Quasispecies development of Helicobacter pylori observed in paired isolates obtained years apart from the same host

Abstract

Helicobacter pylori isolates show greater genetic diversity than other bacterial species studied, but the basis for this phenomenon is unknown. Whether detectable genomic mutation appears within an H. pylori population during persistent colonization was investigated. Paired H. pylori populations obtained across 7- to 10-year intervals from 13 patients were characterized by use of methods including polymerase chain reaction (PCR) genotyping for cagA, vacA, iceA, recA, and IS605; random arbitrarily primed DNA (RAPD)-PCR and amplified fragment length polymorphism (AFLP) analysis; and ELISA, to determine Lewis phenotypes. Genotyping, including recA sequence analysis, revealed that initial and follow-up populations represented the same population in 11 patients (85%). Nevertheless, distinct dissimilarities were shown within each of these 11 pairs by both RAPD-PCR and AFLP analyses. During follow-up, Lewis-y levels, but not Lewis-x levels, decreased significantly. The changes detected by RAPD-PCR and AFLP indicate that genetic drift occurs within H. pylori populations over the course of years of colonization of a single host.

Figure 1

Sequences of 206-bp polymerase chain reaction product derived from Helicobacter pylori recA for 26 paired H. pylori pools. For each pair shown in the dendrogram, original and follow-up pools are designated A and B, respectively. Sequences were identical for initial and follow-up pools of pairs 3–13, each pair having its unique sequence. In contrast, there was 96.7% and 98.0% identity within pairs 1 and 2, respectively. Pools 3A and 3B (*) consisted of mixed populations with silent C/T polymorphism at position 39. In-pair clusters each were supported in majority of bootstrap replicates.

Figure 2

Sequences of 323-bp polymerase chain reaction product derived from Helicobacter pylori IS605 insertion element, for those isolates containing this element (pairs 3, 4, 6, 8, and 13 and second pool of pair 1). Pool designations in this dendrogram are identical to those in figure 1. Intrapair identity was 100%, whereas out-of-pair identity was 95.9% ± 0.03%. In-pair clusters each were supported in majority of bootstrap replicates.

Figure 3

Analysis of random arbitrarily primed DNA (RAPD) polymerase chain reaction profiles of 26 paired Helicobacter pylori pools obtained with primers 1254, 9355, and d11344. Pool designations are identical to those in figure 1. RAPD photographs were digitized, and levels of similarity between patterns were calculated (GelCompar 4.0 software; Applied Maths, Kortrijk, Belgium). The range of intrapair similarity for pairs 3–13 was 65%–95%, but for pairs 1 and 2, similarities were only 46% each.

Figure 4

Analysis of amplified fragment length polymorphism (AFLP) profiles of 26 paired Helicobacter pylori pools. Isolate designations are identical to those in figure 1. Fluorescent-labeled AFLP profiles were scanned and digitized, and levels of similarity between profiles were calculated. The range of intrapair similarity was 83%–96% for pairs 3–13, but similarities for pairs 1 and 2 were only 73% and 74%, respectively.