Genomic and phenotypic diversity of Enterococcus faecalis isolated from endophthalmitis

Enterococcus faecalis are hospital-associated opportunistic pathogens and also causative agents of post-operative endophthalmitis. Patients with enterococcal endophthalmitis often have poor visual outcomes, despite appropriate antibiotic therapy. Here we investigated the genomic and phenotypic characteristics of E. faecalis isolates collected from 13 patients treated at the University of Pittsburgh Medical Center Eye Center over 19 years. Comparative genomic analysis indicated that patients were infected with E. faecalis belonging to diverse multi-locus sequence types (STs) and resembled E. faecalis sampled from clinical, commensal, and environmental sources. We identified known E. faecalis virulence factors and antibiotic resistance genes in each genome, including genes conferring resistance to aminoglycosides, erythromycin, and tetracyclines. We assessed all isolates for their cytolysin production, biofilm formation, and antibiotic susceptibility, and observed phenotypic differences between isolates. Fluoroquinolone and cephalosporin susceptibilities were particularly variable between isolates, as were biofilm formation and cytolysin production. In addition, we found evidence of E. faecalis adaptation during recurrent endophthalmitis by identifying genetic variants that arose in sequential isolates sampled over eight months from the same patient. We identified a mutation in the DNA mismatch repair gene mutS that was associated with an increased rate of spontaneous mutation in the final isolate from the patient. Overall this study documents the genomic and phenotypic variability among E. faecalis causing endophthalmitis, as well as possible adaptive mechanisms underlying bacterial persistence during recurrent ocular infection.

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Genomic and phenotypic diversity of Enterococcus faecalis isolated from endophthalmitis

10.1101/2020.08.05.239061 ◽

2020 ◽

Author(s):

Gayatri Shankar Chilambi ◽

Hayley R. Nordstrom ◽

Daniel R. Evans ◽

Regis P. Kowalski ◽

Deepinder K. Dhaliwal ◽

...

Keyword(s):

Antibiotic Resistance ◽

Enterococcus Faecalis ◽

Biofilm Formation ◽

Phenotypic Diversity ◽

Medical Center ◽

Phenotypic Variability ◽

Comparative Genomic ◽

Mismatch Repair Gene ◽

Ocular Infection ◽

First Case

ABSTRACTEnterococcus faecalis are hospital-associated opportunistic pathogens and also causative agents of post-operative endophthalmitis. Patients with enterococcal endophthalmitis often have poor visual outcomes, despite appropriate antibiotic therapy. Here we investigated the genomic and phenotypic characteristics of E. faecalis isolates collected from 13 patients treated at the University of Pittsburgh Medical Center Eye Center over 19 years. Comparative genomic analysis indicated that patients were infected with E. faecalis of diverse multi-locus sequence types (STs) previously associated with clinical, commensal, and environmental sources. We identified known E. faecalis virulence factors and antibiotic resistance genes in each genome, including genes conferring resistance to aminoglycosides, erythromycin, and tetracyclines. We assessed all isolates for their cytolysin production, biofilm formation, and antibiotic susceptibility, and observed phenotypic differences between isolates. Fluoroquinolone and cephalosporin susceptibilities were particularly variable, as were biofilm formation and cytolysin production. In addition, we found evidence of E. faecalis adaptation during recurrent endophthalmitis by identifying genetic variants that arose in sequential isolates sampled over eight-months from the same patient. We identified a mutation in the DNA mismatch repair gene mutS that was associated with a hypermutator phenotype in the final isolate from the patient, which was also more resistant to ceftazidime. Overall this study documents the genomic and phenotypic variability among E. faecalis causing endophthalmitis, as well as possible adaptive mechanisms underlying bacterial persistence during recurrent ocular infection.IMPORTANCEBacterial endophthalmitis is a sight-threatening infection of the inside of the eye. Enterococcus faecalis cause endophthalmitis occasionally, but when they do the infections are often severe. Here we investigated the genomes, antibiotic susceptibilities, and virulence-associated traits among E. faecalis collected from 13 patients with post-operative endophthalmitis. We wondered whether there were common bacterial factors that might explain why enterococcal endophthalmitis is so destructive to ocular tissues. Instead we found that E. feacalis isolated from endophthalmitis were genetically and phenotypically diverse; isolates belonged to a variety of genetic lineages and showed varying levels of antibiotic resistance and biofilm formation. We also undertook further characterization of three closely related E. faecalis isolates from a patient with recurrent endophthalmitis, and found that a hypermutator strain emerged during persistent infection. Hypermutators have been found in a variety of other infection contexts; here we describe what we believe is the first case of a hypermutator arising during ocular infection.

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Genetic profiles of hereditary nonpolyposis colorectal cancer.

Journal of Clinical Oncology ◽

10.1200/jco.2011.29.4_suppl.469 ◽

2011 ◽

Vol 29 (4_suppl) ◽

pp. 469-469

Author(s):

C. Therklidsen ◽

G. Jonsson ◽

I. Bernstein ◽

M. Nilbert

Keyword(s):

Colorectal Cancer ◽

Mismatch Repair ◽

Hereditary Nonpolyposis Colorectal Cancer ◽

Gene Mutations ◽

Comparative Genomic ◽

Mismatch Repair Gene ◽

Repair Gene ◽

Hereditary Nonpolyposis ◽

Gains And Losses ◽

Mismatch Repair Gene Mutations

469 Background: With the aim to identify genetic markers of hereditary nonpolyposis colorectal cancer (HNPCC), we applied tiling BAC array-based comparative genomic hybridization (aCGH) to 46 HNPCC-associated colorectal cancer. Methods: 32 k iling BAC arrays were used to generate high-density genomic profiles. Tumors were selected through a case-control design with half of the tumors derived from individuals with disease-predisposing mismatch repair gene mutations and the reminder from phenotypic HNPCC families without identified mutations. In addition, an equal number of sporadic tumors were used for comparison. Results: Tumors with disease-predisposing germline mutations showed frequent gains of chromosomes 1p (39%), 17 (43%), 19 (57%) and 22q (30%). HNPCC associated tumors without mutations did as a group have more complex alterations with the most frequent changes being gains of 20q (70%), 19 (35%), 17 (26%) and loss of 18 (39%). Gains of 1p and 20q and loss of chromosome 18 were identified as significant discriminators between HNPCC tumors with/without germline MMR gene mutations. Conclusions: The aCGH profiles of HNPCC-associated colorectal cancer suggest that specific gains and losses may be used to distinguish between tumors with/without germline mismatch repair gene mutations. No significant financial relationships to disclose.

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Ribonucleotide reductase small subunit M2 serves as a prognostic biomarker and predicts poor survival of colorectal cancers

Clinical Science ◽

10.1042/cs20120240 ◽

2013 ◽

Vol 124 (9) ◽

pp. 567-579 ◽

Cited By ~ 43

Author(s):

Xiyong Liu ◽

Hang Zhang ◽

Lily Lai ◽

Xiaochen Wang ◽

Sofia Loera ◽

...

Keyword(s):

Real Time ◽

Ribonucleotide Reductase ◽

Negative Impact ◽

Medical Center ◽

Distant Metastases ◽

Small Subunit ◽

Colorectal Cancers ◽

Mismatch Repair Gene ◽

Poor Survival ◽

Repair Gene

The overexpression of RRM2 [RR (ribonucleotide reductase) small subunit M2] dramatically enhances the ability of the cancer cell to proliferate and to invade. To investigate further the relevance of RRM2 and CRCs (colorectal cancers), we correlated the expression of RRM2 with the clinical outcome of CRCs. A retrospective outcome study was conducted on CRCs collected from the COH [(City of Hope) National Medical Center, 217 cases] and ZJU (Zhejiang University, 220 cases). IHC (immunohistochemistry) was employed to determine the protein expression level of RRM2, and quantitative real-time PCR was employed to validate. Multivariate logistic analysis indicated that the adjusted ORs (odds ratios) of RRM2-high for distant metastases were 2.06 [95% CI (confidence interval), 1.01–4.30] and 5.89 (95% CI, 1.51–39.13) in the COH and ZJU sets respectively. The Kaplan–Meier analysis displayed that high expression of RRM2 had a negative impact on the OS (overall survival) and PFS (progress-free survival) of CRC in both sets significantly. The multivariate Cox analysis further demonstrated that HRs (hazard ratios) of RRM2-high for OS were 1.88 (95% CI, 1.03–3.36) and 2.06 (95% CI, 1.10–4.00) in the COH and ZJU sets respectively. Stratification analysis demonstrated that the HR of RRM2 dramatically increased to 12.22 (95% CI, 1.62–258.31) in the MMR (mismatch repair) gene-deficient subgroup in the COH set. Meanwhile, a real-time study demonstrated that down-regulation of RRM2 by siRNA (small interfering RNA) could significantly and specifically reduce the cell growth and adhesion ability in HT-29 and HCT-8 cells. Therefore RRM2 is an independent prognostic factor and predicts poor survival of CRCs. It is also a potential predictor for identifying good responders to chemotherapy for CRCs.

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Frequency and prognostic role of tumor infiltrating lymphocytes and MMR status in advanced non-colorectal GI malignancies.

Journal of Clinical Oncology ◽

10.1200/jco.2018.36.5_suppl.89 ◽

2018 ◽

Vol 36 (5_suppl) ◽

pp. 89-89

Author(s):

Uqba Khan ◽

Ameer Hamza ◽

Renny Abraham ◽

Muhammad S Khurram ◽

Tarik H. Hadid ◽

...

Keyword(s):

Checkpoint Inhibitors ◽

Medical Center ◽

Tumor Infiltrating Lymphocytes ◽

Mismatch Repair Gene ◽

High Power Field ◽

Repair Gene ◽

Number Of Patients ◽

Significant Difference ◽

Infiltrating Lymphocytes ◽

The Impact

89 Background: Tumor infiltrating lymphocytes (TILs) and mismatch repair gene mutation (MMR) status are emerging biomarkers in immunotherapy. MMR status and TILs have significant clinical implications with respect to treatment with checkpoint inhibitors. We designed a study to determine the frequency and prognostic utility of TILs and MMR in advanced unresectable non-colorectal GI (NCGI) cancers. Methods: This is a retrospective cohort study of patients who were diagnosed with metastatic or unresectable NCGI cancers between 2009 and 2015 at St. John Hospital and Medical Center. Immunohistochemistry panels were performed on representative tissue sections for MMR testing. TILs were assessed on the hematoxylin and eosin stained slide of the same tissue section. MMR was interpreted as deficient (d) or proficient and TILs were categorized as ≤5 or > 5 per high power field. Descriptive statistics were generated using frequency distributions, medians and means. Kaplan-Meier analysis was performed to determine the impact of TILS and MMR on survival; differences by factor were assessed with the Log_Rank test. Results: We analyzed 132 patients; the mean age at diagnosis was 66.7 years, 62.1% were male. All samples had proficient MMR status. The percentage of patients with TILs ≤ 5 was 46.2. There was no statistically significant difference in median overall survival (OS) by TILs when stratified by stage of tumor. When stratified by type of tumor, median OS by TILs level was significantly different only for hepatocellular cancers (HCC) (≤ 5 TILs, 86 days vs. > 5 TILs 312 days, p = 0.031), table 1. Conclusions: Our study suggests that MMR-d tumors are quite rare in advanced NCGI malignancies. TILs can be present in tumor microenvironment of NCGI malignancies. Though the number of patients in our study was small, there was a statistically significant difference in median OS of patients with HCC when stratified by TILs status.[Table: see text]

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Frequency and prognostic utility of MSI and TILs in advanced noncolorectal GI malignancies.

Journal of Clinical Oncology ◽

10.1200/jco.2018.36.4_suppl.313 ◽

2018 ◽

Vol 36 (4_suppl) ◽

pp. 313-313

Author(s):

Uqba Khan ◽

Ameer Hamza ◽

Muhammad S Khurram ◽

Renny Abraham ◽

Paul Mazzara ◽

...

Keyword(s):

Median Survival ◽

Checkpoint Inhibitors ◽

Medical Center ◽

Adoptive T Cell Therapy ◽

Mismatch Repair Gene ◽

Repair Gene ◽

T Cell Therapy ◽

Number Of Patients ◽

Significant Difference ◽

The Impact

313 Background: Mismatch repair gene mutation status not only has a role in pathogenesis but also has significant clinical implications with respect to treatment with checkpoint inhibitors. Additionally tumor infiltrating lymphocytes (TILs) are also emerging prognostic biomarker and are utilized in adoptive T-cell therapy as well. Methods: This is a retrospective cohort study of patients who were diagnosed with advanced unresectable non-colorectal GI (NCGI) cancers. Biopsy specimens of patients diagnosed between 2009 and 2015 at St. John Hospital and Medical Center were analyzed. Immunohistochemistry panels were performed on a representative tissue sections for microsatellite instability (MSI) testing. TILs were assessed on the hematoxylin and Eosin stained slide of the same tissue section. MSI was interpreted as stable or high and TILs were categorized as ≤5 and > 5 per high power field. Descriptive statistics were generated using frequency distributions, medians and means. Kaplan-Meier analysis was performed to determine the impact of TILs and MSI on survival; differences by factor were assessed with the Log_Rank test. Results: We analyzed 114 patients; the mean age at diagnosis was 66.8 ± 10.7 years, 61.4% were male. All samples were MSI stable. The percentage of patients with TILs ≤ 5 was 46.5. When stratified by tumor stage, overall median survival by TILs level did not differ significantly. When stratified by type of tumor, overall median survival by TILs level was significantly different only for hepatocellular cancers (HCC) (≤ 5 TILs, 86 days vs. > 5 TILs 312 days, p = 0.031) only, (see table). Conclusions: Our study shows that MSI-H tumors are very rare in advanced NCGI malignancies. TILs are definitely present in tumor microenvironment of NCGI malignancies. Though the number of patients of our study was small, there was a statistically significant difference in median overall survival of patients with HCC when stratified by TILs status.[Table: see text]

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In vitro biofilm formation and antimicrobial susceptibility patterns of bacteria isolated from suspected external ocular infected patients attending Jimma University Medical Center eye clinic, southwest Ethiopia

10.21203/rs.2.1780/v1 ◽

2019 ◽

Author(s):

Kuma Diriba ◽

Tesfaye Kassa ◽

Yared Alemu ◽

Sisay Bekele

Keyword(s):

Antimicrobial Susceptibility ◽

Biofilm Formation ◽

Medical Center ◽

Ocular Infection ◽

Bacterial Isolates ◽

Susceptibility Pattern ◽

Southwest Ethiopia ◽

Antimicrobial Susceptibility Pattern ◽

Ocular Infections ◽

Jimma University

Abstract Background: Ocular disease with its complications is a major public health problem which significantly impacts on quality of life in developing countries. An ocular infection due to microbial agents, can lead to reduced vision and blindness. This study was aimed to assess the antimicrobial susceptibility pattern and biofilm forming potential of bacteria isolated from suspected external ocular infected patients attending Jimma University Medical Center (JUMC). Method: A cross sectional facility based study was conducted on 319 suspect patients with external ocular infections from March 2017 to June 2017 at JUMC in Southwest Ethiopia. External ocular specimens were collected and standard operating procedures were followed to handle and culture throughout the study period. Antimicrobial susceptibility pattern of the isolates was determined by disk diffusion method according to CLSI 2015. Microtiter (96 wells) plate method was used to screen biofilm formation by measuring optical density at 570nm. Result: Out of 319 study participants with external ocular infection, the prevalence of bacterial pathogens was 46.1%. The predominant bacterial isolates were Coagulase negative staphylococcus (CoNS) (27.7%) followed by Staphylococcus aureus (19.7%). Among Gram negatives, Pseudomonas aeroginosa (6.8%) was the leading isolate. Increased antimicrobial resistance was observed for tetracycline (64%), erthromycin (66.7%) and penicillin (77.1%). Amoxicillin-clavulanic acid, ciprofloxacin and gentamicin were the most effective drugs for both Gram negative and Gram positive ranging from about 70 to 100% with the later two drugs for external ocular infections. Methicillin resistant S. aureus (MRSA) accounted for 13.8% of S. aureus isolates.. Multidrug resistance (MDR) accounted for 68.7%. The overall biofilm formation rate of bacterial ocular pathogens was 66.1%; with P. aeruginosa (40%), CoNS (34.1%) and S. aureus (31%) formed strong biofilm phenotype. Conclusion: The prevalence of bacterial isolates among external ocular infection was high. Almost all bacterial isolates were resistant to atleast one or more drugs. MDR pathogens were observed increasingly among biofilm formers or vice versa. Therefore, antimicrobial susceptibility testing should be practiced to guide treatment of external ocular cases and to control the emergence of drug resistant bacteria.

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Metabolic Capacity of Sinorhizobium (Ensifer) meliloti Strains as Determined by Phenotype MicroArray Analysis

Applied and Environmental Microbiology ◽

10.1128/aem.00196-09 ◽

2009 ◽

Vol 75 (16) ◽

pp. 5396-5404 ◽

Cited By ~ 43

Author(s):

Emanuele G. Biondi ◽

Enrico Tatti ◽

Diego Comparini ◽

Elisa Giuntini ◽

Stefano Mocali ◽

...

Keyword(s):

Sinorhizobium Meliloti ◽

Evolutionary Dynamics ◽

Phenotypic Diversity ◽

Phenotypic Variability ◽

Natural Populations ◽

Growth Conditions ◽

Comparative Genomic ◽

Sulfur Source ◽

Phenotype Microarray ◽

High Genetic Diversity

ABSTRACT Sinorhizobium meliloti is a soil bacterium that fixes atmospheric nitrogen in plant roots. The high genetic diversity of its natural populations has been the subject of extensive analysis. Recent genomic studies of several isolates revealed a high content of variable genes, suggesting a correspondingly large phenotypic differentiation among strains of S. meliloti. Here, using the Phenotype MicroArray (PM) system, hundreds of different growth conditions were tested in order to compare the metabolic capabilities of the laboratory reference strain Rm1021 with those of four natural S. meliloti isolates previously analyzed by comparative genomic hybridization (CGH). The results of PM analysis showed that most phenotypic differences involved carbon source utilization and tolerance to osmolytes and pH, while fewer differences were scored for nitrogen, phosphorus, and sulfur source utilization. Only the variability of the tested strain in tolerance to sodium nitrite and ammonium sulfate of pH 8 was hypothesized to be associated with the genetic polymorphisms detected by CGH analysis. Colony and cell morphologies and the ability to nodulate Medicago truncatula plants were also compared, revealing further phenotypic diversity. Overall, our results suggest that the study of functional (phenotypic) variability of S. meliloti populations is an important and complementary step in the investigation of genetic polymorphism of rhizobia and may help to elucidate rhizobial evolutionary dynamics, including adaptation to diverse environments.

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Genotypic and phenotypic characterization of Streptococcus mutans strains isolated from patients with dental caries

Iranian Journal of Microbiology ◽

10.18502/ijm.v13i4.6968 ◽

2021 ◽

Author(s):

Mohammad Shahadat Hossain ◽

Sadab Alam ◽

Yead Morshed Nibir ◽

Tahrima Arman Tusty ◽

Sayyeed Mahmud Bulbul ◽

...

Keyword(s):

Dental Caries ◽

Streptococcus Mutans ◽

Biofilm Formation ◽

Acid Production ◽

Phenotypic Diversity ◽

Phenotypic Variability ◽

Clinical Isolates ◽

Phenotypic Characterization ◽

Specific Gene ◽

Polymerase Chain

Background and Objectives: The oral cavity harbors numerous Streptococcus mutans strains which display remarkable genotypic and phenotypic diversity. This study evaluated the genotypic and phenotypic diversity of 209 S. mutans strains isolated from 336 patients with dental caries and compared with the universal reference strain, UA159. Materials and Methods: Selective cultivation on mitis-salivaries-bacitracin agar and species-specific polymerase chain re- action (PCR) was carried out to isolate and identify the 209 S. mutans isolates from 336 patients with dental caries. Arbitrari- ly primed polymerase chain reaction (AP-PCR), PCR amplification of specific gene, acid production and biofilm formation capacity were performed to evaluate the genotypic and phenotypic variation. Student’s t-test and Chi-square test were used for analysis of variables and a probability (P) of <0.05 was considered as significant. Results: Our study revealed a high degree of genotypic and phenotypic variability among the clinical strains. We observed significant differences in colony morphology, generation time, biofilm formation, and acid production while growing in cul- ture medium. All the clinical isolates were able to lower pH while growing in Todd-Hewitt broth. Consistent with phenotypic variations, we also observed genotypic variation by AP-PCR and gene specific PCR. AP-PCR analysis suggested that most of the patients with dental caries have distinct type of S. mutans strains. Genes related to various two component systems were highly conserved among the isolated strains, however, bacteriocin encoding genes such as nlmAB, nlmC were absent in nearly half of the clinical isolates. Conclusion: Our results support that S. mutans clinical isolates have wide genotypic diversity and show variation in growth kinetics, acid production, acid tolerance and biofilm formation capacity and indicates the presence of diverse mechanism to initiate and establish the biofilm lifestyle which leads to tooth decay.

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