Microbiota Diversity in Non-Small Cell Lung Cancer Gut and Mouth Cavity Microbiota Diversity in Non-Small Cell Lung Cancer Patients.

Lung malignancies have a substantial impact on cancer incidence and mortality worldwide. Even though many factors involved in the development of the disease are known, many questions remain unanswered. Previous studies suggest that the intestinal microbiota may have a role in developing malignant diseases. According to some findings, the microbiota has proven to be a key modulator of carcinogenic processes and the immune response against cancer cells, potentially influencing the effectiveness of immunotherapy. In our study, we characterized culturable microorganisms associated with non-small cell lung cancer (NSCLC) that can be recovered from rectal swabs and mouthwash. In addition, we also explored differences in the culturable microbiota with two main types of NSCLC - adenocarcinoma (ADC) and squamous cell carcinoma (SCC). With 141 patients included in the study (86 ADC and 55 SCC cases), a significant difference was observed between the two types in seven bacterial species (Collinsella, Corynebacterium, Klebsiella, Lactobacillus, Neisseria, Rothia, and Streptococcus), including the site of origin. The relationship between microbial dysbiosis and lung cancer is poorly understood; future research could shed light on the links between gut microbiota and lung cancer development.

[68Ga]Ga-Ornibactin for Burkholderia cepacia complex Infection Imaging Using Positron Emission Tomography.

Bacteria from the Burkholderia cepacia complex are generally considered to be non-pathogenic to the healthy population. However, some of these species may cause serious nosocomial infections in immunocompromised patients; as such, it is essential to diagnose these infections rapidly so that adequate treatment can be initiated. We report here the use of a radiolabeled siderophore, ornibactin (ORNB), for positron emission tomography imaging. We successfully radiolabeled ORNB with gallium-68 with high radiochemical purity and proved that the resulting complex has optimal in vitro characteristics. In mice, the complex did not show excessive accumulation in organs and was excreted in the urine. We demonstrated that the [68Ga]Ga-ORNB complex accumulates at the site of Burkholderia multivorans infection, including pneumonia, in two animal infection models. These results suggest that [68Ga]Ga-ORNB is a promising tool for the diagnosis, monitoring, and evaluation of the therapeutic response to B. cepacia complex infection.

Which bacterial toxins are worthy of validation as markers in colorectal cancer screening? A critical review.

Appropriate screening of early asymptomatic cases can reduce the disease burden and mortality rate of sporadic colorectal cancer (CRC) significantly. Currently, fecal occult blood testing (FOBT) is able to detect up to 80% of asymptomatic cases in the population aged 50+. Therefore, there is still a demand for new screening tests that would complement FOBT, mainly by detecting at least a part of the FOBT-negative CRC and adenoma cases, or possibly by identifying person at increased risk of sporadic CRC in order to offer them tailored follow-up. Among the potential markers studied, our knowledge has advanced at most in toxigenic gram-negative bacteria. In this review, we assess their potential critically and recommend those best suited for prospective evaluation of their true ability to increase the sensitivity of FOBT when combined during general population screening. In our opinion, colibactin and Bacteroides fragilis toxin are the best candidates, possibly complemented by the cytotoxic necrotizing factor (CNF).

Cyclomodulins and Hemolysis in E. coli as Potential Low-Cost Non-Invasive Biomarkers for Colorectal Cancer Screening.

The frequent occurrence of E. coli positive for cyclomodulins such as colibactin (CLB), the cytotoxic necrotizing factor (CNF), and the cytolethal distending factor (CDT) in colorectal cancer (CRC) patients published so far provides the opportunity to use them as CRC screening markers. We examined the practicability and performance of a low-cost detection approach that relied on culture followed by simplified DNA extraction and PCR in E. coli isolates recovered from 130 CRC patients and 111 controls. Our results showed a statistically significant association between CRC and the presence of colibactin genes clbB and clbN, the cnf gene, and newly, the hemolytic phenotype of E. coli isolates. We also observed a significant increase in the mean number of morphologically distinct E. coli isolates per patient in the CRC cohort compared to controls, indicating that the cyclomodulin-producing E. coli strains may represent potentially preventable harmful newcomers in CRC patients. A colibactin gene assay showed the highest detection rate (45.4%), and males would benefit from the screening more than females. However, because of the high number of false positives, practical use of this marker must be explored. In our opinion, it may serve as an auxiliary marker to increase the specificity and/or sensitivity of the well-established fecal immunochemical test (FIT) in CRC screening.

68Ga-labelled desferrioxamine-B for bacterial infection imaging.

PURPOSE: With the increase of especially hospital-acquired infections, timely and accurate diagnosis of bacterial infections is crucial for effective patient care. Molecular imaging has the potential for specific and sensitive detection of infections. Siderophores are iron-specific chelators recognized by specific bacterial transporters, representing one of few fundamental differences between bacterial and mammalian cells. Replacing iron by gallium-68 without loss of bioactivity is possible allowing molecular imaging by positron emission tomography (PET). Here, we report on the preclinical evaluation of the clinically used siderophore, desferrioxamine-B (Desferal®, DFO-B), radiolabelled with 68Ga for imaging of bacterial infections. METHODS: In vitro characterization of [68Ga]Ga-DFO-B included partition coefficient, protein binding and stability determination. Specific uptake of [68Ga]Ga-DFO-B was tested in vitro in different microbial cultures. In vivo biodistribution was studied in healthy mice and dosimetric estimation for human setting performed. PET/CT imaging was carried out in animal infection models, representing the most common pathogens. RESULTS: DFO-B was labelled with 68Ga with high radiochemical purity and displayed hydrophilic properties, low protein binding and high stability in human serum and PBS. The high in vitro uptake of [68Ga]Ga-DFO-B in selected strains of Pseudomonas aeruginosa, Staphylococcus aureus and Streptococcus agalactiae could be blocked with an excess of iron-DFO-B. [68Ga]Ga-DFO-B showed rapid renal excretion and minimal retention in blood and other organs in healthy mice. Estimated human absorbed dose was 0.02 mSv/MBq. PET/CT images of animal infection models displayed high and specific accumulation of [68Ga]Ga-DFO-B in both P. aeruginosa and S. aureus infections with excellent image contrast. No uptake was found in sterile inflammation, heat-inactivated P. aeruginosa or S. aureus and Escherichia coli lacking DFO-B transporters. CONCLUSION: DFO-B can be easily radiolabelled with 68Ga and displayed suitable in vitro characteristics and excellent pharmacokinetics in mice. The high and specific uptake of [68Ga]Ga-DFO-B by P. aeruginosa and S. aureus was confirmed both in vitro and in vivo, proving the potential of [68Ga]Ga-DFO-B for specific imaging of bacterial infections. As DFO-B is used in clinic for many years and the estimated radiation dose is lower than for other 68Ga-labelled radiopharmaceuticals, we believe that [68Ga]Ga-DFO-B has a great potential for clinical translation.

Candida albicans culture from a rectal swab can be associated with newly diagnosed colorectal cancer.

Plenty of metagenomic studies have suggested possible associations between microbiome composition and colorectal cancer (CRC). However, these techniques are not economic enough for routine use so far. Therefore, we explored the possibility to detect species associated with colorectal cancer by conventional culture from rectal swab. Fifty-two patients newly diagnosed for adenoma/CRC and 52 age-matched controls were recruited and sampled. Rectal swabs were inoculated on several types of plates and incubated appropriately under both aerobic and anaerobic conditions. All colonial morphotypes were subcultured and identified using MALDI-ToF MS. Although no bacterial species was significantly associated with CRC in our study, we surprisingly observed a strong and significant overrepresentation of the yeast Candida albicans in cases (P = 0.0066, odds ratio 5.444 [95% CI 1.449-20.462]). Potential confounding factors were associated neither with CRC (history of CRC in first-degree relatives, a personal history of appendectomy and cholecystectomy, increased BMI (body mass index), and the percentage of males) nor with C. albicans presence (preexisting diabetes and PPI medication) in our cohort. A growing body of evidence supports the view that C. albicans does cause cancer in humans. We hypothesize that presence of C. albicans in the gut may induce or facilitate some part of the sporadic CRC cases. Our observation should be a strong incentive to verify the potential usefulness of the easily culturable C. albicans yeast as a screening marker for patients at risk of CRC or those suffering an early asymptomatic stage of CRC.

Antibiotic susceptibility and production of endotoxin by Ochrobactrum anthropi isolated from environment and from patients with cystic fibrosis.

The aim of this work was to compare production of endotoxin and to determine susceptibility to antibiotics in two groups of specimens-wild-type strains Ochrobactrum anthropi isolated from the environment and the strains isolated from patients with cystic fibrosis. The determination of the endotoxin produced by the test strains was carried on by using a limulus amebocyte lysate test (LAL test). Determination of ATB sensitivity was accomplished by means of a broth dilution method in a microtiter plate (MIC). No significant difference was found between the group of ochrobacters isolated from the environment and the group of ochrobacters isolated from cystic fibrosis patients. Antibiotic sensitivity testing has indicated that the resistance to tigecycline, trimethoprim/sulfamethoxazole, and gentamicin was slightly higher in strains isolated from cystic fibrosis patients in comparison with strains isolated from the environment. In general, most of the test strains were sensitive to most of the antibiotics tested. Significant resistance has been demonstrated for cefotaxime. Resistance was also found for gentamicin in strains number 4 and 7. The MIC was equal to the breakpoint for this antibiotic (8000 mg/L).

Genome sequence of the opportunistic human pathogen Magnusiomyces capitatus.

The yeast Magnusiomyces capitatus is an opportunistic human pathogen causing rare yet severe infections, especially in patients with hematological malignancies. Here, we report the 20.2 megabase genome sequence of an environmental strain of this species as well as the genome sequences of eight additional isolates from human and animal sources providing an insight into intraspecies variation. The distribution of single-nucleotide variants is indicative of genetic recombination events, supporting evidence for sexual reproduction in this heterothallic yeast. Using RNAseq-aided annotation, we identified genes for 6518 proteins including several expanded families such as kexin proteases and Hsp70 molecular chaperones. Several of these families are potentially associated with the ability of M. capitatus to infect and colonize humans. For the purpose of comparative analysis, we also determined the genome sequence of a closely related yeast, Magnusiomyces ingens. The genome sequences of M. capitatus and M. ingens exhibit many distinct features and represent a basis for further comparative and functional studies.

Imaging of Pseudomonas aeruginosa infection with Ga-68 labelled pyoverdine for positron emission tomography.

Pseudomonas aeruginosa is an increasingly prevalent opportunistic pathogen that causes a variety of life-threatening nosocomial infections. Novel strategies for the development of new antibacterial treatments as well as diagnostic tools are needed. One of the novel diagnostic strategies for the detection of infection could be the utilization of siderophores. Siderophores are low-molecular-weight chelators produced by microbes to scavenge essential iron. Replacing iron in siderophores by suitable radiometals, such as Ga-68 for positron emission tomography (PET) imaging, opens approaches for targeted imaging of infection. Here we report on pyoverdine PAO1 (PVD-PAO1), a siderophore produced by P. aeruginosa, labelled with Ga-68 for specific imaging of Pseudomonas infections. PVD-PAO1 was labelled with Ga-68 with high radiochemical purity. The resulting complex showed hydrophilic properties, low protein binding and high stability in human serum. In vitro uptake of 68Ga-PVD-PAO1 was highly dependent on the type of microbial culture. In normal mice 68Ga-PVD-PAO1 showed rapid pharmacokinetics with urinary excretion. PET imaging in infected animals displayed specific accumulation of 68Ga-PVD-PAO1 in infected tissues and better distribution than clinically used 18F-fluorodeoxyglucose (18F-FDG) and 68Ga-citrate. Ga-68 labelled pyoverdine PAO1 seems to be a promising agent for imaging of P. aeruginosa infections by means of PET.

Are we any closer to screening for colorectal cancer using microbial markers?A critical review.

The role of gut microbiota in the development of sporadic colorectal cancer (CRC) is supported by a number of studies, however, the conclusiveness of published metagenomic studies is questioned by technical pitfalls and limited by small cohort sizes. In this review, we evaluate the current knowledge critically and outline practical solutions. We also list candidate CRC risk markers that are - in our opinion - well supported by available data and thus deserve clinical validation. Last but not least, we summarise available knowledge useful for improving care for patients immediately.

NAS agar is more suitable than McKay agar for primary culture of Streptococcus milleri group (SMG) fastidious bacteria, S. intermedius in particular.

Streptococcus milleri group (SMG) is a group of three streptococcal species (S. anginosus, intermedius and constellatus) that act as opportunist pathogens, among others in cystic fibrosis. Due to their fastidious character, they are both difficult to cultivate and to differentiate from less pathogenic streptococcal species, therefore being most probably underdiagnosed. Semi-selective McKay agar and NAS agar were developed to facilitate SMG recovery from clinical samples; however, direct comparison of recovery rates has not been published yet. We tested the performance of both media on 123 patient samples and demonstrated general superiority of NAS agar for SMG recovery during primary cultivation convincingly. This observation was also confirmed by quantitative drop tests during subculture. Despite the undisputed overall superiority of NAS agar over McKay agar, a smaller fraction of strains grew better on McKay agar. Inter-strain differences were the most probable explanation. Therefore, when economic conditions are not limiting and maximum recovery rate is desirable, both plates are advised to be used in parallel for primary cultivation of clinical samples.

McRAPD unlike MALDI-TOF MS is a suitable candidate for routine discrimination of new Haemophilus influenzae strain acquisition in chronic obstructive pulmonary disease (COPD) and cystic fibrosis.

BACKGROUND AND AIMS: Haemophilus influenzae new strain acquisition has been demonstrated to increase the relative risk of acute exacerbation fourfold in contrast to colonisation or chronic infection by the same strain in chronic obstructive pulmonary disease (COPD). Unfortunately, molecular typing techniques are not suitable for routine use due to cost, labour-intensity and need for special expertise. We tested two techniques potentially useful for routine typing, namely the newly available MALDI-TOF MS and the modified McRAPD compared to MLST as the gold standard. METHODS: In 10 patients (10.8%) suffering from COPD or cystic fibrosis, H. influenzae isolates were recovered repeatedly at different timepoints from the same patient during the study period. This allowed for thirteen pairwise comparisons of typing results in isolates recovered consecutively from the same patient to test the ability of the techniques to uncover new strain acquisition. RESULTS: MLST detected 9 cases of new strain acquisition among the 13 pairwise comparisons. However, MALDI-TOF MS reported all 13 pairs as different and thus new. In contrast, McRAPD was able to differentiate all the new strain acquisitions from pre-existing ones, both by visual inspection of melting profiles and by Relative Significant Difference values. CONCLUSIONS: Unlike MALDI-TOF MS, McRAPD appears to be a suitable candidate for routine discrimination of new strain acquisitions because of its accuracy and, rapid, easy and economic performance.

[Prolonged culture in a humid chamber increases the yields of pathogenic bacteria from the respiratory tract samples of patients suffering from cystic fibrosis].

Current standards of care for cystic fibrosis (CF) patients lack unequivocal recommendations concerning the duration of primary culture of bacteriological samples. With the exception of Burkholderia cepacia (5 days), the minimum recommended duration of primary culture varies between 48 and 72 hours. Our aim was to evaluate the effect of an extended 10-day period of primary culture in a humid chamber in samples acquired from the respiratory tract of patients suffering from CF. Compared to standard culture, prolonged culture in a humid chamber yielded 1.85 times more isolates of pathogenic species in pharyngeal swabs (76 versus 41 isolates) and 1.4 times more isolates in sputum samples (116 versus 82), but only 1.14 times more isolates in nasal swabs (25 versus 22). Prolonged culture was most beneficial for Achromobacter spp. (6 versus 0), Stenotrophomonas maltophilia (16 versus 5) and Pseudomonas aeruginosa (69 versus 49), whereas there was little or no benefit at all for Staphylococcus aureus (87 versus 73) and Moraxella catarrhalis (10 versus 10). Therefore, prolonged culture in a humid chamber may definitely be recommended for pharyngeal swabs and sputum samples obtained from patients suffering from CF to achieve the maximum recovery rate of pathogenic bacteria, in particular non-fermenting Gram-negative rods.

The Streptococcus milleri group in chronic obstructive pulmonary disease.

BACKGROUND AND AIMS: S. anginosus, constellatus and intermedius, also known as the Streptococcus milleri group (SMG) are three streptococcal species more frequently detected in cases of invasive disease, abscesses and empyema in particular. Recent research suggests they play a role in exacerbations of cystic fibrosis (CF). Owing to poor recovery on standard culture media and difficult differentiation from non-pathogenic streptococci, SMG may be underdiagnosed in routine settings. We aimed to establish the incidence of SMG in chronic obstructive pulmonary disease (COPD) patients compared to CF patients and to examine possible links of SMG to exacerbations that plays a key role in progression of COPD. METHODS: Altogether, 90 respiratory tract samples of patients suffering from CF or COPD were examined during the period from July 2012 to December 2013. Semi-selective McKay agar was used for primary cultivation of SMG and MALDI TOF MS was used for species identification that was confirmed by biochemical profiling and specific PCR. RESULTS: We confirmed the presence of SMG in CF (17.6% incidence in adult patients) and newly established its presence in COPD (10.3% incidence). In COPD, SMG was detected in 4 cases of acute exacerbations, where no other bacterial pathogen was detected. In 3/4 cases, increased CRP level indicated bacterial infection as a cause of the exacerbation and in all 3 cases, patients recovered during antibiotic treatment. CONCLUSIONS: Our data indicate SMG may act as opportunist pathogens able to cause exacerbations in COPD.

Performance of pyrosequencing versus MALDI-TOF MS in bacteria identification in chronic lung disease.

Rapid identification of the etiological agent in bacterial infection is necessary for correct diagnosis and appropriate therapy. In general, identification of pure cultures of bacteria using conventional phenotyping techniques requires 4-24 hours. Recently available new molecular technologies offer the potential of same day species identification once pure culture is available. Our aim was to evaluate the performance of rDNA V1 hypervariable region pyrosequencing, and the whole cell MALDI-TOF MS protein profiling in routine species identification. During the period from June 2012 to June 2014, 1.140 pure culture isolates were recovered from 402 samples from 126 patients suffering cystic fibrosis, chronic obstructive pulmonary disease or bronchiectasis. All the isolates were subjected to species identification by both techniques. Unfortunately, pyrosequencing was able to reach the species level in 43.2% of isolates only, whereas MALDI-TOF was clearly superior with 96.8% respectively. The overall sensitivity values also clearly underlined the superiority of MALDI-TOF MS with 96.8% compared to 85.1% achieved by pyrosequencing. Generally, MALDI-TOF MS turned out to be the best suitable technique in routine bacterial identification, whereas pyrosequencing could be recommended as the method of choice particularly in situations where MALDI-TOF MS fails to identify rare species.

Usefulness of PCR-HRMA in identification of non-fermentative Gram-negative rods recovered from patients suffering from cystic fibrosis or chronic obstructive pulmonary disease.

Adequate treatment of microbial infections requires rapid and accurate identification of the etiological agent. In routine diagnostics, identification of bacteria conventionally relies on phenotypic testing, which can be hindered by phenotypic variations. Therefore, genotyping techniques should perform faster and more accurately. Recently, the technique of high-resolution melting analysis (HRMA) of PCR amplicons promises to provide a convenient and economic tool of genotypic identification. In our study, we performed prospective routine testing of a PCR-HRMA system that was recently published in a proof-of-the-principle study. The system was evaluated by analysing 275 clinical isolates of bacteria acquired from 65 patients suffering from cystic fibrosis or chronic obstructive pulmonary disease. Our results show that its routine use may result in partial worsening of its discriminatory power; however, it still outmatched conventional phenotyping in the group of non-fermentative Gram-negative rods. Moreover, when supplemented by rapid, simple and economic oxidase test, it can be even simplified for more economic performance.

The CRZ1/SP1-like gene links survival under limited aeration, cell integrity and biofilm formation in the pathogenic yeast Cryptococcus neoformans.

AIMS: Limited aeration has been demonstrated to cause slowdown in proliferation and delayed budding, resulting eventually in a unique unbudded G2-arrest in the obligate aerobic pathogenic yeast Cryptococcus neoformans. Also, the ability to adapt to decreased oxygen levels during pathogenesis has been identified as a virulence factor in C. neoformans. The aim of this study was to identify and characterize genes that are necessary for the proliferation slowdown and G2-arrest caused by limited aeration. METHODS: Random mutants were prepared and screened for lack of typical slowdown of proliferation under limited aeration. The CNAG_00156.2 gene coding for a zinc-finger transcription factor was identified in mutants showing most distinctive phenotype. Targeted deletion strain and reconstituted strain were prepared to characterize and confirm the gene functions. This gene was also identified in a parallel studies as homologous both to calcineurin responsive (Crz1) and PKC1-dependent (SP1-like) transcription factors. RESULTS: We have confirmed the role of the cryptococcal homologue of CRZ1/SP1-like transcription factor in cell integrity, and newly demonstrated its role in slowdown of proliferation and survival under reduced aeration, in biofilm formation and in susceptibility to fluconazole. CONCLUSIONS: Our data demonstrate a tight molecular link between slowdown of proliferation during hypoxic adaptation and maintenance of cell integrity in C. neoformans and present a new role for the CRZ1 family of transcription factors in fungi. The exact positioning of this protein in cryptococcal signalling cascades remains to be clarified.

Possibilities in identification of genomic species of Burkholderia cepacia complex by PCR and RFLP.

The strains belonging to Burkholderia cepacia complex are important opportunistic pathogens in immunocompromised patients and cause serious diseases. It is possible to obtain isolates from soil, water, plants and human samples. Taxonomy of this group is difficult. Burkholderia cepacia complex consists of seventeen genomic species and the genetic scheme is based on recA gene. Commonly, first five genomovars occurre in humans, mostly genomovars II and III, subdivision IIIA. Within this study we tested identification of first five genomovars by PCR with following melting analysis and RFLP. The experiments were targeted on eubacterial 16S rDNA and specific gene recA, which allowed identification of all five genomovars. RecA gene appeared as more suitable than 16S rDNA, which enabled direct identification of only genomovars II and V; genomovars I, III and IV were similar within 16S rDNA sequence.

Usefulness of McRAPD for typing and importance of biofilm production in a case of nosocomial ventriculoperitoneal shunt infection caused by Candida lusitaniae.

A case report of ventriculoperitoneal shunt infection caused by Candida lusitaniae in a 6-year-old patient with cerebral astrocytoma and obstructive hydrocephalus is presented briefly with emphasis on the course of antifungal treatment. Seven isolates recovered subsequently from the cerebrospinal fluid were studied retrospectively. To confirm identity, isolates were typed using pulsed-field gel electrophoresis and melting curve of random amplified polymorphic DNA (McRAPD). Further, the ability to form biofilm and its susceptibility to systemic antifungals were evaluated. Using McRAPD, identity of C. lusitaniae isolates showing slight microevolutionary changes in karyotypes was undoubtedly confirmed; successful application of numerical interpretation of McRAPD for typing is demonstrated here for the first time. The strain was also recognized as a strong biofilm producer. Moreover, minimum biofilm inhibitory concentrations were very high, in contrast to low antifungal minimum inhibitory concentrations of isolates. It can be concluded that McRAPD seems to be a simple and reliable method not only for identification but also for typing of yeasts. A ventriculoperitoneal shunt colonized by C. lusitaniae was revealed as the source of this nosocomial infection, and the ability of the strain to form biofilm on its surface likely caused treatment failure.