistant. Among patients infected with these isolates, 92.3 % had pulmonary infections, and the 30-day mortality rate was 61.5 %. The analysis revealed that not all strains are highly virulent. Whole-genome sequencing (WGS) identified the sequence types as ST136, ST208, ST381, ST195, and ST281, and the capsular types as KL77, KL7, KL33, KL2, and KL3. The ST208 and KL7 isolates exhibited higher virulence and greater biofilm formation, with KL7 isolates also showing higher capsule production. Despite these differences, no significant variations in virulence genes were observed among the mucoid isolates, except for biofilm-associated and quorum-sensing genes. The highly virulent ST208/KL7 strains (AB276, AB313, and AB552) lacked biofilm-associated genes (csuA/BABCDE), indicating these genes do not directly cause differences in biofilm formation. ConclusionThe mucoid A. baumannii isolates were extensively drug-resistant, and infections caused by these isolates could lead to higher mortality. However, not all strains had high virulence, with variations likely related to specific sequence types (ST) and K locus (KL) types.

In vivo and in vitro analysis of the role of the Prc protease in inducing mucoidy in Pseudomonas aeruginosa.

After colonizing the lungs of individuals with cystic fibrosis, P. 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.

Various mutations in icaR, the repressor of the icaADBC locus, occur in mucoid Staphylococcus aureus isolates recovered from the airways of people with cystic fibrosis

Staphylococcus aureus is one of the major pathogens isolated from the airways of people with cystic fibrosis (pwCF). Recently, we described a mucoid S. aureus phenotype from respiratory specimens of pwCF, which constitutively overproduced biofilm that consisted of polysaccharide intercellular adhesin (PIA) due to a 5bp-deletion (5bp-del) in the intergenic region of the intercellular adhesin (ica) locus.Since we were not able to identify the 5bp-del in mucoid isolates of two pwCF with long-term S. aureus persistence and in a number of mucoid isolates of pwCF from a prospective multicenter study, these strains were (i) characterized phenotypically, (ii) investigated for biofilm formation, and (iii) molecular typed by spa-sequence typing. To screen for mutations responsible for mucoidy, the ica operon of all mucoid isolates was analyzed by Sanger sequencing. Whole genome sequencing was performed for selected isolates.For all mucoid isolates without the 5 bp-del, various mutations in icaR, which is the transcriptional repressor of the icaADBC operon. Mucoid and non-mucoid strains belonged to the same spa-type. Transformation of PIA-overproducing S. aureus with a vector expressing the intact icaR gene restored the non-mucoid phenotype.Altogether, we demonstrated a new mechanism for the emergence of mucoid S. aureus isolates of pwCF.

Urine-mediated suppression of Klebsiella pneumoniae mucoidy is counteracted by spontaneous Wzc variants altering capsule chain length.

Klebsiella pneumoniae is a hospital-associated pathogen primarily causing urinary tract infections (UTIs), pneumonia, and septicemia. Two challenging lineages include the hypervirulent strains, causing invasive community-acquired infections, and the carbapenem-resistant classical strains, most frequently isolated from UTIs. While hypervirulent strains are often characterized by a hypermucoid phenotype, classical strains usually present with low mucoidy. Since clinical UTI isolates tend to exhibit limited mucoidy, we hypothesized that environmental conditions may drive K. pneumoniae adaptation to the urinary tract and select against mucoid isolates. We found that both hypervirulent K. pneumoniae and classical Klebsiella UTI isolates significantly suppressed mucoidy when cultured in urine without reducing capsule abundance. A genetic screen identified secondary mutations in the wzc tyrosine kinase that overcome urine-suppressed mucoidy. Over-expressing Wzc variants in trans was sufficient to boost mucoidy in both hypervirulent and classical Klebsiella UTI isolates. Wzc is a bacterial tyrosine kinase that regulates capsule polymerization and extrusion. Although some Wzc variants reduced Wzc phospho-status, urine did not alter Wzc phospho-status. Urine does, however, increase K. pneumoniae capsule chain length diversity and enhance cell-surface attachment. The identified Wzc variants counteract urine-mediated effects on capsule chain length and cell attachment. Combined, these data indicate that capsule chain length correlates with K. pneumoniae mucoidy and that this extracellular feature can be fine-tuned by spontaneous Wzc mutations, which alter host interactions. Spontaneous Wzc mutation represents a global mechanism that could fine-tune K. pneumoniae niche-specific fitness in both classical and hypervirulent isolates. IMPORTANCE Klebsiella pneumoniae is high-priority pathogen causing both hospital-associated infections, such as urinary tract infections, and community-acquired infections. Clinical isolates from community-acquired infection are often characterized by a tacky, hypermucoid phenotype, while urinary tract isolates are usually not mucoid. Historically, mucoidy was attributed to capsule overproduction; however, recent reports have demonstrated that K. pneumoniae capsule abundance and mucoidy are not always correlated. Here, we report that human urine suppresses K. pneumoniae mucoidy, diversifies capsule polysaccharide chain length, and increases cell surface association. Moreover, specific mutations in the capsule biosynthesis gene, wzc, are sufficient to overcome urine-mediated suppression of mucoidy. These Wzc variants cause constitutive production of more uniform capsular polysaccharide chains and increased release of capsule from the cell surface, even in urine. These data demonstrate that K. pneumoniae regulates capsule chain length and cell surface attachment in response host cues, which can alter bacteria-host interactions.

P242 Small colony variants of Staphylococcus aureus often exhibit a mucoid phenotype in the airways of people with cystic fibrosis

In a recent multicenter study, we determined the prevalence (cross-sectional) and the persistence of mucoid Staphylococcus aureus in people with cystic fibrosis (pwCF) in a one-year prospective study: from 41 of 451 S. aureus-positive pwCF from 13 centers (9.1%) mucoid S. aureus was cultured (unpublished observation). SCVs have been shown to be associated with more severe lung disease. It is not known whether mucoid isolates also exhibit the small colony variant (SCV) phenotype. Therefore, we analyzed the study isolates according to the SCV phenotype.

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.

Mechanism of resistance to phagocytosis and pulmonary persistence in mucoid Pseudomonas aeruginosa.

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. 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. 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.

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.

Synergy Between Pseudomonas aeruginosa Filtrates And Voriconazole Against Aspergillus fumigatus Biofilm Is Less for Mucoid Isolates From Persons With Cystic Fibrosis.

Persons with cystic fibrosis (CF) frequently suffer from Pseudomonas aeruginosa and Aspergillus fumigatus co-infections. There is evidence that co-infections with these interacting pathogens cause airway inflammation and aggravate deterioration of lung function. We recently showed that P. aeruginosa laboratory isolates synergistically interact with the anti-fungal azole voriconazole (VCZ), inhibiting biofilm metabolism of several A. fumigatus laboratory strains. Interaction was usually mediated via pyoverdine, but also via pyocyanin or pyochelin. Here we used planktonic filtrates of 7 mucoid and 9 non-mucoid P. aeruginosa isolates from CF patients, as well as 8 isolates without CF origin, and found that all of these isolates interacted with VCZ synergistically at their IC50 as well as higher dilutions. CF mucoid isolates showed the weakest interactive effects. Four non-mucoid P. aeruginosa CF isolates produced no or very low levels of pyoverdine and did not reach an IC50 against forming A. fumigatus biofilm; interaction with VCZ still was synergistic. A VCZ-resistant A. fumigatus strain showed the same level of susceptibility for P. aeruginosa anti-fungal activity as a VCZ-susceptible reference strain. Filtrates of most Pseudomonas isolates were able to increase anti-fungal activity of VCZ on a susceptible A. fumigatus strain. This was also possible for the VCZ-resistant strain. In summary these data show that clinical P. aeruginosa isolates, at varying degrees, synergistically interact with VCZ, and that pyoverdine is not the only molecule responsible. These data also strengthen the idea that during co-infections of A. fumigatus and P. aeruginosa lower concentrations of VCZ might be sufficient to control fungal growth.

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.

Mucoid bovine mastitis isolates of Streptococcus uberis - difficulties in identification

Highly mucoid Streptococcus uberis were isolated on blood agar from five milk samples originating from two dairy cow farms. All the isolates were CAMP test and esculin hydrolysis negative. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) following prior extraction with 70% formic acid was used for identification to the species level. Given that it is plausible that such strains could be isolated in the future from dairy cows by laboratories in Serbia and the Balkans, we consider this case report to be a useful instruction for veterinary bacteriology laboratories.

Microbiological features of Pseudomonas aeruginosa in children with Cystic Fibrosis

The purpose of the study was to determine the frequency rate of Pseudomonas aeruginosa infection among children with cystic fibrosis (CF) in Dnipro region and to provide microbiological characteristics of the obtained isolates. The study was conducting from January 2019 to December 2020. Children with genetically confirmed CF diagnosis were enrolled. The main research method was bacteriological with identification of microorganisms by biochemical properties; antimicrobial susceptibility was determined by disk-diffusion method. Biological material: mucus from a deep smear from the posterior pharyngeal wall, sputum and tracheobronchial lavage waters. The Leeds criteria were used to define persons with chronic infection. The study involved 21 children. We collected 183 respiratory samples with 49 isolates of P. aeruginosa. The most important co-existing pathogens were Staphylococcus aureus, Aspergillus spp. and Candida spp. In our study, P. aeruginosa was associated with Aspergillus spp. (χ2=20.952; df=1; p˂0.001). Mucoid isolates were found in 24.49% of cases. P. aeruginosa showed variable sensitivity to different groups of antimicrobial agents, but the highest resistance was to penicillins. Mucoid P. aeruginosa was more resistant to penicillins (p˂0.001) and cephalosporins (p=0.036). Infection P. aeruginosa is frequent among children with CF; there were three children with chronic bronchopulmonary infection P. aeruginosa in Dnipro region in the end of 2020. The likelihood of Aspergillus spp. infection was higher in the case of current P. aeruginosa infection. P. aeruginosa showed variable susceptibility to different groups of antimicrobial agents, but mucoid isolates were more resistant.

Automated antimicrobial susceptibility testing of slow-growing Pseudomonas aeruginosa strains in the presence of tetrazolium salt WST-1

Slow growing, mucoid isolates of Pseudomonas aeruginosa require adaptation of the protocol used for automated antimicrobial susceptibility testing (AST). In the present study we used a water soluble tetrazolium salt WST-1 (4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate) in combination with menadione for possibly improving AST of slow growing and biofilm-forming P. aeruginosa isolates from cystic fibrosis (CF) patients. WST-1 and menadione addition ensures sensitive detection of microbial growth increase in the presence of antibiotics that may remain undetected with the automated VITEK® 2 method. We observed that 32.8% of P. aeruginosa isolates from CF and bronchiectasis patients produced an elevated absorbance signal intensity thereby increasing the sensitivity while maintaining the accuracy of VITEK 2. Our study merits future investigation with other slow growing pathogenic bacterial species.

Association of Oral Microbiota Properties on Hyaluronidase and Biofilm Formation

Correlation between hyaluronidase (Hyl) activity and biofilm detachment in a few bacterial species was found. However, it is unclear if this association applies to bacterial species or for more general bacterial characteristics. This study determined the association between biofilm production and Hyl activity among bacterial isolates from the oral cavity of healthy subjects, and its association with Gram staining group, colony surface morphology and bacteria shape. The swab was taken from the tongue, cheek and entire teeth surfaces of 35 subjects, and tested for biofilm through modified microtiter plate assay while Hyl production was screened through HA rapid plate method. Forty-four isolates were found, each 50% are Gram-positive, and Gram-negative bacteria, with the majority are cocci and non-mucoid colony. More than 70% of isolates are moderate and strong; (n= 17, 38.6%) and (n=15, 34.1%) respectively for biofilm production; and 68.2% are Hyl producer. A significant association was found between Hyl and bacterial shape (p=0.018) and colony morphology (p=0.018), while other association is not significantly measured, including between Hyl and biofilm (p=0.659). This study showed that biofilm production is not affected by the characteristics of the bacteria to produce or not produce hyaluronidase. Meanwhile, Hyl production is prone in rod shape and mucoid isolates which need further investigations.

Molecular epidemiological characterization in mucoid-type Streptococcus pneumoniae isolates obtained from invasive pneumococcal disease patients in Japan

IntroductionStreptococcus pneumoniae with a mucoid-type capsule is associated with invasive pneumococcal diseases (IPDs). Despite the introduction of pneumococcal vaccines, IPDs caused by mucoid-type isolates are still prevalent. The present study aimed to characterize mucoid-type S. pneumoniae isolated from IPD patients throughout Japan in 2017 (post-vaccination era). MethodsA total of 225 mucoid-type isolates were collected. The serotype, antimicrobial susceptibility, and multilocus sequence type of these isolates were determined. ResultsThe prevalence of IPDs caused by mucoid-type isolates was high in adults, especially in the elderly (≥65 years of age), and prognosis in these patients was significantly poor. Of the mucoid-type isolates, the predominant serotype was serotype 3 (84.4%), and the remaining were serotypes 37 (15.1%) and 8 (0.4%). Antimicrobial susceptibility showed that most mucoid isolates exhibited the penicillin-intermediate resistant S. pneumoniae genotype (gPISP). However, the serotype 3 isolate exhibited the penicillin-resistant S. pneumoniae genotype (gPRSP). This gPRSP isolate was classified into ST166, which is related to serotypes 9 V and 11 strains. Sequence analysis of the capsule-coding regions and its flanking regions indicated that recombination occurred upstream and downstream of the capsule-coding region, suggesting that gPRSP (serotype 9 V/ST166) obtaining the type-3 capsule gene cluster resulted in the emergence of gPRSP (serotype 3/ST166). ConclusionsOur findings indicated that IPDs caused by mucoid-type S. pneumoniae are still a serious concern and mucoid-type S. pneumoniae with novel phenotype could emerge via capsular switching in response to environmental changes such as introduction of vaccines and improper use of antimicrobial agents.

Bacterial killing is enhanced by exogenous administration of lysozyme in the lungs

ObjectiveLysozyme, a 14-kDa protein, is one of the most abundant antimicrobials in the lungs. Its concentration in airway surface sufficient to kill several bacterial pathogens in vitro. The purpose of this study was to determine if administration of exogenous lysozyme would further enhance bacterial killing in vivo. MethodsTo assess the effect of acute lung infection on endogenous lysozyme protein levels, mice were infected by intratracheal instillation of Pseudomonas aeruginosa and bronchoalveolar (BAL) fluid assessed for lysozyme concentration and for muramidase activity. In order to inform in vivo testing, species-specific bacterial killing efficacy was determined by incubating mucoid P. aeruginosa with 2×105 units of chicken lysozyme, human lysozyme or with vehicle at 37°C for 2hours. Subsequently, mice challenged with intratracheally-administered mucoid P. aeruginosa, were reintubated and injected with 2×105 Units of native human lysozyme, recombinant human lysozyme or with vehicle. Lung bacterial burden was enumerated subsequently. ResultsThe concentration of lysozyme protein in BAL fluid from mice challenged with mucoid clinical isolate of P. aeruginosa was increased 4-fold at 6hours post-infection. Quantitative culture showed that the number of recoverable bacteria was significantly decreased by both chicken and human lysozyme compared to vehicle but human lysozyme was significantly more effective than chicken egg lysozyme. In vivo, 24hours post-infection quantitative culture of lung homogenates showed that the number of viable bacteria recovered from mice treated with either native or recombinant lysozyme was decreased with 0.76±0.25×104 and 0.84±0.16×104, respectively, vs. 7.0±2.52×104 CFU/g protein in mice treated with HBSS, both P<0.05. ConclusionsThese results indicate that endogenous lysozyme is increased during acute lung infection and that early administration of exogenous lysozyme further enhances bacterial killing in vivo.

The prevalence of Staphylococcus aureus with mucoid phenotype in the airways of patients with cystic fibrosis—A prospective study

Background: Staphylococcus aureus is one of the most frequently isolated pathogens in the respiratory tract of CF patients. Recently, we characterized peculiar mucoid S. aureus isolates, which are excessive biofilm formers and which carried a 5bp-deletion within the intergenic region of the ica operon. In this prospective study, we determined the prevalence of mucoid S. aureus-isolates in the airways of CF-patients during a 3-months period.Methods: We analyzed specimens (sputa, throat swabs) from 81 CF patients who attended two CF centers in Münster, Germany. Ten S. aureus isolates were randomly picked from every S. aureus-positive airway specimen and evaluated for mucoidy using Congo Red agar and phenotypic tests. Mucoid isolates were characterized by spa sequence typing, biofilm production and sequencing of the intergenic region of the ica operon to screen for the 5bp-deletion.Results: In 7 of 81 examined patients (8.6%), we detected mucoid S. aureus phenotypes (37 out of 1050 isolates; 3.5%). Twenty-five mucoid isolates carried the 5bp-deletion. Mucoid isolates produced excessive biofilm and were significantly more resistant to certain antibiotics.Conclusions: In our prospective study, mucoid S. aureus was present in 8.6% of S. aureus–positive CF-patients. In 6 of 7 patients, mucoid isolates carried the 5bp-deletion, indicating that also other so far not identified mechanisms cause excessive biofilm formation. Further studies are necessary to ascertain the clinical impact of mucoid S. aureus phenotypes on the severity of the CF disease.

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.