Next-generation sequencing technologies and their application to the study and control of bacterial infections.

BACKGROUND: With the efficiency and the decreasing cost of next-generation sequencing, the technology is being rapidly introduced into clinical and public health laboratory practice. AIMS: The historical background and principles of first-, second- and third-generation sequencing are described, as are the characteristics of the most commonly used sequencing instruments. SOURCES: Peer-reviewed literature, white papers and meeting reports. CONTENT AND IMPLICATIONS: Next-generation sequencing is a technology that could potentially replace many traditional microbiological workflows, providing clinicians and public health specialists with more actionable information than hitherto achievable. Examples of the clinical and public health uses of the technology are provided. The challenge of comparability of different sequencing platforms is discussed. Finally, the future directions of the technology integrating it with laboratory management and public health surveillance systems, and moving it towards performing sequencing directly from the clinical specimen (metagenomics), could lead to yet another fundamental transformation of clinical diagnostics and public health surveillance.

Proposed model for the high rate of rearrangement and rapid migration observed in some IncA/C plasmid lineages.

IncA/C plasmids are a class of plasmids from the Enterobacteriaceae that are relatively large (49 to >180 kbp), that are readily transferred by conjugation, and that carry multiple antimicrobial resistance genes. Reconstruction of the phylogeny of these plasmids has been difficult because of the high rate of remodeling by recombination-mediated horizontal gene transfer (HGT). We hypothesized that evaluation of nucleotide polymorphisms relative to the rate of HGT would help to develop a clock to show whether anthropic practices have had significant influences on the lineages of the plasmid. A system was developed to rapidly sequence up to 191 known open reading frames from each of 39 recently isolated IncA/C plasmids from a diverse panel of Salmonella enterica and Escherichia coli strains. With these data plus sequences from GenBank, we were able to distinguish six distinct lineages that had extremely low numbers of polymorphisms within each lineage, especially among the largest group designated as group 1. Two regions, each about half the plasmid in size, could be distinguished with a separate lineal pattern. The distribution of group 1 showed that it has migrated extremely rapidly with fewer polymorphisms than can be expected in 2,000 years. Remodeling by frequent HGT was evident, with a pattern that appeared to have the highest rate just upstream of the putative conjugation origin of transfer (oriT). It seems likely that when an IncA/C plasmid is transferred by conjugation there is an opportunity for plasmid remodeling adjacent to the oriT, which was also adjacent to a multiple antimicrobial resistance gene cassette.

Ribosomal operon intergenic sequence region (ISR) heterogeneity in Campylobacter coli and Campylobacter jejuni.

AIMS: The intergenic sequence regions (ISR) between the 16S and 23S genes of Campylobacter jejuni and Campylobacter coli are markedly different for each species. However, in the genomic sequence for Camp. coli RM2228, two rRNA operons have an ISR that is characteristic of Camp. coli, and the third operon is characteristic of Camp. jejuni. The aim of this study was to determine the prevalence of ISR heterogeneity in these organisms. METHODS AND RESULTS: PCR primers were designed to yield a 327-base pair (bp) product for Camp. coli and 166-bp product for Camp. jejuni. A strain like Camp. coli RM2228 should yield products of both sizes. DNA from a panel of Camp. coli (n=133) and Camp. jejuni (n=134) isolates were tested. All of the isolates yielded products of the predicted size for the species. To verify the data for Camp. coli RM2228, each ribosomal operon from the isolate was individually amplified by PCR and tested with the ISR primer pair. Products of both sizes were produced as predicted. CONCLUSIONS: The cross-species heterogeneity of the ISR seen in Camp. coli RM2228 is uncommon. SIGNIFICANCE AND IMPACT OF THE STUDY: The heterogeneity must have been caused by horizontal gene transfer at a frequency lower than predicted from housekeeping gene data. Thus, it can be expected that species identification based on the ISR can be confused in rare isolates.

Triple balloon-on-a-wire or "ménage à trois" coronary angioplasty.

We have described here the triple balloon or "ménage à trois" technique of coronary angioplasty of trifurcation lesions or closely approximated bifurcation lesions. Although the need for this technique is uncommon on anatomic grounds, it can facilitate excellent angiographic results.

Brachial approach directional coronary atherectomy of right coronary artery saphenous vein bypass grafts.

We describe here a technique for performing directional coronary atherectomy to right coronary artery saphenous vein bypass grafts from the brachial approach using a long introducer sheath system. This technique has the advantages of 1) avoiding femoral artery trauma and 2) ease of access into the bypass graft, avoiding occasional problems with guide catheter kinking or non-coaxial alignment.

Regulation of the estrogen receptor in MCF-7 cells by estradiol.

The role of estradiol in the regulation of its cognate receptor in MCF-7 cells was investigated in this study. After treatment with 10(-9) M estradiol, the level of receptor protein was measured using an enzymeimmunoassay. By 6 h, the receptor protein declined by about 60% from a level of approximately 3.6 to 1.2 fmol/micrograms DNA. The level of receptor remained suppressed for 24-48 h. Similar results were obtained with an estrogen receptor (ER) binding assay. The steady state level of ER mRNA was determined by an RNase protection assay. Estrogen treatment resulted in a maximum suppression of mRNA by 6 h. Receptor mRNA remained depressed for 48 h. Transcription run on experiments demonstrated a transient decrease of about 90% in ER transcription after 1 h. By 3-6 h transcription increased approximately 2-fold and remained elevated for at least 48 h. These data suggest that estrogen down-regulates ER mRNA by inhibition of ER gene transcription at early times and by a posttranscriptional effect on receptor mRNA at later times.

Cardiac transplantation: first-year experience in a community hospital.

The issue of decentralizing heart transplant services, formerly restricted to a few large medical centers, is currently under review by federal and state governments. We present the results of the first year of cardiac transplantation at a 385-bed community hospital. Twelve patients were selected according to generally accepted criteria from a pool of 24 referrals, all from within 75 miles of our institution. All patients were in New York Heart Association Class IV preoperatively. The one-year survival rate was found to be 82%, which is equivalent to that reported by established centers. All surviving patients were fully rehabilitated. Rates of infection and rejection were lower than expected, and costs were about half the national average. This series, in all likelihood, tests the limits to which the decentralization of cardiac transplant services can be taken. We conclude that cardiac transplantation can be accomplished at a community hospital with results, even for the first patients undergoing transplantation, comparable to those obtained by established programs at major medical centers.