Norwegian patients and retail chicken meat share cephalosporin-resistant Escherichia coli and IncK/blaCMY-2 resistance plasmids.

OBJECTIVES: In 2012 and 2014 the Norwegian monitoring programme for antimicrobial resistance in the veterinary and food production sectors (NORM-VET) showed that 124 of a total of 406 samples (31%) of Norwegian retail chicken meat were contaminated with extended-spectrum cephalosporin-resistant Escherichia coli. The aim of this study was to compare selected cephalosporin-resistant E. coli from humans and poultry to determine their genetic relatedness based on whole genome sequencing (WGS). METHODS: Escherichia coli representing three prevalent cephalosporin-resistant multi-locus sequence types (STs) isolated from poultry (n=17) were selected from the NORM-VET strain collections. All strains carried an IncK plasmid with a blaCMY-2 gene. Clinical E. coli isolates (n=284) with AmpC-mediated resistance were collected at Norwegian microbiology laboratories from 2010 to 2014. PCR screening showed that 29 of the clinical isolates harboured both IncK and blaCMY-2. All IncK/blaCMY-2-positive isolates were analysed with WGS-based bioinformatics tools. RESULTS: Analysis of single nucleotide polymorphisms (SNP) in 2.5 Mbp of shared genome sequences showed close relationship, with fewer than 15 SNP differences between five clinical isolates from urinary tract infections (UTIs) and the ST38 isolates from poultry. Furthermore, all of the 29 clinical isolates harboured IncK/blaCMY-2 plasmid variants highly similar to the IncK/blaCMY-2 plasmid present in the poultry isolates. CONCLUSIONS: Our results provide support for the hypothesis that clonal transfer of cephalosporin-resistant E. coli from chicken meat to humans may occur, and may cause difficult-to-treat infections. Furthermore, these E. coli can be a source of AmpC-resistance plasmids for opportunistic pathogens in the human microbiota.

Liposome encapsulation of the internal control for whole process quality assurance of nucleic acid amplification-based assays.

A system intended for whole process quality assurance of nucleic acid amplification assays was developed based on the use of liposomes as cell-mimicking vehicles for the internal control, allowing introduction of the internal control directly into the crude biological specimens. By the proof of principle testing, the Roche Cobas Amplicor CT assay was chosen as model system and the Roche CT/NG Internal Control was thus loaded into the liposomes. The liposome/DNA particles were spiked into a Chlamydia trachomatis-positive urine specimen. A quantitative "in-house" duplex real-time C. trachomatis PCR assay showed that liposomes having Blue Dextran 2000 polysaccharide co-entrapped were the most suited particles as they were efficiently deposited by the centrifugation carried out according to the Roche urine specimen preparation procedure. Furthermore, it was demonstrated that the liposome/DNA particles might be used for whole process quality assurance of Amplicor assay without major modifications of the assay protocol. An additional feature of the use of these liposomes was that the pellet became blue coloured and that might facilitate a thorough removal of the urine supernatant without increasing the risk of disturbance of the pellet. Principally, the liposome/internal control system is versatile and seems to be applicable for whole process quality control of amplification-based assays for detection of various pathogens.

Detection of GB virus C RNA by GBV-C LCx and two PCR assays with primers from the 5' non-coding and NS5B region.

The objective of this study was to compare the sensitivity of three different reverse transcriptase-polymerase chain reaction (RT-PCR) based tests, for detection of GB virus C (GBV-C) RNA. One commercial and two 'in house' RT-PCR assays were employed in the testing of serum samples from 114 chronic hepatitis C infected individuals. A part of the 5' non-coding region (5'NCR) of the GBV-C genome was amplified by the GBV-C LCx assay (Abbott) and one of the 'in house' RT-PCR tests. In the other 'in house' RT-PCR a segment of the NS5B region was amplified. The 'in house' assays included the use of internal controls that were co-amplified with use of the same outer PCR primers as the virus targets. The GBV-C LCx from Abbott and 5'NCR 'in house' PCR tests detected 28 and 27 GBV-C positive individuals, respectively. The sample positive only in the LCx test was confirmed by the 'in house' 5'NCR RT PCR using an increased virus input. In comparison, the NS5B 'in house' PCR test detected 24 of the GBV-C positive samples. One sample showed no amplification of internal controls/virus target in the 5'NCR 'in house' PCR and another samples was amplification negative in the NS5B PCR. The PCR assays with primers from the 5'NCR of the virus genome e.g. the GBV-C LCx, were more sensitive compared with RT-PCR using primers from the NS5B region. The GBV-C LCx seemed to be the most sensitive and robust assay. Internal controls included in the 'in house' assays identified two samples with failure of the amplification.

False-negative results of a ligase chain reaction assay to detect Chlamydia trachomatis due to inhibitors in urine.

The aim of this study was to assess the presence of inhibitors in urine specimens causing false-negative results in a commercial Chlamydia trachomatis gap-filling ligase chain reaction (Gap-LCR) assay. On testing of urine samples by the Gap-LCR assay and urethral swab specimens by cell culture, 73 (19%) Chlamydia trachomatis positive subjects were detected among 382 men attending a clinic for sexually transmitted diseases. In 56 subjects, the agent was detected in both the urine and the urethral samples, while 309 subjects were negative in both tests. In seven subjects urine samples were Gap-LCR positive (confirmed by a different Gap-LCR assay), but the corresponding urethral swab samples were cell culture-negative. In another ten subjects the urethral swab samples were cell culture positive, but their urine samples were Gap-LCR negative. Subsequent re-analysis of the urine samples including the addition of external Chlamydia trachomatis DNA indicated full or partial inhibition in nine of the cell culture-positive Gap-LCR negative subjects. When urine preparations were freeze-thawed and diluted prior to testing, Chlamydia trachomatis was detected in six of the ten initially Gap-LCR-negative samples. Gap-LCR inhibitors were present in at least nine (12%) of the 73 urine preparations from the Chlamydia trachomatis positive individuals. Identification of samples containing Gap-LCR inhibitors and subsequent processing to reduce the inhibition increased the sensitivity of the test from 86% to 95%.

Sequence analysis of HIV-1 group O from Norwegian patients infected in the 1960s.

Three Norwegians, a couple and their daughter, died from AIDS in 1976 after up to 10 years of clinical manifestations of HIV infection (Lindboe et al., 1986, Acta Pathol. Microbiol, Immunol. Scand. 94, 117-123; Frøland et al., 1988, Lancet i, 1344-1345). We here demonstrate the presence of HIV DNA in autopsy materials from the father and the daughter. In phylogenetic analysis, the obtained sequences of the HIV pol and vif genes clustered with the HIV-1 group O clade. The genotyping was confirmed by detection of antibodies against HIV-1 group O in blood samples from the father and the mother. That these and other early isolates of HIV-1 are very similar to the presently circulating viruses and not intermediates between the present subtypes, verifies that the latest common ancestor of HIV-1 existed long before the emergence of the present epidemic. The presence of HIV-1 group O 30 years ago suggests that the limited spread of these viruses, compared to HIV-1 group M viruses, is not due to a later emergence of the group O viruses.

Hybrid PCR sequencing: sequencing of PCR products using a universal primer.

We describe a general method for making template DNA for sequencing of PCR products. The procedure may be particularly useful for PCR products where minimal sequence information is known or as an alternative to primer walking when sequencing long PCR products. A cassette containing the hybridization site for the M13 sequencing primer is ligated to a sample PCR product. Using one phosphorylated primer specific for the cassette together with one primer specific for the sample PCR product, subsequent PCR amplifies one hybrid construct directionally. This allows utilization of the universal M13 primer when sequencing of one strand after the removal of the complementary strand using lambda-exonuclease.

Characterization of the COL2A1 VNTR polymorphism.

The variable number of tandem repeat (VNTR) region 3' to the collagen type II gene (COL2A1) was amplified in vitro by the polymerase chain reaction. Subsequent high-resolution gel electrophoresis showed that the five earlier reported alleles could be further subtyped. A total of 17 allelic variants with a heterozygosity of 73.0% were found in 202 unrelated Norwegians. DNA sequencing of 19 COL2A1 alleles has been performed. The internal organization of the VNTR was common for all alleles, as previously shown for a few alleles. Moreover, the polymorphism in the COL2A1 locus is mainly due to variation in the numbers of copies of two repeat units, containing 34 and 31 bp, respectively, and/or to small deletions in either of the two units. DNA sequencing of alleles with the same electrophoretic size revealed no heterogeneity such as an alternating order of the different units, a feature that might have been expected to be the result of unequal crossing-over events. The observed ordered structure of the VNTR and the possibility of single-stranded DNA from the cores in the VNTR forming hairpins and loops suggest that the COL2A1 polymorphism may have evolved mainly by replication slippage mechanisms.

Use of DNA amplification (PCR) and direct DNA sequencing in the characterization of C4 alleles.

A procedure for detailed characterization of individual C4 alleles has been developed. DNA containing the two polymorphic clusters of C4 was amplified in the polymerase chain reaction (PCR). Direct DNA sequencing of amplified DNA was then performed by a modification of previously described techniques. The results were confirmed by M13 sequencing. Single C4A3 and C4B1 allele sequences were in accordance with previous reports. An individual typed C4A3B1 revealed double bands in the autoradiogram in the positions corresponding to the polymorphic nucleotides. We did not find the reported thymine in position 3641 specific for the C4A4 allele in an individual typed C4A4B2.

Genetic analysis of C4 polymorphism by use of DNA amplification (PCR), allele-specific oligonucleotide probes and allele-specific restriction enzymes.

In vitro DNA amplification allows multiplication of selected gene segments thereby improving the sensitivity in DNA analysis. Different allelic variants in the amplified DNA may be disclosed either by subsequent hybridization with allele-specific oligonucleotides or by subsequent allele-specific digestion with selected restriction endonucleases, followed by separation in agarose gel electrophoresis. The genes that code for human complement component C4 are polymorphic. Presently we demonstrate that allelic differences in C4, involving one base pair only, can be efficiently identified in the amplified DNA by each of the two techniques. A combination of both techniques may also be employed. The DNA amplification procedure may give access to selected 'haploid' fragments for individual DNA studies.