Amplified fragment length polymorphism and whole genome sequencing: a comparison of methods in the investigation of a nosocomial outbreak with vancomycin resistant enterococci

Background Recognition of nosocomial outbreaks with antimicrobial resistant (AMR) pathogens and appropriate infection prevention measures are essential to limit the consequences of AMR pathogens to patients in hospitals. Because unrelated, but genetically similar AMR pathogens may circulate simultaneously, rapid high-resolution molecular typing methods are needed for outbreak management. We compared amplified fragment length polymorphism (AFLP) and whole genome sequencing (WGS) during a nosocomial outbreak of vancomycin-resistant Enterococcus faecium (VRE) that spanned 5 months. Methods Hierarchical clustering of AFLP profiles was performed using unweighted pair-grouping and similarity coefficients were calculated with Pearson correlation. For WGS-analysis, core single nucleotide polymorphisms (SNPs) were used to calculate the pairwise distance between isolates, construct a maximum likelihood phylogeny and establish a cut-off for relatedness of epidemiologically linked VRE isolates. SNP-variations in the vanB gene cluster were compared to increase the comparative resolution. Technical replicates of 2 isolates were sequenced to determine the number of core-SNPs derived from random sequencing errors. Results Of the 721 patients screened for VRE carriage, AFLP assigned isolates of 22 patients to the outbreak cluster. According to WGS, all 22 isolates belonged to ST117 but only 21 grouped in a tight phylogenetic cluster and carried vanB resistance gene clusters. Sequencing of technical replicates showed that 4–5 core-SNPs were derived by random sequencing errors. The cut-off for relatedness of epidemiologically linked VRE isolates was established at ≤7 core-SNPs. The discrepant isolate was separated from the index isolate by 61 core-SNPs and the vanB gene cluster was absent. In AFLP analysis this discrepant isolate was indistinguishable from the other outbreak isolates, forming a cluster with 92% similarity (cut-off for identical isolates ≥90%). The inclusion of the discrepant isolate in the outbreak resulted in the screening of 250 patients and quarantining of an entire ward. Conclusion AFLP was a rapid and affordable screening tool for characterising hospital VRE outbreaks. For in-depth understanding of the outbreak WGS was needed. Compared to AFLP, WGS provided higher resolution typing of VRE isolates with implications for outbreak management.

Microbial Community Structure Analysis of Blood Pond Hell Hot Spring in Japan and Search for Metal-Reducing Microbes

Blood Pond Hell (Chinoike Jigoku) is located in the hot spring town of Beppu, in Kyushu Island of Japan, and features a unique hot, acidic, red-coloured pond. This study aimed to investigate the microbial diversity in this unique extreme environment and eventually to isolate useful acidophilic microbes. The initial PCR (using bacteria-or archaea-specific primers) on environmental samples detected the presence of bacteria, but not archaea. The following random sequencing analysis confirmed the presence of a large bacterial diversity at the site (123 clones comprising 18 bacterial and 1 archaeal genera), including those closely related to known autotrophic and heterotrophic acidophiles (Acidithiobacillussp.,Sulfobacillussp.,Alicyclobacillussp.). Nonetheless, successive enrichment cultivation with Fe (III) under oxygen depletion lead to isolation of previously non-detected archaeal (Sulfolobussp.) colonies on solid media. Two isolates showing Fe (III) reduction ability were namedSulfolobussp. GA1 and GA2. The isolates were also found to reduce highly toxic Cr (VI) to less toxic/soluble Cr (III), demonstrating their potential utility in metal bioremediation.

Cluster-Based Multipolling Sequencing Algorithm for Collecting RFID Data in Wireless LANs

With the growing use of RFID (Radio Frequency Identification), it is becoming important to devise ways to read RFID tags in real time. Access points (APs) of IEEE 802.11-based wireless Local Area Networks (LANs) are being integrated with RFID networks that can efficiently collect real-time RFID data. Several schemes, such as multipolling methods based on the dynamic search algorithm and random sequencing, have been proposed. However, as the number of RFID readers associated with an AP increases, it becomes difficult for the dynamic search algorithm to derive the multipolling sequence in real time. Though multipolling methods can eliminate the polling overhead, we still need to enhance the performance of the multipolling methods based on random sequencing. To that extent, we propose a real-time cluster-based multipolling sequencing algorithm that drastically eliminates more than 90% of the polling overhead, particularly so when the dynamic search algorithm fails to derive the multipolling sequence in real time.

Bottom-Up Driven Involuntary Auditory Evoked Field Change: Constant Sound Sequencing Amplifies But Does Not Sharpen Neural Activity

The capability of involuntarily tracking certain sound signals during the simultaneous presence of noise is essential in human daily life. Previous studies have demonstrated that top-down auditory focused attention can enhance excitatory and inhibitory neural activity, resulting in sharpening of frequency tuning of auditory neurons. In the present study, we investigated bottom-up driven involuntary neural processing of sound signals in noisy environments by means of magnetoencephalography. We contrasted two sound signal sequencing conditions: “constant sequencing” versus “random sequencing.” Based on a pool of 16 different frequencies, either identical (constant sequencing) or pseudorandomly chosen (random sequencing) test frequencies were presented blockwise together with band-eliminated noises to nonattending subjects. The results demonstrated that the auditory evoked fields elicited in the constant sequencing condition were significantly enhanced compared with the random sequencing condition. However, the enhancement was not significantly different between different band-eliminated noise conditions. Thus the present study confirms that by constant sound signal sequencing under nonattentive listening the neural activity in human auditory cortex can be enhanced, but not sharpened. Our results indicate that bottom-up driven involuntary neural processing may mainly amplify excitatory neural networks, but may not effectively enhance inhibitory neural circuits.