Genetic Diversity of Uropathogenic Escherichia coli Isolated from Human T Lymphotropic Virus Type I (HTLV-I) Infected Individuals

Urinary tract infection (UTI) is a frequent pathology among HTLV-I+ individuals being capable of severely compromising the kidneys and bladder. Molecular characteristics of uropathogenic Escherichia coli (UPEC) from HTLV-I+ infected individuals are unknown. UPEC isolates from HTVL-I+ individuals, with and without clinical symptoms of myelopathy, were submitted to genetic typing seeking to infer bacterial diversity and potential virulence. 71 bacterial isolates were characterized according to random amplified polymorphic DNA and phylotypes. Phylotyping classified E. coli into four phylogenetic groups: A (18.3%), B1 (16.9%), B2 (39.4%), and D (25.3%) and 8 phylotypes according to the presence of the genetic sequences chuA, yjaA and the DNA fragment TSPE4.C2: −−− (5.6%) and −+− (12.6%) in phylogroup A, −−+ (7.0%) and −++ (9.8%) in B1, +++ (32.3%) and ++− (7.0%) in B2, +−− (15.4%) and +−+ (9.8%) in D. The B2 phylogroup was the most prevalent among HTLV− associated myelopathy and asymptomatic individuals. RAPD-PCR typing revealed a high degree of bacterial polymorphism indicating a non-clonal origin. Genotypes were not found to be distributed according to clinical status or epidemiological features. Our results lead us to suggest that the neurological impairment in HTLV-I+ individuals can be a risk factor for urinary infections due E. coli which are caused by distinct bacterial lineages.


Introduction
The Human T-lymphoytropic type I (HTLV-I) is a retrovirus associated with a chronic myelopathy known as HTLV-I-Associated Myelopathy or Tropical Spastic Paraparesis (HAM/TSP) [1].HAM/TSP is a progressive neurologic disorder characterized by leg weakness, diffuse hyperreflexia, clonus, loss of vibration sense, and detrusor insufficiency leading to bladder dysfunction.Central Africa, Japan, the Caribbean, South America and Melanesia are the principal endemic areas for HTLV-I [2].The majority of HTLV-I-infected individuals are clinically asymptomatic and less than 5% of seropositive subjects develop HAM/TSP [1] [3] [4].Urologic manifestations are present in up 90% of patients with HAM/TSP and are characterized by frequency, urgency and urge incontinence [1] [5] [6] [7].Epidemiological studies have described that HTLV-I+ individuals have a higher prevalence and incidence of urinary symptoms than seronegative controls [5] [6] [8] [9].Urinary tract infection (UTI) is an extremely frequent pathology among HTLV-I+ individuals due to the occurrence of bladder dysfunctions favoring colonization by bacteria [6] [8] [9] [10].These urinary infections can be clinically relevant, being capable of severely compromising the kidneys and bladder [11].
However, bladder impairment depends on the stage of development of the illness [10].
With the advancement of the degree of motor impairment, the exacerbation of urinary symptoms has been observed, with the development of systematic complications even being possible [5] [6] [9].Studies which investigate the etiology of UTIs in HTLV-I+ patients found that Escherichia coli is a bacterial species relevant in urinary infections; however, the genetic characteristics of these microorganisms isolated in this group of individuals are little known [9].Recent studies have shown a great variety of molecular methodologies utilized as diagnostic tools, as well as for typing purposes, allowing the detection of diversity, inference of genetic interrelation, and correlation with the clinical-epidemiological context.Among the molecular techniques used for the study of diversity in E. coli populations, the technique of random amplification of polymorphic DNA (RAPD-PCR) has received special attention as a typing method due to its simplicity of execution, low cost, flexibility, discriminatory power, and reproducibility under standardized experimental conditions.These studies have allowed the detection of the circulation of particular genetic lineages and revealed that E. coli epidemiologically correlated share urovirulence properties which are relevant for the determination and evolution of the clinical conditions [12] [13].Phylogenetic analysis has significantly contributed to the elucidation of the pathogenic potential of E. coli populations.This molecular characterization has allowed the designation of the main phylogenetic groups: A, B1, B2, and D [14].The microorganisms belonging to these distinct phylogroups are genetically diverse and differ according to metabolic properties, ecological niches, and the capacity to cause disease [15] [16].E. coli belonging to group B2, and to a lesser extent from D, have frequently been associated with extra-intestinal infections, while commensal E. coli and diarrheagenic strains are normally found as members from groups A, B1, and D [15] [16] [17] [18].
In this study, we characterized uropathogenic E. coli (UPEC) isolates from HTVL-I+ individuals, with and without clinical myelopathy symptoms, by the random amplification of polymorphic DNA (RAPD) and PCR-multiplex seeking to infer bacterial diversity, genetic interrelations and potential virulence.

Bacterial Samples and Participants
A total of 71 Escherichia coli isolates from adult individuals treated at the Laboratório de Pesquisa em Neuroinfecções do Instituto Nacional de Infectologia (INI/Fiocruz), with positive urine culture, and HTLV-I+ serology were included in this study.E. coli was isolated in the Laboratório de Bacteriologia e Bioensaios (INI/Fiocruz).For patients with clinical symptoms of myelopathy, neurological impairment was determined based on the Expanded Scale of the State of Incapacity (ESSI) [19].Out of all of the E. coli isolates, 35 were obtained from HTLV-I associated myelopathy patients (HAM/ TSP), presenting different degrees of neurological impairment (EDSS >0 to EDSS < 7) and, 36 isolates were recovered from individuals without clinical myelopathy symptoms (EDSS = 0, asymptomatic) (Table 1).Clinical-epidemiological data were obtained from the electronic records.The study protocol was approved by the Instituto Nacional de Infectologia and Escola Nacional de Saúde Pública Sergio Arouca Ethics Committee on Human Research, Fundação Oswaldo Cruz (CAEE 0071.0.009.031-07/CAAE 0022.0.031.000-10).
RAPD profiles were inspected visually and defined according to the presence or absence and intensity of polymorphic bands.A 1 Kb DNA ladder was used as a molecular weight marker (Invitrogen).Semi automated analysis used the UVI Soft Image Acquisition and Analysis Software, program UVIProBandmap, version 11.9 (Uvitec, Cambridge).Cluster analysis was done by using the unwieghted pair group method with arithmetric averages (UPGMA) of the Image Analysis System.The percentages of similarity were estimated by Dice coefficient.The reproducibility of RAPD amplifications was assessed using the selected primers with different DNA samples isolated independently from the same strain and amplified at different times.Numerical index of the discriminatory ability of RAPD primers was calculated by applying Simpson's Index of Diversity equation [21].

PCR-Triplex for E. coli Phylotyping
PCR assay was performed using phylogenetic group specific primers for the genes chuA.1/chuA.2,yjaA.1/yjaA.2 and the DNA fragment TSPE4C2.1/TSPE4C2.2 [14].A, B1, B2 and D phylogroups and respective allelic variants (phylotypes) were determined based on the presence or absence of bands according to previously defined criteria in the triplex amplification assay.To estimate the size of the fragments a 100 bp DNA

Statistical Analysis
Results were compared and analyzed using the Fisher exact test, Epi Info program, version 3.5.1.P-value ≤ 0.05 was considered statistically significant.

Results and Discussion
Our study investigated the genetic diversity and the phylogenetic groups of 71 E. coli uropathogenic isolates recovered from HTLV-I+ individuals, with and without clinical myelopathy symptoms.RAPD typing revealed high levels of polymorphism that are consistent with previously reported observations for E. coli [22].Escherichia coli has been recognized as a genetically diverse species with a complex phylogeny, broad virulence factors armament and significant genomic plasticity, comprising non-pathogenic gut commensals and strains responsible for intestinal and extra-intestinal disease [22] [23]   levant for the RAPD-PCR types distribution and the genetic relatedness of E. coli isolates.The overall chromosomal analysis of UPEC isolates from HTLV-associated myelopathy and asymptomatic individuals, showed distinct genetic background which is consistent with previous observations with nonrelated epidemiological strains and thus characterizing distinct evolutionary lineages [22].
Clermont genotyping was used to assign the diversity and to characterize the potential virulence within E. coli isolates (Table 2, Figure 3).The results obtained classified the E. coli into four phylogenetic groups: A (18.3%), B1 (16.9%),B2 (39.4%), and D (25.3%).The B2 phylogroup was the most common among the bacterial isolates as much in the HAM/TSP group of patients as with asymptomatic patients, corresponding    urinary infection, as much in the HAM/TSP patients as in the asymptomatic patients, showed a wide distribution of the groups.The B2 group however, was the most prevalent.In the HAM/TSP patients with symptomatic UTI, 33.3% belonged to the B2 group, 23.8% to the A and B1 groups, and 19.1% to the D group.Among bacterial isolates from individuals with asymptomatic UTI, the B2 and D groups were the highest represented, corresponding to 33.3%, followed by groups A and B1, with 25%, and 8.3% respectively.Epidemiological studies have shown that in patients with classic myelopathy, the greater majority present urinary symptomatology [5] [6] [26].In patients without clinical symptoms for myelopathy (asymptomatic) the phylogenetic groups B2, B1, D, and A were detected in 50%, 25%, 18.6%, and 6.3% from the isolates recovered from patients with symptomatic UTI, respectively.Among patients without UTI symp-toms, the isolates showed the following distribution: B2 (45%), D (30%), A (20%), and B1 (5%).The E. coli isolates from recurrent infections with symptomatic and asymptomatic UTI conditions were characterized as belonging to the B2 and A/B1 groups, respectively.Our results confirm previous observations, highlighting the relevance of B2 phylogroups in urinary infections, reinforcing in this manner the role of this pathogenic lineage in the establishment of these infections.In the group of patients with more compromised neurological functioning (EDSS > 6), the higher frequency was for isolates belonging to the A and B1 groups.These findings lead us to suggest that the vulnerability of the host could contribute to the establishment of the infection, given that these phylogenetic groups do not constitute typical agents for urinary infection.2).In the whole population studied, the phylotype +++ was the most prevalent in the B2 group.
In the phylogenetic D group, the phylotype +−− was the most prevalent among the HAM/TSP patients, and the +−+ allelic variant was the most common among the asymptomatic individuals.No statistical significance was observed in terms of the distribution of the phylotypes.The −++ variant of the B1 phylogroup had not been described in previous studies into pathogenic populations of E. coli [15] [16].The phylogenetic characterization of the UPEC from HTLV-I+ patients was not previously reported; however, we mainly classified the isolates as belonging to the B2 phylogenetic group, where typically is classified uropathogenic populations of E. coli.The phylogenetic classification together with the diversity analysis permitted the elucidation of the clonal structure and the pathogenic potential of these microorganisms.Our results contributed to the molecular epidemiology of UPEC among HTLV-I+ individuals, showing that urinary infections due to E. coli, either recurrently manifested or not, are owed themselves to distinct bacterial lineages.Urinary symptoms have been described to negatively influence the quality of life of individuals infected by HTLV-I.Investments on therapeutic resources in order to decrease the risks of urinary infection, and preservation of a functional urinary system have been stimulated [8].Studies seeking to investigate properties of virulence and resistance to antimicrobials of these uropathogens are currently under way in our laboratory.The data set obtained will be applicable to support epidemiological measures and public health monitoring, especially, together with therapeutic and preventative measures for these infections, which place at risk and compromise quality of life for these individuals.

Figure 1 ,Figure 1 .
Figure 1.Dendrogram generated by the Dice coefficient and clustering by unweighted pair group method with arithmetric averages and respective RAPD numeric profiles (primer 1254) of E. coli isolates from HLTV-I seropositive individuals with myelopathy.

Figure 3 .
Figure 3. Representative electrophoretic profiles of E. coli carrying phylogenetic markers.

Table 2 .
Phylotype distribution among E. coli isolates recovered from individuals with and without HTLV-associated myelopathy.