Detection of enteroaggregative Escherichia coli by loop-mediated isothermal amplification.

A novel gene amplification method, loop-mediated isothermal amplification (LAMP), has been recently developed as a rapid, specific diagnostic method for various infectious diseases. We have investigated whether LAMP can be used to detect small numbers of enteroaggregative Escherichia coli (EAEC) cells contaminated in food samples. Primers for LAMP reaction were designed with EAEC aggR gene sequences (available in GenBank). LAMP specificity with these primers was the same as that of PCR in a study of 37 EAEC and 42 non-EAEC bacterial strains. The sensitivity of the LAMP method was better than that of PCR in a study of serially diluted EAEC cells. The LAMP method was significantly more effective than was PCR in detecting EAEC-contaminated food samples (Fisher's exact test, P < 0.05). Therefore, the LAMP method described here should be useful for detecting small numbers of EAEC cells in food samples.

Variable number of tandem repeats and pulsed-field gel electrophoresis cluster analysis of enterohemorrhagic Escherichia coli serovar O157 strains.

Ninety-five enterohemorrhagic Escherichia coli serovar O157 strains, including 30 strains isolated from 13 intrafamily outbreaks and 14 strains isolated from 3 mass outbreaks, were studied by pulsed-field gel electrophoresis (PFGE) and variable number of tandem repeats (VNTR) typing, and the resulting data were subjected to cluster analysis. Cluster analysis of the VNTR typing data revealed that 57 (60.0%) of 95 strains, including all epidemiologically linked strains, formed clusters with at least 95% similarity. Cluster analysis of the PFGE patterns revealed that 67 (70.5%) of 95 strains, including all but 1 of the epidemiologically linked strains, formed clusters with 90% similarity. The number of epidemiologically unlinked strains forming clusters was significantly less by VNTR cluster analysis than by PFGE cluster analysis. The congruence value between PFGE and VNTR cluster analysis was low and did not show an obvious correlation. With two-step cluster analysis, the number of clustered epidemiologically unlinked strains by PFGE cluster analysis that were divided by subsequent VNTR cluster analysis was significantly higher than the number by VNTR cluster analysis that were divided by subsequent PFGE cluster analysis. These results indicate that VNTR cluster analysis is more efficient than PFGE cluster analysis as an epidemiological tool to trace the transmission of enterohemorrhagic E. coli O157.

[The risk of pertussis and diphtheria infections among pediatric healthcare workers in Japan].

For infection control in pediatric hospitals, we investigated the risk of pertussis and diphtheria infections among pediatric healthcare workers. Forty-nine Japanese pediatric healthcare workers in 12 general hospitals were screened for antibodies of pertussis toxin (PT), filamentous hemagglutinin (FHA), and diphtheria toxin (DT). The seropositive rates of anti-PT IgG (protective level, > 10 U/mL), anti-FHA IgG (> 10 U/ mL), and anti-DT (> 0.11 U/mL) were 50, 82, and 59%, respectively. During this survey period (Oct. 2003-Feb. 2004), 16 (33%) of the healthcare workers were in contact with pertussis-infant (s). However, all culture and PCR tests for Bordetella pertussis were negative. One of the 16 exposed healthcare workers, a male pediatrician, had serological evidence of a pertussis infection, but no disease symptomatic of pertussis. Our observations indicate that i) 50 and 41% of Japanese pediatric healthcare workers were seronegative for pertussis (anti-PT IgG) and diphtheria antibodies, respectively, and ii) although the healthcare workers had a high rate of contact with pertussis-infant (s), the infection rate was low. For pertussis and diphtheria infection control in pediatric hospitals, it is important for healthcare workers to be aware of their own protection levels against these diseases.

Comparison between agarose gel electrophoresis and capillary electrophoresis for variable numbers of tandem repeat typing of Mycobacterium tuberculosis.

Variable numbers of tandem repeat (VNTR) typing of Mycobacterium tuberculosis was performed on 54 strains including 23 strains derived from 9 outbreaks. PCR amplicon sizes of 12 mycobacterial interspersed repetitive unit tandem repeat loci were measured using both agarose gel electrophoresis and capillary electrophoresis. Similarities using agarose gel electrophoresis of Euclidian distances among the 23 strains derived from the 9 outbreaks were significantly lower than that using capillary electrophoresis (Wilcoxon signed ranks test, P < 0.01). By clustering analysis using unweighted pair group method using arithmetic averages, all of the 23 strains derived from the 9 outbreaks were each clustered with more than 90% similarities based on the distance using capillary electrophoresis. In contrast, differential clusters with more than 90% similarity were observed with only 7 strains derived from 3 outbreaks when analyzed by agarose gel electrophoresis. These results indicated that measurement of PCR amplicon size of tandem repeat loci should be carried out using capillary electrophoresis and that agarose gel electrophoresis is not suitable for clustering analysis of M. tuberculosis VNTR typing.

[Molecular epidemiological analysis of an outbreak of Campylobacter jejuni using restriction enzyme double-digestion technique for genotyping of isolates by pulsed-field gel electrophoresis].

In an outbreak of gastroenteritis in elementary school students and their families in Chiba Prefecture, Japan, Campylobacter jejuni was isolated from the stools of 14 patients who developed diarrheal illness after a one-day bus trip. C. jejuni was also isolated from the stools of 3 patients not going on the bus trip. Pulsed-field gel electrophoresis (PFGE) analysis was done on 17 isolates of C. jejuni to study genetic relationships among them. PFGE profiles of isolates treated with restriction enzymes Sma I, Ksp I and Kpn I were separated into 9, 10, and 10 types, but the relationship between PFGE profiles and epidemiological profiles was unclear. Dendrograms of PFGE of isolates double-digested with both Sma I and Ksp I were typed into D1, D2, D3 and D4, and profiles compared to profiles of serotyping and flagellin typing of isolates and epidemiological profiles to evaluate genetical and epidemiological relationships. Thirteen isolates of PFGE type D1 possessed serotype G and flagellin type Al and were isolated from patients going on the bus trip. Type D2 isolated from a student going on the bus trip and type D3 isolates from two students not going on the bus trip had serotype B and flagellin type A2. C. jejuni of PFGE type D4, serotype UT, and flagellin type A3 was also isolated from a student not going on the trip. Our results show that at least two outbreaks of C. jejuni occurred simultaneously in people related to the school. Restriction enzyme double-digestion PFGE was thus useful in the molecular epidemiological analysis of the C. jejuni outbreak.

[Comparison between biotype of Vibrio cholerae O1 and genotype using polymerase chain reaction].

To compare between biotype of Vibrio cholerae O1 and genotype using polymerase chain reaction (PCR), 9 classical and 81 El Tor biovar strains were investigated for hemolysis, agglutination of avian erythrocytes, VP test reactivity, sensitivity to both polymyxin B and classical phage IV, and genotype using PCR amplification of hlyA, tcpA, rtxA and rtxC. One classical biovar strain showed atypical reaction upon agglutination of avian erythrocytes. Eighteen El Tor biovar strains showed atypical reactions, with the exception of sensitivity to polymyxin B. By PCR detection of hlyA, rtxA and rtxC amplifications, all classical biovar strains possessed only classical type hlyA, while all El Tor biovar strains possessed El Tor type hlyA, rtxA and rtxC. By PCR analysis of amplicons, all classical biovar strains possessed classical type tcpA. One ctx-negative El Tor biovar strain possessed degenerated classical type tcpA and 4 ctx-negative El Tor biovar strains had no detectable tcpA. These results indicated that genotype of V. cholerae O1 using PCR detection of hlyA, rtxA and rtxC was consistent with biotype of the organism, suggesting that analysis of the genotype of the organism was as effective as by biochemical properties. However, PCR detection of hlyA is most appropriate for the biotyping of V. cholerae O1, as compared to biochemical properties, since El Tor biovar was originally distinguished from classical biovar strains by the hemolytic reaction.

[Cluster analysis of restriction fragment length polymorphism patterns of Mycobacterium tuberculosis isolated in Chiba prefecture].

Methods for cluster analysis of IS6110 based restriction fragment length polymorphism (RFLP) patterns of Mycobacterium tuberculosis isolates were studied for an epidemiological investigation in Chiba prefecture. To normalize patterns, external size markers were adopted instead of typical internal size markers used in the standard method. RFLP patterns were run on 1.4% agarose gels and external markers were applied to outside and middle lanes on each gel for precise comparison. The resulting RFLP patterns of 74 isolates were clustered by similarity. Similarity was calculated with the Dice coefficient using parameter settings at 0.8% tolerance and 0.5% optimization. Patterns of 19 isolates from 8 outbreaks showed high similarity within each outbreak. Cluster analysis, as described here, provides insights into epidemiological tracing of tuberculosis in Chiba prefecture.

Clade analysis of enterohemorrhagic Escherichia coli serotype O157:H7/H- strains and hierarchy of their phylogenetic relationships.

Enterohemorrhagic Escherichia coli serotype O157:H7/H-(O157) strains isolated in Chiba prefecture, Japan, during 2002-2009 were studied by lineage, subgroup, cluster, and clade analysis. Lineage analysis of 470 O157 strains with no known epidemiological relationships using lineage specific polymorphism assay-6 showed that there were 242 lineage I strains, 160 lineage I/II strains, 67 lineage II strains, and 1 atypical strain. Clade analysis of these strains by single nucleotide polymorphism in eight loci showed that lineage I contained all the clade 1, clade 2, and clade 3 strains, and some of the clade 4/5 strains. In contrast, clade 7, clade 8, and the remaining clade 4/5 strains were divided between lineage I/II and II, and clade 6 was in lineage I/II, suggesting paraphyletic evolution of these lineages. Cluster and subgroup analysis of the stx phage insertion site showed that all lineage I strains were cluster 3 and all lineage I/II and II strains, with the exception of clade 9, were in cluster 1. Clade analysis also indicated that there were three phylogenetic groups of clade 4/5 strains: ancestral groups containing lineage I/IIand II strains and a descendant group containing lineages I. Analysis of stx2c gene distribution showed that stx2c was in ancestral clade 4/5 strains but not in descendant 4/5 strains, suggesting that the ancestral group may be clade 4 as reported by Manning et al. The results with the markers used in this study suggested that the hierarchy of O157 phylogenetic relationships was lineage as the upper level, followed by subgroup and then cluster, and clade as the lowest level. The need for refinement of clade definition and modification of the model of the O157 evolution have been discussed.

Biased distribution of IS629 among strains in different lineages of enterohemorrhagic Escherichia coli serovar O157.

The distribution of insertion sequence (IS) 629 among strains of enterohemorrhagic Escherichia coli serovar O157 (O157) was investigated and compared with the strain lineages defined by lineage specific polymorphism assay-6 (LSPA-6) to demonstrate the effectiveness of IS629 analysis for population genetics analysis. Using pulsed-field gel electrophoresis and variable-number tandem repeat typing, 140 strains producing both VT1 and VT2 and 98 strains producing only VT2 were selected from a total of 592 strains isolated from patients and asymptomatic carriers in Chiba Prefecture, Japan, during 2003-2008. By LSPA-6 analysis, six strains had atypical amplicon sizes in their Z5935 loci and five strains had atypical amplicon sizes in their arp-iclR intergenic regions. Sequence analyses of PCR amplified DNAs showed that five of the six loci used for LSPA-6 analysis had tandem repeats and the allele changes were due to changes in the number of tandem repeats. Subculturing and long-term incubation was found to have no detectable effect on the lineages defined by LSPA-6 analysis, demonstrating the robustness of LSPA-6 analysis. Minimum spanning tree analysis reconstruction revealed that strains in lineage I, I/II, and II clustered on separate branches, indicating that the distribution of IS629 was biased among O157 strains in different lineages. Strains with LSPA-6 codes 231111, 211113, and 211114 had atypical amplicon sizes and were clustered in lineage I/II branch, and strains with LSPA-6 codes 212114, 221123, 221223, 222123, 222224, 242123, 252123, and 242222 had atypical amplicon sizes and clustered in lineage II branches. Linkage disequilibrium was observed in strains in every lineage when the standardized index of association was calculated using IS629 distribution data. Therefore, the distribution analysis of IS629 may be effective for population genetics analysis of O157 due to the biased IS629 distribution among strains in the three O157 lineages.