Michael Hogardt

Adaptation of Pseudomonas aeruginosa during persistence in the cystic fibrosis lung.

The long-term persistance of P. aeruginosa in the cystic fibrosis (CF) lung is characterized by the selection of a variety of genotypes and phenotypes that typically descend from one infecting P. aeruginosa clone, a process known as adaptive radiation. This adaptation process of P. aeruginosa includes complex physiological changes that likely confer a selective advantage to better thrive in the diverse niches and microenvironments of the inflamed and hostile CF airways. The occurrence of P. aeruginosa variants is fixed by mutation and selection. Common loss-of-function mutations in genes such as lasR, mucA and mexT lead to a general adaptation pattern and P. aeruginosa variants with increased antimicrobial resistance, alginate overproduction, reduced acute virulence, and improved metabolic fitness. Strikingly, several virulence-associated traits and immunostimulatory components of P. aeruginosa are turned off. In contrast, other cellular factors are positively selected such as the outer membrane protein OprF, the blue copper protein azurin, the cytochrome c peroxidase c551, and the enzymes of the arginine deiminase pathway ArcA-ArcD. These metabolic components probably are required for the optimal anaerobic or microaerobic growth and viability of P. aeruginosa within CF airways. Besides these common adaptations found by the comparison of P. aeruginosa isolates from different CF patients, the overall diversity of isogenic isolates from one CF patient is extended by variable changes in the expression of regulatory-, transport-, metabolic-, and virulence-associated genes. A better understanding of the microevolution of P. aeruginosa towards niche specialists according the selection pressure in the CF lung is a prerequisite to develop new strategies for the detection of P. aeruginosa variants, the antipseudomonal treatment, the prediction of the infectious disease state, and the development of efficient vaccines.

Stage-Specific Adaptation of Hypermutable Pseudomonas aeruginosa Isolates during Chronic Pulmonary Infection in Patients with Cystic Fibrosis

BACKGROUND Pseudomonas aeruginosa (PA) strains with defective DNA mismatch repair genes generate numerous bacterial variants because of high mutation rates. In cystic fibrosis (CF), such mutator strains may lead to the rapid selection of survivors that are specifically adapted to the hostile environment of the inflamed CF lung. METHODS Genotypes and phenotypes of 111 PA variants descending from 3 distinct mutator strains obtained from 3 patients with CF were systematically characterized. RESULTS We demonstrated that PA mutS isolates accumulated in the CF lung during the observation period of 3-6 years, with dominance during the final stage of the disease. Mutator strains from the final stage of disease were multiresistant and had lost a set of established virulence-associated traits, including cytotoxicity for bronchial epithelial cells (Calu-3) and macrophages (J774). This pathoadaptation was associated with the loss of survival capacity in a typical environmental habitat, such as tap water. Strikingly, nonmutator strains that maintained their virulence potential persisted as a minority, probably with a preference for the lower airways. CONCLUSIONS Mutator strains may evolve from the initially infecting PA strain and generate numerous variants with a loss of destructive virulence factors, probably because of selection for improved survival in the deteriorated CF lung but at the expense of the ability to live freely.

Molecular evidence of nosocomial Pneumocystis jirovecii transmission among 16 patients after kidney transplantation.

ABSTRACT In recent years, clusters of Pneumocystis jirovecii (formerly Pneumocystis carinii) pneumonia (PCP) among immunocompromised individuals have been reported. Mostly, the source of infections was suspected to be within the clinical settings when transplant recipients and PCP patients shared hospital facilities. We report on a cluster of 16 renal transplant recipients positive for P. jirovecii. None of them received anti-Pneumocystis prophylaxis prior to P. jirovecii detection. Epidemiological studies revealed that 15 of them had received kidney transplants at a German university hospital and attended the same inpatient and outpatient clinic from January through September 2006. Multilocus sequence typing (MLST) was performed on the following genes: ITS1, β-tub, 26S, and mt26S. P. jirovecii DNA was available from 14 patients and showed identical MLST types among these renal transplant recipients. Surprisingly, one patient who was treated at a different nephrological center and reported no personal contact with patients from the renal transplantation cluster harbored an identical P. jirovecii MLST type. Three HIV-positive patients and one bone-marrow-transplanted hematologic malignancy patient—treated at different medical centers—were used as controls, and different MLST types were revealed. Interestingly, in three of the four previously described regions, new alleles were detected, and one new polymorphism was observed in the mt26S region. The epidemiological data and the genotyping results strongly suggest a nosocomial patient-to-patient transmission of P. jirovecii as the predominant transmission route. Therefore, strict segregation and isolation of P. jirovecii-positive/suspected patients in clinical settings seems warranted.

Microevolution of Pseudomonas aeruginosa to a Chronic Pathogen of the Cystic Fibrosis Lung

Pseudomonas aeruginosa is the leading pathogen of chronic cystic fibrosis (CF) lung infection. Life-long persistance of P. aeruginosa in the CF lung requires a sophisticated habitat-specific adaptation of this pathogen to the heterogeneous and fluctuating lung environment. Due to the high selective pressure of inflamed CF lungs, P. aeruginosa increasingly experiences complex physiological and morphological changes. Pulmonary adaptation of P. aeruginosa is mediated by genetic variations that are fixed by the repeating interplay of mutation and selection. In this context, the emergence of hypermutable phenotypes (mutator strains) obviously improves the microevolution of P. aeruginosa to the diverse microenvironments of the CF lung. Mutator phenotypes are amplified during CF lung disease and accelerate the intraclonal diversification of P. aeruginosa. The resulting generation of numerous subclonal variants is advantegous to prepare P. aeruginosa population for unpredictable stresses (insurance hypothesis) and thus supports long-term survival of this pathogen. Oxygen restriction within CF lung environment further promotes persistence of P. aeruginosa due to increased antibiotic tolerance, alginate production and biofilm formation. Finally, P. aeruginosa shifts from an acute virulent pathogen of early infection to a host-adapted chronic virulent pathogen of end-stage infection of the CF lung. Common changes that are observed among chronic P. aeruginosa CF isolates include alterations in surface antigens, loss of virulence-associated traits, increasing antibiotic resistances, the overproduction of the exopolysaccharide alginate and the modulation of intermediary and micro-aerobic metabolic pathways (Hogardt and Heesemann, Int J Med Microbiol 300(8):557-562, 2010). Loss-of-function mutations in mucA and lasR genes determine the transition to mucoidity and loss of quorum sensing, which are hallmarks of the chronic virulence potential of P. aeruginosa. Metabolic factors that are positively selected in response to the specific environment of CF lung include the outer membrane protein OprF, the microaerophilic oxidase Cbb3-2, the blue copper protein azurin, the cytochrome c peroxidase c551 and the enzymes of the arginine deiminase pathway ArcA-ArcD. These metabolic adaptations probably support the growth of P. aeruginosa within oxygen-depleted CF mucus. The deeper understanding of the physiological mechanisms of niche specialization of P. aeruginosa during CF lung infection will help to identify new targets for future anti-pseudomonal treatment strategies to prevent the selection of mutator isolates and the establishment of chronic CF lung infection.

Flagellin Induces Myeloid-Derived Suppressor Cells: Implications for Pseudomonas aeruginosa Infection in Cystic Fibrosis Lung Disease

Pseudomonas aeruginosa persists in patients with cystic fibrosis (CF) and drives CF lung disease progression. P. aeruginosa potently activates the innate immune system, mainly mediated through pathogen-associated molecular patterns, such as flagellin. However, the host is unable to eradicate this flagellated bacterium efficiently. The underlying immunological mechanisms are incompletely understood. Myeloid-derived suppressor cells (MDSCs) are innate immune cells generated in cancer and proinflammatory microenvironments and are capable of suppressing T cell responses. We hypothesized that P. aeruginosa induces MDSCs to escape T cell immunity. In this article, we demonstrate that granulocytic MDSCs accumulate in CF patients chronically infected with P. aeruginosa and correlate with CF lung disease activity. Flagellated P. aeruginosa culture supernatants induced the generation of MDSCs, an effect that was 1) dose-dependently mimicked by purified flagellin protein, 2) significantly reduced using flagellin-deficient P. aeruginosa bacteria, and 3) corresponded to TLR5 expression on MDSCs in vitro and in vivo. Both purified flagellin and flagellated P. aeruginosa induced an MDSC phenotype distinct from that of the previously described MDSC-inducing cytokine GM-CSF, characterized by an upregulation of the chemokine receptor CXCR4 on the surface of MDSCs. Functionally, P. aeruginosa–infected CF patient ex vivo–isolated as well as flagellin or P. aeruginosa in vitro–generated MDSCs efficiently suppressed polyclonal T cell proliferation in a dose-dependent manner and modulated Th17 responses. These studies demonstrate that flagellin induces the generation of MDSCs and suggest that P. aeruginosa uses this mechanism to undermine T cell–mediated host defense in CF and other P. aeruginosa–associated chronic lung diseases.

Detection of Differences in the Nucleotide and Amino Acid Sequences of Diphtheria Toxin from Corynebacterium diphtheriae and Corynebacterium ulcerans Causing Extrapharyngeal Infections

ABSTRACT While Corynebacterium ulcerans can mimic classical diphtheria, extrapharyngeal infections are extremely rare. Sequencing of the diphtheria toxin (DT)-encoding tox gene of two C. ulcerans isolates from extrapharyngeal infections revealed differences from C. diphtheriae DT sequences, mainly in the translocation and receptor-binding domains. C. ulcerans supernatants were much less potent than supernatant from C. diphtheriae. A C. ulcerans DT-specific PCR is described below.

Pneumocystis carinii Carriage among Cystic Fibrosis Patients, as Detected by Nested PCR

ABSTRACT A total of 137 sputa from 95 consecutive cystic fibrosis (CF) patients undergoing routine bacteriological surveillance were analyzed for Pneumocystis carinii colonization using nested PCR. Seven of 95 patients (7.4%) were PCR positive, suggesting thatP. carinii carriage may exist among CF patients due to their underlying pulmonary disease.

Multidrug-resistant organisms detected in refugee patients admitted to a University Hospital, Germany June‒December 2015

Multidrug-resistant Gram-negative bacteria (MDR GNB) were found to colonise 60.8% (95% confidence interval: 52.3-68.9) of 143 refugee patients mainly from Syria (47), Afghanistan (29), and Somalia (14) admitted to the University Hospital Frankfurt, Germany, between June and December 2015. This percentage exceeds the prevalence of MDR GNB in resident patients four-fold. Healthcare personnel should be aware of this and the need to implement or adapt adequate infection control measures.

Hospital-wide outbreak of Burkholderia contaminans caused by prefabricated moist washcloths

We experienced a hospital outbreak of Burkholderia contaminans (Burkholderia cepacia Group K) in a German university hospital with two campuses. Cases were defined as the microbiological detection of B. cepacia complex (BCC) in any clinical specimen sent to the laboratory during 30 June to 21 October 2008. Species identification of BCC was performed by recA gene sequencing, followed by pulsed-field gel electrophoresis (PFGE; SpeI digest) for clonal identity. In total, 61 BCC-positive cases were diagnosed at the two campuses. At least nine patients contracted a ventilator-associated pneumonia with BCC. One patient suffered an infection of a pacing wire insertion site and four patients had septicaemia. Sixteen patients died in hospital, none thought to be due to the outbreak strain. BCC was eventually found in packages of moist prefabricated washcloths used for intensive care patients. German healthcare authorities were informed and a Europe-wide alarm (RAPEX) was initiated through the systems to prevent infections in other hospitals. PFGE proved clonal identity between isolates from clinical specimens and washcloths of both campuses. After elimination of the contaminated washcloths no further cases occurred. This example of a relatively newly introduced product raises the question of whether current regulations are adequate to protect consumers. For critically ill patients, care products should be carefully evaluated. In case of infections due to contaminated products, immediate communication to healthcare authorities is required, including RAPEX warning if products are sold across Europe.

Transfer of the Core Region Genes of the Yersinia enterocolitica WA-C Serotype O:8 High-Pathogenicity Island to Y. enterocolitica MRS40, a Strain with Low Levels of Pathogenicity, Confers a Yersiniabactin Biosynthesis Phenotype and Enhanced Mouse Virulence

ABSTRACT The high-pathogenicity island (HPI) of yersiniae encodes an iron uptake system represented by its siderophore yersiniabactin (Ybt). The HPI is present in yersiniae with high levels of pathogenicity—i.e., Yersinia pestis, Y. pseudotuberculosis, and Y. enterocolitica biogroup (BG) 1B—but absent in Y. enterocolitica strains with low (BG 2 to 5) and no (BG 1A) levels of pathogenicity and has been shown to be an important virulence factor. Comparison of the HPI in Y. enterocolitica (Yen-HPI) and that in Y. pestis and Y. pseudotuberculosis revealed that, in contrast to genes of the variable region, genes of the core region (genes irp9 to fyuA) are highly homologous. In the present work the Yen-HPI core genes were rescued from the chromosome of Y. enterocolitica WA-C (BG 1B, serotype O:8) using the FRT-FLP recombinase system. Transfer of the resulting plasmid pCP1 into the siderophore-deficient strain Y. enterocolitica NF-O (BG 1A) led to no halo on siderophore indicator chrome azurol S (CAS) agar. Transfer of pCP1 into the Y. enterocolitica strain MRS40 (serotype O:9, BG 2; phenotype, CAS negative) led to a CAS halo larger than that of parental strain WA-C, indicating high Ybt production. pCP1 was highly unstable in iron-deficient medium, and no enhanced mouse virulence conferred by MRS40 carrying pCP1 could be detected. To overcome the problem of instability, pCP1 was integrated into the chromosome of MRS40, leading to the formation of a CAS halo comparable to that seen with WA-C and correspondingly to increased mouse virulence. Thus, the core genes of Yen-HPI are sufficient to confer a positive CAS phenotype and mouse virulence to Y. enterocolitica MRS40, BG 2, but are insufficient to confer this phenotype to Y. enterocolitica NF-O, BG 1A.