Mere Contaminants Research Articles

Pantoea agglomerans: A rare infectious outbreak affecting maintenance hemodialysis patients in a tertiary care hospital.

Pantoea agglomerans is an environmental pathogen known to cause infection in immunocompromised individuals, particularly after thorn injuries. However, previous data showed few cases of human disease caused by contaminated medical products such as parenteral nutrition, anesthetic agents, blood, and peritoneal dialysis solutions. Infection in hemodialysis patients is rare. In this study, we presented a detailed account of several hemodialysis patients infected with this contagious pathogen and compared them with noninfected dialysis patients. We retrospectively reviewed the hospital records of 105 hemodialysis patients. Seventeen of 105 patients were diagnosed with P. agglomerans infection. We carefully analyzed their entire in-hospital course. Among infected patients, 52.9% were male with a median age of 49 (IQR: 32-66) years. Compared to the noninfected patients, age below 50 years, prior kidney transplantation, prior immunosuppression and antibiotics use, and dialysis via a tunneled vascular catheter were the significant epidemiological features. Despite negative microbiological investigations, we suspect the possible infectious spread via infected central venous catheter was the likely infectious source. Most importantly, all patients responded well to intravenous antibiotics. Only two patients required the removal of the tunneled catheter. Their mortality rate was 0%. P. agglomerans infection, although considered rare, is becoming increasingly prevalent among dialysis patients. Its occurrence must be appraised as an infectious outbreak rather than mere contamination. Prompt treatment, source identification, and early implementation of preventive strategies should always be the goal to curtail this infection at an early stage.

A rare case of Candida parapsilosis pneumonia in an immunocompetent patient

Introduction: The presence of fungus in an immunocompetent host is usually disregarded as a mere contaminant, as it can be a commensal organism of the skin, gastrointestinal, urogenital, and respiratory tract. Hence, its growth in cultures has to be interpreted within a clinical context. This case illustrates the challenges experienced when diagnosing Candida parapsilosis necrotizing pneumonia, and the importance for considering candida pneumonia as a differential diagnosis for an immunocompetent patient. After a thorough literature review, we would like to present the first case report of C. parapsilosis causing necrotizing pneumonia in an immunocompetent patient. Case Report: We present a case involving a middle-aged smoking male who presented with respiratory and metabolic abnormalities and was found to have necrotizing pneumonia. He was managed for severe sepsis with lactic acidosis, respiratory failure, and severe acute kidney injury (AKI), which improved with broad spectrum antibiotics and fluids. These conditions improved; however, his respiratory distress did not despite a prolonged course of antibiotics. This led to a workup for other causes of necrotizing pneumonia, after which cultures revealed the growth of C. parapsilosis. He was then started on antifungals and subsequently improved. Conclusion: Candida necrotizing pneumonia is a rare disease for an immune-competent individual; however, chronic lung damage in the setting of a smoking history may make individuals more susceptible. This case illustrates the challenges associated when dealing with such a case, and it is the team’s hope that publishing this case will add to awareness. Additionally, this can contribute to improved antibiotic stewardship and earlier diagnosis which will hopefully lead to a shorter hospital stay and improved morbidity and mortality.

The genus Ralstonia: The new kid on the block.

The genus Ralstonia comprises of aerobic, gram-negative, oxidase positive, nonfermentative, largely environmental organisms. They are an emerging pathogen in the hospital setting and are increasingly associated with opportunistic infections and outbreaks. We hereby present a case series of six patients diagnosed with bacteraemia caused by Ralstonia spp. and a brief review of literature. These cases highlight that isolation of a nonfermenting gram-negative bacillus from blood culture of a patient admitted in critical care setting should not be ignored as mere contaminant. Clinicians and microbiologists need to work as a team to combat this novel bug.

Corynebacterium bovis surgical site infection and brain abscess: The first case report and literature review

Corynebacterium bovis is a mainly zoonotic pathogen, a common cause of bovine mastitis. It is however rarely pathogenic in humans, with only few cases reported in the literature. We present the first reported case of neurosurgical site infection due to c.bovis, resulting in a brain abscess. A 75-year-old female presented with dysphasia resulting from lung metastases. She underwent surgical resection, and four months later presented with swelling, tenderness and crusted exudate over the surgical site. Mri revealed surgical site infection and brain abscess, therefore the patient underwent urgent surgery. C.bovis was isolated from all specimens sent from different locations. The patient received appropriate antibiotic treatment without sequela. C. Bovis is being increasingly reported as a cause of various human infections, and should not automatically be considered to be a mere contaminant. It is imperative to be certain, prior to the antibiotics treatment, that this particular isolate is likely to be the pathogen, as it can be evident when there are multiple positive cultures of this pathogen from several locations.

Virulence Characterization of Coagulase-Negative Staphylococci Isolated from Sexually Active Women with Symptomatic Genital Tract Infection, In Comparison with Staphylococcus aureus Isolated from the Same Cases

This study included 33 coagulase-negative staphylococci (CNS) and 35 Staphylococcus aureus isolated from sexually active females with symptomatic genital tract infection (64 pregnant and 110 non-pregnant). Comparison of phenotypic characteristics showed that the difference is not significant between CNS and S. aureus isolates for hemolytic activity (63.6% and 54.2%, respectively); lipolytic activity (87.8% and 88.5%, respectively); protease production (18.1% and 31.4%, respectively). and biofilm formation ( 96.9% and 94.2%, respectively). Whereas the difference is significant (P≤ 0.05) for urease production (18.1% and 42.8%, respectively). With the exception of lipolytic activity and biofilm formation, co-expression of other factors showed that there was an opposite expression of hemolytic and proteolytic activities of both CNS and S. aureus isolates. For CNS only 3/33 (9%) of the isolates were hemolytic and preoteolytic while for S. aureus only 2/35 (5.7%) isolates had this characteristics. Also for S. aureus isolates, ureolytic activity was more correlated with hemolytic (20%) and proteolytic (11.4%) activities than in CNS (6.0% and 3.0%, respectively). We concluded that, for this study included factors, the virulence of CNS isolated from females' genital tract infection is comparable to that of the potential females' genital tract pathogen, S. aureus. So that, CNS can be opportunistic pathogens in these cases rather than mere contaminants.

Antimicrobial Resistance and Virulence Genes in Staphylococci Isolated from Aviary Capercaillies and Free-living Birds in South-eastern Poland.

The current study characterises Staphylococcus bacteria recovered from dead free-living birds and captive capercaillies kept in south-eastern Poland. The results provide novel information about the antimicrobial resistance phenotype/genotype and the virulence profile of these bacteria. Samples of internal organs were taken from dead birds. Staphylococcus strains were identified by matrix-assisted laser desorption/ionisation-time-of-flight mass spectrometry. Susceptibility to 13 antibiotics was tested using a standard disc diffusion method on Mueller-Hinton agar. All isolates were screened for the presence of antibiotic resistance genes and staphylococcal enterotoxins (A to E), toxic shock syndrome toxin 1, exfoliative toxins A and B and Panton-Valentine leukocidin. A total of 129 bacterial strains belonging to 19 species of the Staphylococcus genus were isolated. A relatively high percentage of them resisted fluoroquinolones, tetracyclines, macrolides and β-lactams to a significant degree and harboured the tetK, tetM, ermC, mphC and mecA genes. Strains of the coagulase-negative S. sciuri, S. xylosus and S. cohnii were isolated with genes encoding enterotoxin A and toxic shock syndrome toxin. Both coagulase-positive and coagulase-negative staphylococci isolated from aviary capercaillies and free-living birds have significant pathogenic potential, and greater attention must be paid to the coagulase-negative species, which are still often considered mere contaminants. Virulence factors associated with resistance to antimicrobials, this being multiple in some strains, seem most important because they can be easily transferred between animals, especially those living in a given area.

48. Bacterial presence in lumbar discs–subclinical infection or contamination? Insights from metabolomics

<h3>BACKGROUND CONTEXT</h3> The accumulating evidence associating subclinical infection with degenerative disc disease (DDD) and the controversy of contamination vs infection mandates further understanding of the microbial activity in the disc and host-microbiome interaction. <h3>PURPOSE</h3> To utilize a novel approach of metabolomics to probe the presence of bacterial metabolites involved in colonization, survival and replication in human lumbar intervertebral discs (LIVD). <h3>STUDY DESIGN/SETTING</h3> An observational case-control study. <h3>PATIENT SAMPLE</h3> Nucleus pulposus from the LIVD of three brain-dead voluntary organ donors (MRI normal and classified as controls) and of three patients undergoing surgery for degenerative disc disease (DDD) (cases) were utilized. <h3>OUTCOME MEASURES</h3> To prove presence of live and metabolically active bacteria in intervertebral disc. <h3>METHODS</h3> Untargeted metabolite profiling was carried out in six discs (3 controls and 3 cases) after extraction using methanol: acetonitrile: water (2 2:1) solvent system and acquired through HPLC-MS/MS platform using C18 reversed-phase column. From the total IVD metabolome, microbial metabolites were filtered by mapping against HMDB, ChEBI, SigMol, Siderophore database, ecdmb database, and PaMet databases. Biological functions of the metabolites were then studied by MSEA pipeline from Metaboanalyst, and the enrichment ratio, p-value, and Variably Importance Projection scores (VIPS) of the metabolites calculated. Degeneration responsive changes in the abundance of the microbial metabolites were calculated based on the peak intensities between the control and cases. <h3>RESULTS</h3> Mass spectrometry identified a total of 17601 and 15003 metabolites, respectively, in the control and diseased discs. Preliminary mapping of the above metabolites against HMDB indicated the multiple sources and of these, 64 metabolites were of microbial origin, accounting for 1.6% of the total IVD metabolome. Principle Component Analysis (PCA) and Orthogonal Partial Least Square-Discriminant Analysis (OPLSD-DA) showed distinct clustered patterns between control and disease, indicating a strong variation in concentration, peak and spectral values of the 64 metabolites between controls and cases. After exclusion of metabolites that were also associated with humans, drugs and food, 39 metabolites specific to bacteria were isolated. Nine were primary related to bacterial growth and survival, and the remaining 30 were secondary related to different environmental stress response activities. The three significant pathways (p 1.0) was found for nine metabolites which included (S)-14-methyilhexadecanoic acid related to P.acnes, 9-OxoODE, and 13-OxoODE related to gut flora, vibriobactin - a siderophore, tuberculosinol and iso-tuberculosinol, virulence factors of M.tuberculosis. There was also upregulation of autoinducer- 2, an important "Quorum sensing molecule" involved in bacterial cross-talk. <h3>CONCLUSIONS</h3> We identified several bacterial-specific metabolites participating in pathways involved in bacterial growth, survival and cross-talk. These were found in both groups but upregulated in diseased discs. Presence of quorum sensing molecules and cell-cell interactions provides firm proof of colonization and growth. These findings indicate that bacterial presence is not mere contamination but a colonization with possible role in infection-mediated inflammaging in DDD. <h3>FDA DEVICE/DRUG STATUS</h3> This abstract does not discuss or include any applicable devices or drugs.

Fatal pneumonia in an immunocompetent individual

This case clearly demonstrate the need for increased awareness of the potential for B. cereus to cause serious systemic infections, even in patients who appear to be otherwise healthy. Unless there is good liaison between the pathologist and clinicians, their role in genuine infection can be easily overlooked. Knowing that Bacillus species can cause life-threatening infections in such apparently normal hosts should prevent clinicians from regarding these organisms as mere contaminants when they are recovered from seriously ill patients. Initial empirical treatment with β- lactam antibiotics in patients showing aerobic gram positive rods may be avoided which may preclude the treatment of B.cereus.

Bacteria in human lumbar discs – subclinical infection or contamination? Metabolomic evidence for colonization, multiplication, and cell-cell cross-talk of bacteria

The accumulating evidence associating sub-clinical infection with disc degeneration (DD) and the controversy of contamination versus infection mandates a further understanding of the microbial activity in the disc and host-microbiome interaction. To utilize a novel approach of metabolomics to probe the presence of bacterial metabolites involved in colonization, survival, and replication in human lumbar intervertebral discs (LIVD). An observational case-control study. Nucleus pulposus from the LIVD of three brain-dead voluntary organ donors (MRI normal and classified as controls) and of three patients undergoing surgery for disc degeneration (DD) (cases) were utilized. Untargeted metabolite profiling was carried out in six discs (3-controls and 3-cases) after extraction using methanol: acetonitrile: water (2:2:1) solvent system and acquired through HPLC-MS/MS platform using C18 reversed-phase column. From the total IVD metabolome, microbial metabolites were filtered by mapping against HMDB, ChEBI, SigMol, Siderophore database, ecdmb database, and PaMet databases. The biological functions of the metabolites were then studied by MSEA pipeline from Metaboanalyst, and the enrichment ratio, p-value, and Variably Importance Projection scores of the metabolites were calculated. Degeneration responsive changes in the abundance of the microbial metabolites were calculated based on the peak intensities between the control and cases. Mass spectrometry identified a total of 17601 and 15003 metabolites, respectively, in the control and degenerated discs. Preliminary mapping of the above metabolites against HMDB indicated the multiple sources, and of these, 64 metabolites were of microbial origin, accounting for 1.6% of the total IVD metabolome. Principle Component Analysis and Orthogonal Partial Least Square-Discriminant Analysis (OPLS-DA) showed distinct clustered patterns between control and disc degene`ration, indicating a strong variation in concentration, peak, and spectral values of the 64 metabolites between controls and cases. After the exclusion of metabolites that were also associated with humans, drugs, and food, 39 metabolites specific to bacteria were isolated. Nine were primary metabolites related to bacterial growth and survival, and the remaining 30 were secondary metabolites related to different environmental stress response activities. The three significant pathways (p<.001) which were predominant in the bacterial metabolites were autoinducer-2 biosynthesis, peptidoglycan biosynthesis, and chorismate pathway. In addition, a significant fold change of >1.0 was found for nine metabolites which included (S)-14-Methyilhexadecanoic acid related to P. acnes, 9-OxoODE, and 13-OxoODE related to gut flora, vibriobactin - a siderophore, tuberculosinol and iso-tuberculosinol, virulence factors of M. tuberculosis. There was also upregulation of Autoinducer- 2, an important "Quorum sensing molecule" involved in bacterial cross-talk. We identified several bacterial-specific metabolites participating in bacterial growth, survival, and cross-talk pathways. These were found in both groups but up-regulated in degenerated discs. The presence of Quorum sensing molecules and cell-cell interactions provides firm proof of colonization and growth. These findings indicate that the bacterial presence may not be mere contamination but could be colonization with a possible role in infection-mediated inflammation in DD. Proof of subclinical infection as an initiator of DD and documentation of exact germ and drug sensitivity will change the way millions of patients with non-specific low back pain (NSLBP) are treated across the world.

FUNGAL DETERIORATION OF LEMON (CITRUS LIMON BURN F.) AND VITAMIN C CONTENT OF INFECTED FRUITS FROM KEFFI, NASARAWA STATE

This study was carried out in Keffi Metropolis to evaluate fungi associated with the deterioration of lemon (Citrus limonBurn F.) and the vitamin C content of the infected fruits. The lemon fruit samples were obtained from four selected marketing centres in Keffi metropolis. These includes Keffii main market, AngwanLambu, AngwanKaje and Angwan Fulani respectively. Out of 48 samples of lemon fruits examined, 34 had fungal species while 14 had no fungal species. The fungal analysis showed that Aspergillus niger, Rhizopus stoloniferand Penicillium digitatumwere associated with the spoilt lemon fruits (C. limon) with frequencies of occurrence of 61.76%, 17.65% and 20.59% respectively. Some fresh lemon fruits stored at a temperature of 25oC and 30oC showed no sign of decay. The incidence of fungal species in lemon fruits from the different sampling markets include Keffi main market (26.47%), AngwanLambu (20.59%), AngwanKaje (29.41%) and Angwan Fulani (23.53%)respectively. There is no significant difference (P&gt;0.05) between species of fungi isolated from different sources (markets) in Keffi. Pathogenicity test showed that Penicillium digitatumwas not a mere contaminant of lemon fruits but a primarily causative organism (80%) followed by Rhizopus stolonifer(60%) and Aspergillus niger(60%). Vitamins C content of both the infected and uninfected lemon fruits showed that infected fruits gave 31.37mg/100ml of vitamin C while the uninfected fruits gave 32.47mg/100ml vitamin C. Consumption of deteriorated lemon fruits should be discouraged.

Nontuberculous mycobacterium: An emerging pathogen: Indian perspective.

Nontuberculous mycobacteria (NTM), considered as mere contaminants, are off late, being reported as potential pathogens through various studies. The infections due to NTM range from pulmonary to extra pulmonary including skin and soft-tissue infections, traumatic and surgical wound infections, and catheter and implant-associated infections. Although extrapulmonary infections are extensively explored, pulmonary infections are scarcely reported due to their misdiagnosis as tuberculosis caused by M. tuberculosis (MTB). Appropriate detection methods are essential in order to facilitate the differential diagnosis of NTM from MTB infections. We aimed to collate the data available on NTM diagnosis and its epidemiology in India in this review. While diagnosis of MTB itself is more challenging in India, for appropriate treatment of NTM, special training and attention is needed for differential diagnosis of the former from latter. Currently, in India, in addition to the available techniques for identification of NTM, line probe assay (Hains life sciences) is proving to be a promising tool for the detection of NTM (common mycobacteria/additional species kit) and their antimicrobial resistance (Genotype NTM-DR VER 1.0). In future, with the price of sequencing steadily coming down, with proper monitoring, whole-genome sequencing could be the test of choice to predict the species, drug resistance, outbreaks in hospitals, and transmission dynamics.

Comparative genome analysis of Corynebacterium species: The underestimated pathogens with high virulence potential.

Non-diphtherial Corynebacterium species or diphtheroids were previously considered as the mere contaminants of clinical samples. Of late, they have been reckoned as the formidable infection causing agents of various diseases. While the scientific database is filled with articles that document whole genome analysis of individual isolates, a comprehensive comparative genomic analysis of diphtheroids alongside Corynebacterium diphtheriae is expected to enable us in understanding their genomic as well as evolutionary divergence. Here, we have analysed the whole genome sequences of forty strains that were selected from a range of eleven Corynebacterium species (pathogenic and non-pathogenic). A statistical analysis of the pan and core genomes revealed that even though the core genome is saturated, the pan genome is yet open rendering scope for newer gene families to be accumulated in the course of evolution that might further change the pathogenic behavior of these species. Every strain had bacteriophage components integrated in its genome and some of them were intact and consisted of toxins. The presence of diversified genomic islands was observed across the dataset and most of them consisted of genes for virulence and multidrug resistance. Moreover, the phylogenetic analysis showed that a diphtheroid is the last common ancestor of all the Corynebacterium species. The current study is a compilation of genomic features of pathogenic as well as non-pathogenic Corynebacterium species which provides insights into their virulence potential in the times to come.

The Microbiome of Leonardo da Vinci's Drawings: A Bio-Archive of Their History.

Seven emblematic Leonardo da Vinci’s drawings were investigated through third generation sequencing technology (Nanopore). In addition, SEM analyses were carried out to acquire photographic documentation and to infer the nature of the micro-objects removed from the surface of the drawings. The Nanopore generated microbiomes can be used as a “bio-archive” of the drawings, offering a kind of fingerprint for current and future biological comparisons. This information might help to create a biological catalog of the drawings (cataloging), a microbiome-fingerprint for each single analyzed drawing, as a reference dataset for future studies (monitoring) and last but not least a bio-archive of the history of each single object (added value). Results showed a relatively high contamination with human DNA and a surprising dominance of bacteria over fungi. However, it was possible to identify typical bacteria of the human microbiome, which are mere contaminants introduced by handling of the drawings as well as other microorganisms that seem to have been introduced through vectors, such as insects and their droppings, visible through the SEM analyses. All drawings showed very specific bio-archives, but a core microbiome of bacteria and fungi that are repeatedly found in this type of material as true degraders were identified, such as members of the phyla Proteobacteria, Actinobacteria, and Firmicutes among bacteria, and fungi belonging to the classes Sordariomycetes and Eurotiomycetes. In addition, some similarities were observed that could be influenced by their geographical location (Rome or Turin), indicating the influence of this factor and denoting the importance of environmental and storage conditions on the specific microbiomes.

IDENTIFICATION AND CHARACTERIZATION OF NONFERMENTATIVE GRAM NEGATIVE BACILLI FROM VARIOUS CLINICAL SAMPLES IN A TERTIARY CARE HOSPITAL, JAIPUR.

Background: Non fermentative gram negative bacilli that were considered to be contaminants in the past have now emerged as important health care associated pathogens.&#x0D; Aim: This study was undertaken to identify and characterize Nonfermenters isolated from various clinical samples.&#x0D; Material and Methods: Present study was conducted in the department of Microbiology, National Institute of Medical Sciences (NIMS) Medical College and hospital, Jaipur from July 2013 to July 2014. A total 150 strains of Nonfermenters were isolated from 1200 various non repetitive clinical samples. The samples were processed and Non fermentative gram negative bacilli (NFGNB) identified as far as possible up to species level as per standard protocols.&#x0D; Results: In the present study, 12.50% of incidence / isolation rate of Nonfermenters in various clinical samples was found. Out of 150 strains of Nonfermenter, Pseudomonas aeruginosa was the most common isolate 134 (89.33%) followed by Acinetobacter baumannii 16 (10.67%). 89 (59.33%) NFGNB were isolated from males and 61 (40.67%) were from females. They were mainly isolated from age group 21-30 Years. Higher rate of isolation of NFGNB were 85 (56.67%) from IPD and 38 (25.33%) from ICU patients. Among all clinical samples Pus and Wound Discharge yield maximum isolates of NFGNB i.e. 54 (36%) followed by Sputum 39 (26.0%).&#x0D; Conclusion: NFGNB should not be ignored as mere contaminant but correlate clinically for its pathogenic potential and identified using standard protocol so as to institute appropriate and timely antibiotic coverage&#x0D; Keywords: Identification, Nonfermenter, Pseudomonas, Acinetobacter

Protein profiling of Acanthamoeba species using MALDI-TOF MS for specific identification of Acanthamoeba genotype.

Acanthamoeba spp. are ubiquitous in the environment and have the potential to cause severe infections. The different genotypes of Acanthamoeba have been shown to influence the severity of the disease and response to therapy. Characterizing Acanthamoeba spp. upto genotype can aid in infection control practices. Twenty-five Acanthamoeba isolates, characterized by 18S rDNA sequencing, were subjected to MALDI-TOF MS analysis by creating a database for the individual genotypes. The differentiating features of the various spectra were observed; the coded samples were then tested against the created database. The results of identification were compared with sequencing. Five different genotypes were obtained-T3, T4, T5, T10, and T11. Spectral analysis revealed genus-specific and genotype-specific peaks. The peak patterns for individual genotype were discrete and reproducible. Clinical isolates produced different peaks from the environmental isolate of the same genotype. A concordance of 92% was obtained with MALDI-TOF MS in comparison with 18sDNA sequencing. MALDI-TOF MS, once optimized, has the potential to reliably identify the genotype of Acanthamoeba spp. and to differentiate clinical isolate from mere contaminant.

Diphtheroids-Important Nosocomial Pathogens.

Diphtheroids are defined as aerobic, non-sporulating, pleomorphic Gram-positive bacilli which are more uniformly stained than Corynebacterium diphtheriae, lack the metachromatic granules and are arranged in a palisade manner. They are usually commensals of the skin and mucous membranes. They differ from C.diphtheriae in biochemical rea-ctions as well as in toxin production. Since, they are usually found as commensals on the skin, they are often considered as mere contaminants when isolated from clinical samples. However, there are increasing reports of these organisms being associated with various infections. Hence, we felt the need to study the common species associated with infections and know the properties of these organisms which are otherwise considered as mere laboratory contaminants. To identify the various species of diphtheroids isolated as pure growth from clinical specimens whose Gram's smear revealed numerous inflammatory cells with Gram positive bacilli and had clinical evidence. A total of 100 isolates of Gram-positive bacilli from 16,242 clinical samples received in the Microbiology Department of Kasturba Medical College were considered for this study from Dec 2013-Dec 2014. Gram-positive bacilli which were seen in the smear along with pus cells, isolated as pure growth and reported as "Corynebacterium spp having clinical significance" were taken for this study while those which were reported as 'Probable skin contaminants' were excluded from this study. Species identification of Gram-positive bacilli was done by biochemical reactions. Antibiotic susceptibility test was done by Kirby-Bauer disk diffusion method. Biofilm production was done by the microtitre plate method of O'Toole and Kolter and statistical analysis was done by using proportion test and Chi-square test. Various species of diphtheroids were isolated from different clinical specimens. C. pseudotuberculosis, C. renale, C. ulcerans, C. striatum, C. minutissimum, Corynebacterium haemolyticum isolated from catheter tips, sputum, tracheostomy secretions and wound infections were highly resistant to many antibiotics while isolates from blood namely C. pseudotuberculosis, C. minutissimum, C. ulcerans and C. renale were nearly sensitive to most of them. It was also interesting to note that there was an increased rate of biofilm production in these isolates. Corynebacterium pseudotuberculosis, Coryne- bacterium ulcerans, Corynebacterium renale, Corynebacterium bovis, Corynebacterium striatum, Corynebacterium minutissimum, Corynebacterium pseudodiphtheriticum and Corynebacterium haemolyticum may survive in the form of biofilms in hospitals and cause multidrug resistant infections. Hence, we need to judiciously identify these organisms, find their antimicrobial susceptibility, treat them and thus prevent infections in hospitals.

Abstract 2009: Flow cytometric analysis, sorting and molecular analysis of dissociated cells from human solid tumors derived from PDX mouse models

Abstract Functional and genetic heterogeneity in tumor tissue has been a well described phenomenon for many decades but only recently emerged as a potentially crucial contributor to cancer development and progression. The correlation between cellular heterogeneity and aggressiveness, metastatic potential and drug susceptibility of a cancerous lesion have led to models in which the existence of multiple clonal cell lineages is a central feature enabling a neoplastic lesion to overcome selective pressures caused by the surrounding tissues’ defensive capabilities as well as therapeutic interventions. In addition, the role of the tumor microenvironment as an integral part of tumorigenesis was recognized and infiltrating leukocytes or tumor associated fibroblasts are no longer viewed as mere contaminants of a solid tumor biopsy. The emerging picture is compared to macroscopic ecosystems and a detailed understanding of the interactions between numerous cell subgroups seems necessary for the complete understanding of cancer pathogenesis. Scarcity of appropriate tools and model systems are an obstacle to the investigation of this heterogeneity at a molecular level but advances over the last few years have led to a significant acceleration in this field. More sensitive and far cheaper methods for collection of genomic and transcriptomic data have revealed a complex picture of the evolution of individual solid tumors. To turn this deeper understanding of tumorigenesis into improved clinical outcomes, routine methods are required to separate complex tumors into subpopulations. This stratification will provide a more comprehensive characterization of the tumor and enable more detailed prediction of disease progression and resistance development. We have developed an integrated workflow for dissociation and flow cytometric analysis and sorting for multiple downstream analysis modalities. Using patient derived xenograft (PDX) mouse models derived from primary human breast cancer biopsies we have demonstrated the ability to identify distinct immunophenotypes for each model and use this analysis to isolate distinct subpopulations. Our successful optimization of a variety of well characterized surface markers (e.g. CD 24, 44, 133, 184, 326 (EpCAM), and CD45) provides a basis for effective fingerprinting of cancer cells from a variety of sources. In an effort to demonstrate the potential of FACS sorting of solid tumor derived cell populations we have interrogated sorted fractions by NGS as well as RT-PCR array analysis and show distinct genotypic as well as gene expression signatures for each subgroup. The evidence provided by our data suggests that the single cell focused approach flow cytometry has traditionally enabled in hematological cancers is accessible for solid tumors as well and may unlock valuable biological insights. Citation Format: Rainer Blaesius, Friedrich Hahn, Eileen Snowden, Warren Porter, Mitchell Ferguson, Frances Tong, Stewart Jurgensen, Chang Chen, Daphne Clancy, Jamal Sirriyah, John Alianti, Perry Haaland, Shannon Dillmore, Jeff Baker, Aaron Middlebrook, Joyce Ruitenberg, Maria Suni, Smita Ghanekar. Flow cytometric analysis, sorting and molecular analysis of dissociated cells from human solid tumors derived from PDX mouse models. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2009. doi:10.1158/1538-7445.AM2015-2009

Abstract A198: Flow cytometric analysis and sorting of dissociated cells from human solid tumors derived from PDX mouse models.

Abstract Functional and genetic heterogeneity in tumor tissue was first observed over 50 years ago. Today, tumor heterogeneity is frequently evoked in describing the pathway from pre-cancerous lesions to aggressive, metastatic cancer. During this progression, multiple clonal lineages are thought to arise, leading to subpopulations of the tumor showing different metastatic profiles and susceptibility to anti-cancer therapy. In addition, the role of the tumor microenvironment became recognized and infiltrating leukocytes or tumor associated fibroblasts are no longer viewed as mere contaminants of a solid tumor biopsy. The emerging picture is increasingly compared to macroscopic ecosystems and a detailed understanding of the interactions between numerous cell subgroups seems necessary for the complete understanding of cancer pathogenesis. Scarcity of appropriate tools and model systems are an obstacle to the investigation of this heterogeneity at a molecular level but advances over the last few years have led to a significant acceleration in this field. More sensitive and far cheaper methods for collection of genomic and transcriptomic data have revealed a complex picture of the evolution of individual solid tumors. To turn this deeper understanding of tumorigenesis into improved clinical outcomes, routine methods are required to separate complex tumors into subpopulations. This stratification will provide a more comprehensive characterization of the tumor and enable more detailed prediction of disease progression and resistance development. We have developed dissociation methods for solid tumor tissue which allows flow cytometric analysis as well as sorting to provide cells for multiple downstream analysis modalities. Using patient derived xenograft (PDX) mouse models derived from primary human breast, colorectal and lung cancer biopsies we have demonstrated efficient dissociation, surface marker analysis and nucleic acid purification from sorted populations. Conditions have been optimized for a range of relevant surface markers (e.g. CD 24, 44, 133, 184, 326 (EpCAM), and CD45) which are suitable to identify cells predicted to have stem cell, endothelial, epithelial or immune cell functions, respectively. Through sequencing of subpopulations identified by their phenotype we have demonstrated the compatibility of our workflow with downstream analysis methods such as Next Generation Sequencing (NGS). Our RNA stability measurements suggest that gene expression analysis is equally feasible. Our data provide a standardized basis for in depth investigation of subpopulations of cells from solid tumors with various molecular techniques. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A198. Citation Format: Rainer Blaesius, Friedrich Hahn, Eileen Snowden, Warren Porter, Mitchell Ferguson, Tina Marmura, Frances Tong, Shannon Dillmore, Aaron Middlebrook, Joyce Ruitenberg, Maria Suni, Smita Ghanekar. Flow cytometric analysis and sorting of dissociated cells from human solid tumors derived from PDX mouse models. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A198.

Urine, Once Thought Sterile, Has Its Own Microbiome

The long-enshrined belief that urine is sterile is giving way to the notion that the urinary tract hosts a bona fide microbiome, according to several investigators who spoke during the symposium, “How Knowledge from the Human Microbiome Projects Will Change the Practice of Clinical Microbiology,” convened last May in Denver during the 2013 ASM General Meeting. Thus, they say, when urine specimens from healthy patients test positive for bacteria, it may no longer be acceptable for clinicians to dismiss those results as mere contamination or “asymptomatic bacteriuria.”

Candida tropicalis in a case of cholangiocarcinoma with cholangitis at a tertiary care hospital in Manipal

Biliary candidiasis is increasing in the hospitalized immunosuppressed individuals. Placement of biliary stents in the cancer patients with obstructive jaundice has been found to be an important factor associated with infectious complications. Positive fungal cultures from bile should not be ignored as mere contamination but should be considered when prescribing treatment for the immunosuppressed with recurrent cholangitis or receiving long-term antibiotic therapy. Here,we report a case of cholangiocarcinoma with cholangitis where Candida tropicalis was the sole pathogen isolated from bile. This is probably the first case of its kind to be reported from Manipal, Karnataka, South India.