Distinguishing importation from diversification of quinolone-resistant Neisseria gonorrhoeae by molecular evolutionary analysis

BMC Evolutionary Biology Distinguishing importation from diversification of quinolone-resistant Neisseria gonorrhoeae by molecular evolutionary analysis Marcos Prez-Losada 2 Keith A Crandall 1 2 Margaret C Bash 0 Michael Dan 5 Jonathan Zenilman 4 Raphael P Viscidi 3 0 Division of Bacterial, Parasitic and Allergenic Products, Center for Biologics Evaluation and Research, United States Food and Drug Administration , Bethesda, MD , USA 1 Department of Microbiology and Molecular Biology, Brigham Young University , Provo, UT , USA 2 Department of Integrative Biology, Brigham Young University , Provo, UT , USA 3 Stanley Division, Department of Pediatrics, Johns Hopkins University School of Medicine , Baltimore MD , USA 4 Division of Infectious Diseases, Department of Medicine, Johns Hopkins University School of Medicine , Baltimore MD , USA 5 Infectious Diseases Unit, Edith Wolfson Hospital , Tel Aviv , Israel Background: Distinguishing the recent introduction of quinolone resistant gonococci into a population from diversification of resistant strains already in the population is important for planning effective infection control strategies. We applied molecular evolutionary analyses to DNA sequences from 9 housekeeping genes and gyrA, parC and porB of 24 quinolone resistant N. gonorrhoeae (QRNG) and 24 quinolone sensitive isolates collected in Israel during 2000-2001. Results: Phylogenetic and eBURST analyses and estimates of divergence time indicated QRNG were introduced on 3 separate occasions and underwent limited diversification by mutation, deletion and horizontal gene transfer. Reconstruction of N. gonorrhoeae demography showed a slowly declining effective strain population size from 1976 to 1993, rapid decline between 1994 and 1999, and an increase from 1999 to 2001. This is partially attributable to declining gonorrhea case rates from 1973 to 1994. Additional contributing factors are selective sweeps of antibiotic resistant gonococci and increased transmission from sex workers. The abrupt decline in the mid-1990s heralded an increased incidence of gonorrhea from 1997 to the present. The subsequent increase in effective strain population size since 1999 reflects the increased gonococcal census population and introduction of quinolone resistance strains. Conclusion: Our study demonstrates the effective use of population genetic approaches to assess recent and historical population dynamics of N. gonorrhoeae. - Background Resistance to fluoroquinolone antibiotics among Neiserria gonorrhoeae strains emerged rapidly in Asia after their introduction in 1989. Recent reports from developed countries have documented clonal spread of quinolone resistant N. gonorrhoeae (QRNG) [1-4]. Understanding the origin of an epidemic and dynamics of its subsequent spread are critical in developing effective control strategies. We recently described a high rate of QRNG in Tel Aviv, Israel during 20002001 [5]. Genetic analysis of porB using molecular probes identified two predominant genotypes among QRNG [6] and pulse field gel electrophoresis demonstrated limited heterogeneity of the resistant strains and clear similarities to resistant strains from southern Israel [7]. These studies suggested recent introduction of QRNG, perhaps from a single source, followed by rapid dissemination through the country. However, not all isolates had the identical genotype raising the question of whether there was diversification of the original strain or whether there were multiple introductions of QRNG strains. Determining genetic relatedness of isolates can provide insights into the source and pattern of spread of N. gonorrhoeae within a community. Previous studies have characterized QRNG by auxotyping/serotyping [3,8], pulse field gel electrophoresis [3,7,9], porB gene typing with molecular probes [6], opa typing [10,11] or multiantigen sequence typing [4,12]. For our study, we used a MultiLocus Sequence Typing (MLST) method [13,14]. MLST is a technique for characterizing bacterial species using sequences of internal fragments of multiple housekeeping genes [15]. The advantage of MLST for population genetic analysis is that housekeeping genes are presumed neutral evolving genetic markers. Additional insights into evolutionary forces structuring bacterial populations can be obtained from examination of loci subject to selection. Therefore, we also sequenced a partial fragment of porB [16], which is under strong positive selection, and segments of gyrA and parC, which are target loci for fluoroquinolone resistance [17,18]. Based on these analyses we inferred the evolutionary history of isolates from phylogenetic, network and eBURST reconstructions and calculated time of divergence from the most recent common ancestor in order to determine if genetically variant strains diverged before or after the putative time QRNG first appeared in Israel. We also estimated selective pressure on each gene and examined the association of selected sites with the ciprofloxacin resistance phenotype. Finally, using a novel analytical approach from population genetics, we estimated the past population dynamics of N. gonorrhoeae in Israel. Results An increase in the incidence of gonorrhea was observed in Israel in 1998, accompanied by the appearance of quinolone resistant N. gonorrhoeae (QRNG) isolates [5]. The incidence of gonorrhea peaked in 2002 and declined sharply thereafter. The rate of isolation of QRNG strains also declined after 2001. During the epidemic period, QRNG strains were detected in several parts of Israel. Pulse field gel electrophoresis analysis of isolates from the Negev region in southern Israel, Jerusalem, Haifa, and Tel Aviv showed that all the QRNG strains were closely related [7]. In the Tel Aviv area, there were 200 cases of gonorrhea in 2000 and 325 cases in 2001. The isolates for the present study were obtained from January 2000 through October 2001 in Tel Aviv, Israel [5]. Of 80 isolates collected during this time period that were previously genotyped by molecular probes for the variable regions of the porB gene [6], we selected 24 fluoroquinolone resistant and 24 sensitive strains for more detailed genotyping by sequencing of fragments of multiple housekeeping genes. Phylogenetic relationships among the ciprofloxacin sensitive and resistant strains based on 9 housekeeping genes were estimated using the statistical parsimony procedure and graphically depicted as a network of gene genealogies (Figure 1). The lines on the network indicate mutational connections among the unique genotypes with the number of substitutions separating these sequences in parentheses adjacent to the line. Genotypes were designated by strain number (CX), ciprofloxacin susceptibility (R = resistance, S = sensitive), contact information (P = paid commercial sex worker, F = non-paid sex worker, U = unknown), and month and year of isolation. There was no clustering of strains by contact information or date of isol (...truncated)