Use of cultivation-dependent and -independent techniques to assess contamination of central venous catheters: a pilot study

<p>Abstract</p> <p>Background</p> <p>Catheters are the most common cause of nosocomial infections and are associated with increased risk of mortality, length of hospital stay and cost. Prevention of infections and fast and correct diagnosis is highly important.</p> <p>Methods</p> <p>In this study traditional semiquantitative culture-dependent methods for diagnosis of bacteria involved in central venous catheter-related infections as described by Maki were compared with the following culture-independent molecular biological methods: Clone libraries, denaturant gradient gel electrophoresis, phylogeny and fluorescence in situ hybridization.</p> <p>Results</p> <p>In accordance with previous studies, the cultivation of central venous catheters from 18 patients revealed that <it>S. epidermidis </it>and other coagulase-negative staphylococci were most abundant and that a few other microorganisms such as <it>P. aeruginosa </it>and <it>K. pneumoniae </it>occasionally were found on the catheters. The molecular analysis using clone libraries and sequencing, denaturant gradient gel electrophoresis and sequencing provided several important results. The species found by cultivation were confirmed by molecular methods. However, many other bacteria belonging to the phyla <it>Proteobacteria, Firmicutes, Actinobacteria </it>and <it>Bacteroidetes </it>were also found, stressing that only a minor portion of the species present were found by cultivation. Some of these bacteria are known to be pathogens, some have not before been described in relation to human health, and some were not closely related to known pathogens and may represent new pathogenic species. Furthermore, there was a clear difference between the bacterial species found in biofilm on the external (exluminal) and internal (luminal) side of the central venous catheter, which can not be detected by Maki's method. Polymicrobial biofilms were observed on most of the catheters and were much more common than the cultivation-dependent methods indicated.</p> <p>Conclusion</p> <p>The results show that diagnosis based on molecular methods improves the detection of microorganisms involved in central catheter-related infections. The importance of these microorganisms needs to be investigated further, also in relation to contamination risk from improper catheter handling, as only in vivo contaminants are of interest. This information can be used for development of fast and more reliable diagnostic tools, which can be used in combination with traditional methods.</p

The impact of different DNA extraction kits and laboratories upon the assessment of human gut microbiota composition by 16S rRNA gene sequencing

Peer reviewedPublisher PD

Identification and quantification of microbial populations in activated sludge and anaerobic digestion processes

Eight different phenotypes were studied in an activated sludge process (AeR) and anaerobic digester AnD) in a full-scale wastewater treatment plant by means of fluorescent in situ hybridization (FISH) and automated FISH quantification software. The phenotypes were ammonia-oxidizing bacteria, nitrite-oxidizing bacteria, denitrifying bacteria, phosphate-accumulating organisms (PAO), glycogen-accumulating organisms (GAO), sulphate-reducing bacteria (SRB), methanotrophic bacteria and methanogenic archaea. Some findings were unexpected: (a) Presence of PAO, GAO and denitrifiers in the AeR possibly due to unexpected environmental conditions caused by oxygen deficiencies or its ability to survive aerobically; (b) presence of SRB in the AeR due to high sulphate content of wastewater intake and possibly also due to digested sludge being recycled back into the primary clarifier; (c) presence of methanogenic archaea in the AeR, which can be explained by the recirculation of digested sludge and its ability to survive periods of high oxygen levels; (d) presence of denitrifying bacteria in the AnD which cannot be fully explained because the nitrate level in the AnD was not measured. However, other authors reported the existence of denitrifiers in environments where nitrate or oxygen was not present suggesting that denitrifiers can survive in nitrate-free anaerobic environments by carrying out low-level fermentation; (e) the results of this paper are relevant because of the focus on the identification of nearly all the significant bacterial and archaeal groups of microorganisms with a known phenotype involved in the biological wastewater treatment.This study was realized with support from the Universitat Politecnica de Valencia, Universitat de Valencia and CONACYT (National Council of Science and Technology of Mexico).Reyes Sosa, MB.; Borrás Falomir, L.; Seco Torrecillas, A.; Ferrer, J. (2015). Identification and quantification of microbial populations in activated sludge and anaerobic digestion processes. Environmental Technology. 36(1):45-53. doi:10.1080/09593330.2014.934745S455336

Microbial community structure elucidates performance of Glyceria maxima plant microbial fuel cell

The plant microbial fuel cell (PMFC) is a technology in which living plant roots provide electron donor, via rhizodeposition, to a mixed microbial community to generate electricity in a microbial fuel cell. Analysis and localisation of the microbial community is necessary for gaining insight into the competition for electron donor in a PMFC. This paper characterises the anode–rhizosphere bacterial community of a Glyceria maxima (reed mannagrass) PMFC. Electrochemically active bacteria (EAB) were located on the root surfaces, but they were more abundant colonising the graphite granular electrode. Anaerobic cellulolytic bacteria dominated the area where most of the EAB were found, indicating that the current was probably generated via the hydrolysis of cellulose. Due to the presence of oxygen and nitrate, short-chain fatty acid-utilising denitrifiers were the major competitors for the electron donor. Acetate-utilising methanogens played a minor role in the competition for electron donor, probably due to the availability of graphite granules as electron acceptors

Phenotypic Variation and Bistable Switching in Bacteria

Microbial research generally focuses on clonal populations. However, bacterial cells with identical genotypes frequently display different phenotypes under identical conditions. This microbial cell individuality is receiving increasing attention in the literature because of its impact on cellular differentiation, survival under selective conditions, and the interaction of pathogens with their hosts. It is becoming clear that stochasticity in gene expression in conjunction with the architecture of the gene network that underlies the cellular processes can generate phenotypic variation. An important regulatory mechanism is the so-called positive feedback, in which a system reinforces its own response, for instance by stimulating the production of an activator. Bistability is an interesting and relevant phenomenon, in which two distinct subpopulations of cells showing discrete levels of gene expression coexist in a single culture. In this chapter, we address techniques and approaches used to establish phenotypic variation, and relate three well-characterized examples of bistability to the molecular mechanisms that govern these processes, with a focus on positive feedback.

Video-supported Analysis of Beggiatoa Filament Growth, Breakage, and Movement

A marine Beggiatoa sp. was cultured in semi-solid agar with opposing oxygen-sulfide gradients. Growth pattern, breakage of filaments for multiplication, and movement directions of Beggiatoa filaments in the transparent agar were investigated by time-lapse video recording. The initial doubling time of cells was 15.7 ± 1.3 h (mean ± SD) at room temperature. Filaments grew up to an average length of 1.7 ± 0.2 mm, but filaments of up to approximately 6 mm were also present. First breakages of filaments occurred approximately 19 h after inoculation, and time-lapse movies illustrated that a parent filament could break into several daughter filaments within a few hours. In >20% of the cases, filament breakage occurred at the tip of a former loop. As filament breakage is accomplished by the presence of sacrificial cells, loop formation and the presence of sacrificial cells must coincide. We hypothesize that sacrificial cells enhance the chance of loop formation by interrupting the communication between two parts of one filament. With communication interrupted, these two parts of one filament can randomly move toward each other forming the tip of a loop at the sacrificial cell

Metabolically active microbial communities in marine sediment under high-CO2 and low-pH extremes

Sediment-hosting hydrothermal systems in the Okinawa Trough maintain a large amount of liquid, supercritical and hydrate phases of CO2 in the seabed. The emission of CO2 may critically impact the geochemical, geophysical and ecological characteristics of the deep-sea sedimentary environment. So far it remains unclear whether microbial communities that have been detected in such high-CO2 and low-pH habitats are metabolically active, and if so, what the biogeochemical and ecological consequences for the environment are. In this study, RNA-based molecular approaches and radioactive tracer-based respiration rate assays were combined to study the density, diversity and metabolic activity of microbial communities in CO2-seep sediment at the Yonaguni Knoll IV hydrothermal field of the southern Okinawa Trough. In general, the number of microbes decreased sharply with increasing sediment depth and CO2 concentration. Phylogenetic analyses of community structure using reverse-transcribed 16S ribosomal RNA showed that the active microbial community became less diverse with increasing sediment depth and CO2 concentration, indicating that microbial activity and community structure are sensitive to CO2 venting. Analyses of RNA-based pyrosequences and catalyzed reporter deposition-fluorescence in situ hybridization data revealed that members of the SEEP-SRB2 group within the Deltaproteobacteria and anaerobic methanotrophic archaea (ANME-2a and -2c) were confined to the top seafloor, and active archaea were not detected in deeper sediments (13–30 cm in depth) characterized by high CO2. Measurement of the potential sulfate reduction rate at pH conditions of 3–9 with and without methane in the headspace indicated that acidophilic sulfate reduction possibly occurs in the presence of methane, even at very low pH of 3. These results suggest that some members of the anaerobic methanotrophs and sulfate reducers can adapt to the CO2-seep sedimentary environment; however, CO2 and pH in the deep-sea sediment were found to severely impact the activity and structure of the microbial community

Study of 16 Portuguese activated sludge systems based on filamentous bacteria populations and their relationships with environmental parameters

A survey in 16 activated sludge waste water treatment plants (WWTP) was conducted to contribute to the knowledge of the environmental parameters that determine the composition of the filamentous community. A total of 128 samples of mixed liquor from municipal WWTP were collected during 2 years, and 22 filamentous morphotypes were identified. The most frequent and abundant filamentous bacteria were, in both cases and by this order, type 0041/0675, type 0092, Microthrix parvicella and 1851, nocardioforms and Haliscomenobacter hydrossis. Concerning dominance, type 1851 was the most frequently dominant morphotype, followed by M. parvicella and types 0092 and 0041/0675. These were also, and by this order, the dominant morphotypes during bulking occurrences. Significant correlations were obtained between the abundance of filamentous bacteria and environmental parameters, but multivariate statistical analysis only confirmed the correlation between type 0092 and Sludge Volume Index (SVI), emphasizing the association of this filament with bulking. The discussion of the results in light of published works was complicated by the random use of terms such as frequency, abundance, and dominance with different and often unclear meanings. This reinforces the need of clarifying these terms when discussing the causes of filamentous overgrowth in WWTP.Portuguese Foundation for Science and Technology (FCT) and the European Community fund FEDER, through Program COMPETE, in the ambit of the Projects FCOMP-01-0124-FEDER-007025 (PTDC/AMB/68393/2006), PEst-OE/EQB/LA0023/2013, RECI/BBB-EBI/0179/2012 (FCOMP-01-0124-FEDER-027462), and the Project BBioEnv - Biotechnology and Bioengineering for a sustainable world,REF. NORTE-07-0124- FEDER-000048, co-funded by the Programa Operacional Regional do Norte (ON.2 – O Novo Norte), QREN, FEDER. PhD grant SFRH/BD/64848/200