Diversity and dynamics of microbial communities in engineered environments and their implications for process stability

The availability of molecular biological tools for studying microbial communities in bioreactors and other engineered systems has resulted in remarkable insights linking diversity and dynamics to process stability. As engineered systems are often more manageable than large-scale ecosystems, and because parallels between engineered environments and other ecosystems exist, the former can be used to elucidate some unresolved ecological issues. For example, the process stability of methanogenic bioreactors containing well-defined trophic groups appears to depend on the diversity of the functional groups within each trophic level as well as on how these functional groups complement each other. In addition to using engineered systems to study general ecological questions, microbial ecologists and environmental engineers need to investigate conditions, processes, and interactions in engineered environments in order to make the ecological engineering of bioreactor design and operation more practicable.     

Advanced HIV Disease at Enrolment in HIV Care: Trends and Associated Factors over a Ten Year Period in Cambodia

Background

Early HIV diagnosis and enrolment in care is needed to achieve early antiretroviral treatment (ART) initiation. Studies on HIV disease stage at enrolment in care from Asian countries are limited. We evaluated trends in and factors associated with late HIV disease presentation over a ten-year period in the largest ART center in Cambodia.

Methods

We conducted a retrospective analysis of program data including all ARV-naïve adults (> 18 years old) enrolling into HIV care from March 2003-December 2013 in a non-governmental hospital in Phnom Penh, Cambodia. We calculated the proportion presenting with advanced stage HIV disease (WHO clinical stage IV or CD4 cell count <100 cells/μL) and the probability of ART initiation by six months after enrolment. Factors associated with late presentation were determined using multivariate logistic regression.

Results

From 2003–2013, a total of 5642 HIV-infected patients enrolled in HIV care. The proportion of late presenters decreased from 67% in 2003 to 44% in 2009 and 41% in 2013; a temporary increase to 52% occurred in 2011 coinciding with logistical/budgetary constraints at the national program level. Median CD4 counts increased from 32 cells/μL (IQR 11–127) in 2003 to 239 cells/μL (IQR 63–291) in 2013. Older age and male sex were associated with late presentation across the ten-year period. The probability of ART initiation by six months after enrolment increased from 22.6% in 2003–2006 to 79.9% in 2011–2013.

Conclusion

Although a gradual improvement was observed over time, a large proportion of patients still enroll late, particularly older or male patients. Interventions to achieve early HIV testing and efficient linkage to care are warranted.     

The Presence of Humic Substances and DNA in RNA Extracts Affects Hybridization Results

RNA extracts obtained from environmental samples are frequently contaminated with coextracted humic substances and DNA. It was demonstrated that the response in rRNA-targeted oligonucleotide probe hybridizations decreased as the concentrations of humic substances and DNA in RNA extracts increased. The decrease in hybridization signal in the presence of humic substances appeared to be due to saturation of the hybridization membrane with humic substances, resulting in a lower amount of target rRNA bound to the membrane. The decrease in hybridization response in the presence of low amounts of DNA may be the result of reduced rRNA target accessibility. The presence of high amounts of DNA in RNA extracts resulted in membrane saturation. Consistent with the observations for DNA contamination, the addition of poly(A) to RNA extracts, a common practice used to prepare RNA dilutions for membrane blotting, also reduced hybridization signals, likely because of reduced target accessibility and membrane saturation effects.     

Feasibility of MR Metabolomics for Immediate Analysis of Resection Margins during Breast Cancer Surgery

In this study, the feasibility of high resolution magic angle spinning (HR MAS) magnetic resonance spectroscopy (MRS) of small tissue biopsies to distinguish between tumor and non-involved adjacent tissue was investigated. With the current methods, delineation of the tumor borders during breast cancer surgery is a challenging task for the surgeon, and a significant number of re-surgeries occur. We analyzed 328 tissue samples from 228 breast cancer patients using HR MAS MRS. Partial least squares discriminant analysis (PLS-DA) was applied to discriminate between tumor and non-involved adjacent tissue. Using proper double cross validation, high sensitivity and specificity of 91% and 93%, respectively was achieved. Analysis of the loading profiles from both principal component analysis (PCA) and PLS-DA showed the choline-containing metabolites as main biomarkers for tumor content, with phosphocholine being especially high in tumor tissue. Other indicative metabolites include glycine, taurine and glucose. We conclude that metabolic profiling by HR MAS MRS may be a potential method for on-line analysis of resection margins during breast cancer surgery to reduce the number of re-surgeries and risk of local recurrence.     

Hernia fibroblasts lack β-estradiol induced alterations of collagen gene expression

Background  

Estrogens are reported to increase type I and type III collagen deposition and to regulate Metalloproteinase 2 (MMP-2) expression. These proteins are reported to be dysregulated in incisional hernia formation resulting in a significantly decreased type I to III ratio. We aimed to evaluate the β-estradiol mediated regulation of type I and type III collagen genes as well as MMP-2 gene expression in fibroblasts derived from patients with or without history of recurrent incisional hernia disease. We compared primary fibroblast cultures from male/female subjects without/without incisional hernia disease.     

Effects of Swine Manure on Macrolide,Lincosamide, and Streptogramin B Antimicrobial Resistance in Soils

Current agricultural practices involve inclusion of antimicrobials in animal feed and result in manure containing antimicrobials and antimicrobial-resistant microorganisms. This work evaluated the effects of land application of swine manure on the levels of tetracycline, macrolide, and lincosamide antimicrobials and on macrolide, lincosamide, and streptogramin B (MLS_B) resistance in field soil samples and laboratory soil batch tests. MLS_B and tetracycline antimicrobials were quantified after solid-phase extraction using liquid chromatography-tandem mass spectrometry. The prevalence of the ribosomal modification responsible for MLS_B resistance in the same samples was quantified using fluorescence in situ hybridization. Macrolide antimicrobials were not detected in soil samples, while tetracyclines were detected, suggesting that the latter compounds persist in soil. No significant differences in ribosomal methylation or presumed MLS_B resistance were observed when amended and unamended field soils were compared, although a transient (<20-day) increase was observed in most batch tests. Clostridium cluster XIVa accounted for the largest fraction of resistant bacteria identified in amended soils. Overall, this study did not detect a persistent increase in the prevalence of MLS_B resistance due to land application of treated swine manure.Treated swine manure contains substantial levels of both antimicrobial-resistant microorganisms (10, 26) and antimicrobials (7, 18, 33). Land application of manure could therefore contribute to public health risks associated with the increasing prevalence of antimicrobial resistance in pathogens both directly, through the dissemination of antimicrobial-resistant pathogens, and indirectly, through the introduction of and selection for antimicrobial resistance genes. Because limited data are available, this connection is largely a theoretical connection, particularly for the indirect effects. However, a recent retrospective study of antimicrobial resistance in soil did support the hypothesis that there is an environmental connection by documenting that there was an increase in the abundance of antibiotic resistance genes in samples collected from 1940 to 2008, during which time antimicrobial production increased dramatically (12).The fate of antimicrobials in amended soils is a function of their sorptive properties, the soil characteristics, and the potential for abiotic and biotic degradation of the antimicrobials. Tetracyclines tend to adsorb to soil (21, 23), which leads to persistence in amended soils (3, 7, 11), although they are also susceptible to degradation (3, 4). The macrolide tylosin frequently is not detected (3, 4, 7, 11, 33) and is likely rapidly degraded in manure and soils (8, 16, 24). However, persistence of tylosin for several months in amended soil has also been reported (6). The differences in degradation rates may be caused by differences in soil characteristics, manure-to-soil ratios, and/or microbial communities (15, 16, 21).Addition of both antimicrobials and antimicrobial-resistant microorganisms might be expected to result in an increase in the levels of resistance. However, most studies have not shown that there is a long-term increase in antimicrobial resistance due to land application of manure at agronomically prescribed rates (5, 9, 26). Transient (i.e., <45-day) increases have been reported (9, 26), as have elevated levels of resistance at sites near manure piles (5). In contrast, another report showed that there were significantly higher levels of tylosin resistance in soils that received animal manure from operations that used subtherapeutic levels of antimicrobials than in soils at sites where there was no use of subtherapeutic levels of antimicrobials (19). One limitation of these studies was their use of culture-based methods to quantify resistance; the results may not be representative of the entire microbial community. The molecular methods that have been used to quantify resistance also have limitations, and the most serious limitation is the inability of these methods to examine the full diversity of known and unknown resistance genes. The previous molecular studies of the impact of land application on resistance were largely restricted to qualitative analyses (10, 25), although quantitative PCR methods for analysis of tetracycline resistance genes have recently been used for cattle and swine lagoons (14, 20). In a retrospective soil study, Knapp et al. (12), who also used quantitative PCR, found multiple site differences, which made it difficult to evaluate the impact of manure application. However, the site with the highest manure application rate did not show the highest levels of antimicrobial resistance, suggesting that there are other factors that have a greater influence on the prevalence of resistance.In the present study, a variation of the fluorescence in situ hybridization (FISH) technique was used to assess the impact of land application of swine manure on the levels of macrolide-lincosamide-streptogramin B (MLS_B) resistance. Although the MLS_B antimicrobials are chemically distinct, methylation or mutation of a single base of the 23S rRNA prevents binding and results in cross-resistance to all three classes (29). The prevalence of MLS_B antimicrobial resistance in the microbial community can therefore be quantified indirectly by hybridization of an oligonucleotide probe to unmethylated, MLS_B-sensitive ribosomes, using either membrane hybridization (1, 10) or FISH (31). These methods do not require culturing or a comprehensive knowledge of the diversity of resistance gene sequences, but they do not detect resistance to specific antimicrobials that results from other mechanisms, such as macrolide efflux.This study focused on evaluating the impact of land application of swine manure on the levels of antimicrobials and the prevalence of antimicrobial resistance in the soil environment. The concentrations of tetracycline, macrolide, and lincosamide antimicrobials and the prevalence of MLS_B resistance were compared for field soils that received no manure, swine manure from farms that did not use antimicrobials (referred to below as organic farms), and swine manure from conventional farms to determine whether land application affects the levels of antimicrobials and MLS_B resistance. The effects of addition of manure, antimicrobials (lincomycin and chlortetracycline), and MLS_B-resistant microorganisms on the prevalence of MLS_B resistance were also compared using soil batch tests.