Mucoid Phenotype Research Articles

Molecular genetic characteristics of the carbapenem resistant <i>Klebsiella pneumoniae KP254</i> strain as a representative of the highly virulent strain evolutionary branch

Here we provide molecular and genetic characteristics of the Klebsiella pneumoniae KP254 clinical strain belonging to clonal group 23 based on the genome-wide sequencing data. It is known that representatives of such clonal group exert highly virulent properties and cause community-acquired infections. Phenotypically, K. pneumoniae KP254 strain is characterized by multidrug resistance, including carbapenems. The determinants of antibiotic resistance (blaSHV-1, oqxAB, fosA) and pathogenicity encoding fimbriae 1, 3 types and the siderophore yersineobactin synthesis were found in the chromosome structure. However, there was uncovered the lack of conjugative element ICEKp1, the pathogenicity island KPHPI208, and the allantoin regulon genes which are often found in highly virulent strains. Analyzing nucleotide sequences in silico allowed to reveal the replicons of incompatibility group plasmids for FII, FIAHI1/FIIK, Col440I, ColpVC, FIBK, FIIpCRY. Combining contigs relative to reference sequences by using the BLASTN service allowed to identify two putative antibiotic resistance plasmids IncFII and IncFIIpCRY as well as one virulence plasmid IncFIBK. The determinants of the aerobactin siderophore, the RmpA2 mucoid phenotype regulator as well as heavy metal resistance genes constitute the virulence plasmid structure. The virulence plasmid nucleotide sequence coverage comprised 93% relative to the virulence plasmid pK2044 with 99.38% identity level; the genomic regions responsible for the salmochelin and RmpA protein synthesis were deleted. The set of antibiotic resistance determinants identified in the mobilome structure includes the genes for beta-lactamase LAP-2 (IncFIIpCRY plasmid) — a TEM-1 analogue, as well as extended-spectrum beta-lactamase CTX-M-55 (IncFII plasmid), both of which are rarely recorded in the Russian Federation. Additionally, widespread genes blaOXA-1, aac(3’)-IIa, ΔcatB4, aac (6’)-Ib-cr, tet(A), qnrSI, sul2, catA2 were also found in the plasmid DNA. The carbapenemase genes are absent in the resistome structure, whereas the examined strain exerts carbapenem resistance. The analysis of the ompK35 and ompK36 porin gene translated sequences revealed mutational changes which resulted in emerged stop codon within the ompK35 gene, whereas OmpK36 amino acid sequence contains a large number of substitutions, insertions, and deletions. The changes identified serve as one of the factors determining the carbapenem resistance. A synergistic effect may be accounted for by activity of the efflux pumps found in the structure of the K. pneumoniae KP254 genome, particularly AcrAB-TolC and KpnEF. Thus, the strain examined by us preserves the most significant signs specific to the highly virulent evolutionary branch Klebsiella strains, and at the same time, acquires the multidrug resistance genetic determinants.

Phage cocktail strategies for the suppression of a pathogen in a cross-feeding coculture.

Cocktail combinations of bacteria-infecting viruses (bacteriophages) can suppress pathogenic bacterial growth. However, predicting how phage cocktails influence microbial communities with complex ecological interactions, specifically cross-feeding interactions in which bacteria exchange nutrients, remains challenging. Here, we used experiments and mathematical simulations to determine how to best suppress a model pathogen, E. coli, when obligately cross-feeding with S. enterica. We tested whether the duration of pathogen suppression caused by a two-lytic phage cocktail was maximized when both phages targeted E. coli, or when one phage targeted E. coli and the other its cross-feeding partner, S. enterica. Experimentally, we observed that cocktails targeting both cross-feeders suppressed E. coli growth longer than cocktails targeting only E. coli. Two non-mutually exclusive mechanisms could explain these results: (i) we found that treatment with two E. coli phage led to the evolution of a mucoid phenotype that provided cross-resistance against both phages, and (ii) S. enterica set the growth rate of the coculture, and therefore, targeting S. enterica had a stronger effect on pathogen suppression. Simulations suggested that cross-resistance and the relative growth rates of cross-feeders modulated the duration of E. coli suppression. More broadly, we describe a novel bacteriophage cocktail strategy for pathogens that cross-feed.

Selection of cereal-sourced lactic acid bacteria as candidate starters for the baking industry.

The quality of sourdough bread mainly depends on metabolic activities of lactic acid bacteria (LAB). The exopolysaccharides (EPS) produced by LAB affect positively the technological and nutritional properties of the bread, while phytases improve the bioavailability of the minerals by reducing its phytate content. In the present study, a pool of 152 cereal-sourced LAB were screened for production of phytases and EPS for potential use as sourdough starter cultures for the baking industry. There was large heterogeneity in the phytase activity observed among the screened isolates, with 95% showing the ability to degrade sodium phytate on plates containing Sourdough Simulation Medium (SSM). The isolates Lactobacillus brevis LD65 and Lactobacillus plantarum PB241 showed the highest enzymatic activity, while the isolates ascribed to Weissella confusa were characterized by low or no phytase activity. Only 18% of the screened LAB produced EPS, which were distinguished as ropy or mucoid phenotypes on SSM supplemented with sucrose. Almost all the EPS producers carried one or more genes (epsD/E and/or epsA) involved in the production of heteropolysaccharides (HePS), whereas the isolates ascribed to Leuconostoc citreum and W. confusa carried genes involved in the production of both HePS and homopolysaccharides (HoPS). Monosaccharide composition analysis of the EPS produced by a selected subset of isolates revealed that all the HePS included glucose, mannose, and galactose, though at different ratios. Furthermore, a few isolates ascribed to L. citreum and W. confusa and carrying the gtf gene produced β-glucans after fermentation in an ad hoc formulated barley flour medium. Based on the overall results collected, a subset of candidate sourdough starter cultures for the baking industry was selected, including Lb. brevis LD66 and L. citreum PB220, which showed high phytase activity and positive EPS production.

Outbreak of KPC-2 Carbapenem-resistant Klebsiella pneumoniae ST76 and Carbapenem-resistant K2 Hypervirulent Klebsiella pneumoniae ST375 strains in Northeast China: molecular and virulent characteristics

BackgroundCarbapenem-resistant hypervirulent Klebsiella pneumoniae strains have recently come into existence worldwide; however, researchers in northeast China are not aware of their clinical features and molecular characteristics.MethodsHere, the molecular and virulent characteristics of 44 carbapenem-resistant K. pneumoniae (CRKP) isolates collected from January 2015 to December 2017 were studied. Multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE) were carried out to define the clonal relatedness among the isolates. PCR and capsular serotyping of the virulence-associated genes, as well as biofilm formation and serum complement-mediated killing assays, were employed to determine the virulent potential. The genomic features and associated mobile genetic elements of JmsCRE57 were detected by whole genome sequencing.ResultsThe only positive isolate was JmsCRE57, which belonged to the ST375 serotype K2 that expressed uge, mrkD, fimH, kpn, aerobactin and rmpA virulence-associated genes and showed strong biofilm formation and serum sensitivity. Sequencing results showed that the JmsCRE57 genome mainly consisted of a circular chromosome, three antimicrobial resistant plasmids and a virulent plasmid. The antimicrobial resistant plasmid expressing blaKPC-2, blaCTX-M-15, aph(3″)-Ib, aph(6)-Id, qnrB1, aac(3)-IIa, aac(6′)-Ib-cr, blaOXA-1, blaTEM-1B, catB4, sul2, dfrA14 and blaSHV-99. The virulent plasmid belonged to the IncHI1B group, which is mainly composed of mucoid phenotype genes and siderophore-associated genes. The remaining CRKP strains that expressed uge, fimH, mrkD and kpn virulence-associated genes were not successfully typed.ConclusionOur results provide new insights on the epidemiology of carbapenem-resistant K2 hypervirulent K. pneumoniae ST375 and CRKP ST76 strains in northeast China, which may help control their future outbreaks.

Genotypic and Phenotypic Diversity of Staphylococcus aureus Isolates from Cystic Fibrosis Patient Lung Infections and Their Interactions with Pseudomonas aeruginosa.

Staphylococcus aureus has recently overtaken Pseudomonas aeruginosa as the most commonly recognized bacterial pathogen that infects the respiratory tracts of individuals with the genetic disease cystic fibrosis (CF) in the United States. Most studies of S. aureus in CF patient lung infections have focused on a few isolates, often exclusively laboratory-adapted strains, and how they are killed by P. aeruginosa Less is known about the diversity of S. aureus CF patient lung isolates in terms of both their virulence and their interaction with P. aeruginosa To begin to address this gap, we recently sequenced 64 clinical S. aureus isolates and a reference isolate, JE2. Here, we analyzed the antibiotic resistance genotypes, sequence types, clonal complexes, spa types, agr types, and presence/absence of other known virulence factor genes of these isolates. We hypothesized that virulence phenotypes of S. aureus, namely, toxin production and the mucoid phenotype, would be lost in these isolates due to adaptation in the CF patient lung. In contrast to these expectations, we found that most isolates can lyse both rabbit and sheep blood (67.7%) and produce polysaccharide (69.2%), suggesting that these phenotypes were not lost during adaptation to the CF lung. We also identified three distinct phenotypic groups of S. aureus based on their survival in the presence of nonmucoid P. aeruginosa laboratory strain PAO1 and its mucoid derivative. Altogether, our work provides greater insight into the diversity of S. aureus isolates from CF patients, specifically the distribution of important virulence factors and their interaction with P. aeruginosa, all of which have implications in patient health.IMPORTANCEStaphylococcus aureus is now the most frequently detected recognized pathogen in the lungs of individuals who have cystic fibrosis (CF) in the United States, followed closely by Pseudomonas aeruginosa When these pathogens are found to coinfect the CF lung, patients have a significantly worse prognosis. While P. aeruginosa has been rigorously studied in the context of bacterial pathogenesis in CF, less is known about S. aureus Here, we present an in-depth study of 64 S. aureus clinical isolates from CF patients, for which we investigated genetic diversity utilizing whole-genome sequencing, virulence phenotypes, and interactions with P. aeruginosa We found that S. aureus isolated from CF lungs are phylogenetically diverse; most retain known virulence factors and vary in their interactions with P. aeruginosa (i.e., they range from being highly sensitive to P. aeruginosa to completely tolerant to it). Deepening our understanding of how S. aureus responds to its environment and other microbes in the CF lung will enable future development of effective treatments and preventative measures against these formidable infections.

Antimicrobial Resistance and Pathogenicity Determination of Community-Acquired Hypervirulent Klebsiella pneumoniae.

Hypervirulent Klebsiella pneumoniae (hvKp) can cause severe invasive infections in healthy and immunocompromised individuals. However, there is still lack of a consensus definition of hvKp. In this study, we solely focused on the clinical isolates cultured from subcutaneous drainage of community-acquired liver abscess cases, and an hvKp strain was defined on the basis of co-harboring virulence gene regulator of mucoid phenotype A (rmpA)/rmpA2, iucA, iroB, and peg-344. A total of 47 nonrepetitive hvKp isolates were collected from January 2015 to December 2017 in a tertiary teaching hospital in Shanghai, China. All isolates were susceptible to the commonly used antibiotics. Only one strain (RJ-Kp24) had the positive detection of blaCTX-M-14 and was resistant to ceftazidime, cefotaxime, and cefepime. S1-pulsed-field gel electrophoresis (PFGE) and southern hybridization confirmed the presence of a roughly 90 kb blaCTX-M-14-carrying plasmid and a roughly 240 kb virulence plasmid. Further analysis revealed that ST23 (n = 17) sequence type and K1 (n = 20) and K2 (n = 9) serotypes were dominant in hvKp, while only 31.9% (15/47) and 46.8% (22/47) of hvKp isolates displayed hypermucoviscosity and resistance to serum killing, respectively. For further evaluation of the pathogenicity of hvKp, six representative strains were randomly selected. Three strains, RJ-Kp10, RJ-Kp28, and RJ-Kp31, displayed a remarkable resistance to serum killing and neutrophil phagocytosis. Mouse lethality assay revealed that these strains had the 50% lethal dose (LD50) of 102-103 cell forming unit (CFU), while others had the LD50 of 104-105 CFU. These results demonstrated that strain virulence differed significantly within these defined hvKp. The convergence of multidrug resistance and enhanced virulence in K. pneumoniae has presented a major infection control challenge.

Crosstalk between the Type VI Secretion System and the Expression of Class IV Flagellar Genes in the Pseudomonas fluorescens MFE01 Strain.

Type VI secretion systems (T6SSs) are contractile bacterial multiprotein nanomachines that enable the injection of toxic effectors into prey cells. The Pseudomonas fluorescens MFE01 strain has T6SS antibacterial activity and can immobilise competitive bacteria through the T6SS. Hcp1 (hemolysin co-regulated protein 1), a constituent of the T6SS inner tube, is involved in such prey cell inhibition of motility. Paradoxically, disruption of the hcp1 or T6SS contractile tail tssC genes results in the loss of the mucoid and motile phenotypes in MFE01. Here, we focused on the relationship between T6SS and flagella-associated motility. Electron microscopy revealed the absence of flagellar filaments for MFE01Δhcp1 and MFE01ΔtssC mutants. Transcriptomic analysis showed a reduction in the transcription of class IV flagellar genes in these T6SS mutants. However, transcription of fliA, the gene encoding the class IV flagellar sigma factor, was unaffected. Over-expression of fliA restored the motile and mucoid phenotypes in both MFE01Δhcp1+fliA, and MFE01ΔtssC+fliA and a fliA mutant displayed the same phenotypes as MFE01Δhcp1 and MFE01ΔtssC. Moreover, the FliA anti-sigma factor FlgM was not secreted in the T6SS mutants, and flgM over-expression reduced both motility and mucoidy. This study provides arguments to unravel the crosstalk between T6SS and motility.

A Frameshift Mutation in wcaJ Associated with Phage Resistance in Klebsiella pneumoniae.

Phage therapy is a potential and promising avenue for controlling the emergence and spread of multidrug-resistant (MDR) Klebsiella pneumoniae, however, the rapid development of anti-phage resistance has been identified as an obstacle to the development of phage therapy. Little is known about the mechanism employed by MDR K. pneumoniae strains and how they protect themselves from lytic phage predation in vitro and in vivo. In this study, comparative genomic analysis shows undecaprenyl-phosphate glucose-1-phosphate transferase (WcaJ), the initial enzyme catalyzing the biosynthesis of colanic acid, is necessary for the adsorption of phage 117 (Podoviridae) to the host strain Kp36 to complete its lytic life cycle. In-frame deletion of wcaJ alone was sufficient to provide phage 117 resistance in the Kp36 wild-type strain. Complementation assays demonstrated the susceptibility of phage 117, and the mucoid phenotype could be restored in the resistant strain Kp36-117R by expressing the wild-type version of wcaJ. Remarkably, we found that bacterial mobile genetic elements (insA and insB) block phage 117 infections by disrupting the coding region of wcaJ, thus preventing phage adsorption to its phage receptor. Further, we revealed that the wcaJ mutation likely occurred spontaneously rather than adapted by phage 117 predation under unfavorable environments. Taken together, our results address a crucial evolutionary question around the mechanisms of phage–host interactions, increasing our current understandings of anti-phage defense mechanisms in this important MDR pathogen.

Material Properties of Interfacial Films of Mucoid and Nonmucoid Pseudomonas Aeruginosa Isolates

Chronic lung infection with bacterial biofilms is a leading cause of death in cystic fibrosis (CF) patients. Among the many species colonizing the lung airways, Pseudomonas aeruginosa can undergo pathoadaptation leading to a mucoid phenotype with interesting material properties. We hypothesize that the surface properties and extracellular materials of mucoid P. aeruginosa cells greatly influence the mechanical behavior of their films at fluid interfaces. In this study, we investigate the interfacial properties of films formed by nonmucoid (PANT) and mucoid (PASL) strains of P. aeruginosa isolated from CF patients. We use pendant drop elastometry to analyze the interfacial response of the films formed by PANT and PASL at the hexadecane-water interface. The dynamic rheological analyses of the films highlight the distinctive signature of the mucoid strains at fluid interfaces. The mucoid films exhibit greater relaxation following a compressive strain than a tensile one, while a full hysteresis response is achieved by the nonmucoid films, indicating that the material properties of films of PANT are conserved under both compression and tension. The wrinkling and shape analysis of the interfacial biological films elucidate that mucoid strain exhibits remarkable viscoelastic properties, enabling remodeling of the living films and dissipation of the compressive stress. The comparative analysis of the material properties of mucoid and nonmucoid P. aeruginosa cells indicates that mucoid switch can play an important role in protecting the bacteria from interfacial stresses. Further characterization of interfacial bacterial films will provide new insights into the development of methods for controlling interfacial films of bacteria.

Draft Genome Sequences of Four Pseudomonas aeruginosa Clinical Strains with Various Biofilm Phenotypes.

Biofilms produced by Pseudomonas aeruginosa present a serious threat to cystic fibrosis patients. Here, we report the draft genome sequences of four cystic fibrosis isolates displaying various mucoid and biofilm phenotypes. The estimated average genome size was about 6,255,986 ± 50,202 bp with a mean G+C content of 66.52 ± 0.06%.

Capsule Thickness, Not Biofilm Formation, Gives Rise to Mucoid Acinetobacter baumannii Phenotypes That are More Prevalent in Long-Term Infections: A Study of Clinical Isolates from a Hospital in China.

BackgroundAcinetobacter baumannii is a nosocomial pathogen of critical importance due to the increasing numbers of antibiotic-resistant isolates. Colonies can have a smooth or matt appearance, but also exhibit slimy, mucoid growth, with the latter being increasingly isolated in patients in recent years.MethodsWe isolated 60 A. baumannii strains from altogether 56 patients and found that all patients were infected by mucoid strains, with four patients having also matt phenotypes in addition to the mucoid ones. The morphology of the colonies and capsules was observed. The antibiotics susceptibilities were tested, and the biofilm formation ability was determined by crystal violet staining. The whole-genome sequencing (WGS) was performed on all the strains, and then the core genome multilocus sequence typing (cgMLST) and drug resistance gene analysis were performed. Finally, a part of isolates were selected to test virulence in a Galleria mellonella model.ResultsWe observed much larger capsules in the mucoid strains compared to the matt isolates. But the mucoid phenotype did not correlate with the amount of biofilm produced by the strain. Almost all mucus-type A. baumannii were multi-drug resistant isolates, containing various antibiotic resistance genes. The main ST types of mucoid-type A.baumannii were ST191 and ST195, of which ST191 isolates were more virulence, while ST195 isolates were weaker.ConclusionThe mucoid A. baumannii had resistance to most antibiotics and some strains had high virulence, which should be paid attention in clinical.

Increase in Invasive Group A Streptococcal Disease and Emergence of Mucoid Strains in a Pediatric Population: February-June 2017.

BackgroundInfection with group A Streptococcus (GAS) can cause severe systemic and locally invasive disease. Invasive group A streptococcal (iGAS) disease incidence varies both seasonally and year-to-year, and it may exhibit clustered outbreaks. We observed an upswing in iGAS cases at a tertiary care Children’s Hospital, prompting further characterization of local iGAS disease.MethodsCases of iGAS disease were abstracted from the medical record by manual chart review of all positive screening tests and cultures for GAS over a 4-year span. Incidence rates per 1000 hospital admissions and per 100 positive GAS tests were calculated and compared. Selected isolates were further characterized by whole-genome sequencing.ResultsSignificant year-to-year differences in per-admission iGAS incidence rate were observed in February and June, although per-positive test incidence rates were not significantly different. Whole-genome sequencing revealed 2 dominant serotypes—emm3 and emm6—with high rates of mucoid phenotype and systemic bacteremia.ConclusionsWe document a significant but transient increase in iGAS disease incidence in 2 months of 2017. Genome sequencing revealed 2 dominant serotypes associated with mucoid phenotypes and severe disease, highlighting the dynamic nature of iGAS disease pattern.

Mucoid Pseudomonas aeruginosa alters sputum viscoelasticity in patients with non-cystic fibrosis bronchiectasis.

Pseudomonas aeruginosa could acquire a mucoid phenotype due to mutations in mucA (mucoid Pseudomonas aeruginosa - mPA) that is a hallmark of poor prognosis in patients with bronchiectasis. Despite the higher prevalence of Pseudomonas aeruginosa in bronchiectasis, how mPA and non-mucoid Pseudomonas aeruginosa (non-mPA) phenotypes could affect viscoelastic properties of sputum is unknown. Our aim was to determine the relationship between Pseudomonas aeruginosa phenotypes isolation, the viscoelastic properties of sputum and the clinical outcomes in patients with bronchiectasis. A cross-sectional study was conducted of sputum samples obtained by spontaneous expectoration and sent for microbiology and rheology analysis. Elasticity and viscosity were measured at two oscillatory frequencies (1 and 100 rad/s). Socio-demographic and clinical data were recorded. We analyzed 17 patients with mPA, 14 with non-mPA and 17 with no organism reported (NOR). Compared with the NOR group, the mPA group showed higher elasticity (median 10.30 vs. 5.70, p = 0.023), viscosity (2.40 vs. 1.50, p = 0.039), and stiffness (10.70 vs. 6.00, p = 0.024). Values in the mPA group tended to be higher compared with non-mPA. Clinically, the mPA group showed greater hospitalizations during the previous year and greater affected lobes than the non-mPA and NOR groups. The mPA phenotype is associated with increased elasticity, viscosity and stiffness of bronchiectatic sputum. Viscoelastic properties could be used as a marker of poor mucociliary clearance in mPA, with potentially important clinical implications.

Discovery of anti-mucoviscous activity of rifampicin and its potential as a candidate antivirulence agent against hypervirulent Klebsiella pneumoniae

A recent increase in the incidence of hypervirulent Klebsiella pneumoniae (hvKP) infections, especially those caused by a sublineage of clonal group CG23 (CG23-I), is raising serious health concerns worldwide. The high virulence of hvKP is, at least in part, attributed to the overproduction of capsular polysaccharide (CPS), which is triggered by a positive regulator of capsular polysaccharide synthesis (cps) genes, named rmpA (regulator of mucoid phenotype A). Although extensive research has been conducted on the mechanisms of hvKP virulence, no study has focused on the development of antivirulence therapeutics. This study attempted to identify and validate an antimicrobial agent able to suppress hvKP hypermucoviscosity. A total of 18 commercially available antimicrobial agents, including β-lactams, quinolones and aminoglycosides, were tested. Rifampicin (RFP) was found to have strong anti-mucoviscous activity against CG23-I hvKP even at subinhibitory concentrations. Polysaccharide extracts from hvKP showed substantially lowered viscosity when cells were grown with RFP. Moreover, microscopic observations demonstrated that RFP treatment results in a drastic reduction in the thickness of the CPS layer around hvKP cells. RFP treatment decreased transcript levels of rmpA and rmpA-regulated cps genes, indicating that RFP suppresses mucoviscosity of hvKP through inhibition of rmpA transcription. These data suggest that RFP may serve as a potential antivirulence agent for refractory hvKP infection.

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.

Antibiotic Resistance and Clinical Presentation of Health Care-Associated Hypervirulent Klebsiella pneumoniae Infection in Korea.

Hypervirulent Klebsiella pneumoniae typically presents as a community-acquired infection causing a liver abscess. It is often associated with rmpA and magA genes and confers a mucoid phenotype. Our aim was to evaluate the clinical presentation and antibiotic resistance of hypervirulent K. pneumoniae (hvKP) of health care-associated origin. The study was performed on 414 K. pneumoniae isolates recovered from patients that visited our hospital from December 2013 through November 2015. Hypervirulence was determined by the presence of a hypermucoviscous phenotype, in 155 isolates (37.4%). We compared health care-associated hvKP infections with community-acquired infections. The hypermucoviscous phenotype was 60 isolates (24.2%) in health care-associated infection and 90 isolates (53.8%) in community-acquired infection. Respiratory infection was the most common source of health care-associated K. pneumoniae. Antibiotic resistance was higher in health care-associated hvKP infections, which were more frequently associated with non-K1/K2 serotypes and less frequently associated with rmpA gene. In our study, 38% of hvKP was health care associated. Pneumonia is the most common infection, besides intraabdominal infection, in community-originating strains. These results suggest that health care-associated hvKP may have different microbiological characteristics from classical community-associated infections. Further investigation is needed for other virulent factors associated with health care-associated hvKP.

Characterisation of Virulence Genes Associated with Pathogenicity in Klebsiella pneumoniae

AbstractPurpose: This study was undertaken to characterise the virulence factors in clinical strains of Klebsiella pneumoniae and analyse their association with various infections caused and also to determine the association between virulence factors and antimicrobial resistance profile. Materials and Methods: A total number of 370 clinically significant, non-duplicate isolates of K. pneumoniae isolated from both hospitalised patients and patients attending clinics were included in this study. Polymerase chain reaction (PCR) was carried out for the detection of various virulence genes such as mucoviscosity-associated gene A (magA), gene associated with allantoin metabolism (allS), Klebsiella ferric iron uptake(Kfu), capsule-associated gene A (K2A), regulator of mucoid phenotype A (rmpA), enterobactin (entB), yersiniabactin (YbtS), aerobactin, Fimbrial adhesin (FimH) and uridine-diphosphate galacturonate 4-epimerase (uge). Antimicrobial susceptibility testing and PCR-based detection of beta-lactamase-encoding genes such as extended-spectrum beta-lactamases, AmpCs and carbapenemases were performed. Univariate analysis was done to find the association between virulence genes and mortality. Results: The siderophore, entB, was present in most (90.5%) of the isolates. Of the 370 isolates, 345 carried multiple virulence genes; 15 harboured single virulence genes and 10 did not harbour any of the studied virulence genes. The most common combination of occurrence was entB and FimH. A mortality rate of 12.75% (38/298) was observed among hospitalised patients. None of the virulence genes had any significant association with mortality. Conclusion: Pathogenic K. pneumoniae can harbour single to multiple virulence genes. Invasive infection with even a single virulence gene-harbouring K. pneumoniae can lead to poor outcomes. Both multidrug-resistant (MDR) and non-MDR K. pneumoniae can harbour a variety of virulence genes. None of the virulence genes have a significant association with mortality.

Susceptibility Testing of Exophiala dermatitidis to Inform Peri-Operative Prophylaxis at Time of Lung Transplantation

Purpose Exophiala dermatitidis is a black fungus that frequently colonises the lungs of people with cystic fibrosis (CF). Mucoid and non-mucoid variants can be detected from the same sputum for some patients, but their role in chronic CF lung disease is not fully understood and attempts at early eradication are not routinely performed. Invasive infections with this species are rare; however in 2014 we reported our first case of fatal, invasive infection with a highly mucoid strain of E. dermatitidis in a 34 year old post lung transplant patient with CF. This prompted us to include peri-operative antifungal prophylaxis for all patients colonised with this species. To inform the choice of optimal antifungal, we performed susceptibility testing of 38 isolates from 34 patients including lung transplant patients. The ability of these isolates to produce biofilm was also investigated. Methods The minimum inhibitory concentration (MIC) of 8 antifungal agents was measured using a commercial broth microdilution assay. Biofilm formation was assessed using a standard in vitro assay using staining with crystal violet. Results Using breakpoints for Candida albicans, all isolates were susceptible to voriconazole and amphotericin B with most isolates also susceptible to itraconazole (89% of isolates) and posaconazole (71% of isolates). In contrast, all isolates were non-susceptible to caspofungin and most isolates (92%) non-susceptible to fluconazole. All strains were inhibited by ketoconazole at ≤0.25 mg/ L whereas MICs of 5-fluorocytosine ranged from 1->64 mg/ L. 31 of 38 isolates were biofilm producers including 4 isolates that were classified as strong biofilm producers. We did not find a correlation between mucoid phenotype and biofilm production. Conclusion E. dermatitidis is commonly found to colonise the lungs of patients with CF, typically produces biofilm, and may rarely produce serious infection post transplantation. We demonstrate in vitro susceptibility to voriconazole and amphotericin B. For patients colonised with this species, we now elect to include voriconazole as part of the peri-operative prophylactic regimen.

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.