Characterized traits in studied UPEC isolates – prevalence and distribution among phylogenetic groups.
UTIs are one of the most frequently acquired bacterial infections and
Of serious concern and an increasing health problem, on a global scale, is the appearance and spread of antimicrobial resistance. One of the major health care concerns is emergence of multidrug resistant bacteria and clinical microbiologists increasingly agree that multidrug resistant Gram-negative bacteria pose the greatest risk to public health (Kumarasamy et al., 2010). Therefore, it is essential to determine susceptibility of pathogenic strains for antimicrobial agents and association of antimicrobial resistance with virulence genes.
One of the means for acquiring specific virulence factor genes and antimicrobial resistance genes, is via mobile DNA (
The search for alternative antibacterial agents is of great importance and colicins, toxic, narrow killing spectrum exhibiting proteins produced by colicinogenic
In summary, to diminish the burden of UPEC, using effective preventive measures, data on phylogenetic groups, serogroups, virulence factor prevalence, antimicrobial resistance, presence of mobile DNA, colicin production and colicin resistance among
In Slovenia in 2002, we collected 110
2. Phylogenetic groups and subgroups
Analysis of our collection of 110 UPEC isolates showed that 55 (50%) belonged to group B2, 28 (25%) to A, 21 (19%) to D, and 6 (5%) to the B1 group (Rijavec et al., 2006). When subgroups were considered the distribution of the isolates was: 4 (4%) of the studied isolates belonged to the subgroup A0, 24 (22%) to A1, 6 (5%) to B22, 49 (45%) to B23, 16 (15%) to D1 and 5 (5%) to D2 (our unpublished data).
The obtained distribution of the studied isolates into the phylogenetic groups, with the large majority classifying to the B2 group was expected since, it is known that ExPEC isolates mainly belong to the B2 phylogenetic group (Picard et al., 1999) however, the disproportionate distribution into the B22 (5%) and B23 (45%), and A0 (4%) and A1 (22%) subgroups was surprising.
Serotyping of the 110 uropathogenic strains revealed that 77 (70%) were O-antigen typable, 19 (17%) were O-nontypable, and 14 (12.7%) were rough. Sixty-three (57%) of the examined strains were H-antigen typable, 41 (37%) were H-antigen negative, and 6 (5%) were H-antigen nontypable. The O-typable strains were distributed into 31 serogroups. Nevertheless, the most frequent were O2 (9 isolates) and serotype O6:H1 (10 isolates). Five common serotypes were identified in three or more strains: O2:HNT (n = 3), O2:H6 (n = 3),
O6:H1 (n = 10), O7:HNT (n = 3), and O74:H39 (n = 4) accounting for 30% of the serotypable isolates. A number of other serotypes were detected in one or two strains (Rijavec et al., 2006).
Serotype analysis of the studied strains revealed that they belonged to diverse serogroups. However, the most frequent were O2 and O6, which are well established as associated with urinary tract infections. The large majority, 96%, of the O2 and O6 isolates were assigned to the B2 phylogenetic group (Rijavec et al., 2006).
4. Virulence factors
Any component of a microbe that is required for, or potentiates its ability to cause disease is designated as a virulence factor. Many different virulence factors exist however, they can all be placed in one of the four major groups of virulence factors: adhesins, toxins, iron uptake systems and host immunity evading systems. Hence, virulence factors facilitate colonization and invasion of the host, avoidance or disruption of host defence mechanisms, injury to host tissue, and/or stimulation of a noxious host inflammatory response (Johnson and Steel, 2000).
Among the first virulence factors that come into play during establishment of an infection are adhesins. Besides their primary role as adhesin molecules, they can also function as invasins, promoters of biofilm formation and transmitors of signals to epithelial cells resulting in inflammation. Various adhesins have been identified and studied (Zhang & Foxman, 2003). In our analysis we focused on the four mostly studied: type 1 fimbriae, P fimbriae, S fimbriae and the Afa/Dr family of adhesins (Starčič Erjavec & Žgur-Bertok, 2008).
Type 1 fimbriae are the most common adhesive organelles of
P fimbriae are among the best studied fimbrial adhesive fibres of UPEC strains. The P fimbrial adhesin molecule (PapG) recognizes globoseries of glycolipids as receptors (Zhang & Foxman, 2003). In our study the
S fimbriae bind to sialyl galactosides. Studies showed that
The Afa/Dr family consists of 13 known adhesins that all bind to the Dra blood group antigen present on the complement regulatory molecule CD55, also known as decay-accelerating factor (DAF) (Bower et al., 2005). The
Among the tested adhesin genes in the studied UPEC isolates (Table 1), the type 1 fimbriae were the most prevalent - the
Analysis of the distribution of adhesin gene sequences among phylogenetic groups revealed that adhesin gene sequences were differently distributed (Table 1):
Further, a very high, statistically significant, prevalence of S fimbriae in the B2 group was detected, 45% of the strains belonging to the B2 group harboured
Toxins affect an astonishing variety of fundamental eukaryotic processes and thereby harm the host (Kaper, 2004) and are important virulence factors in a variety of
Well known toxins are also invasins, the Ibe proteins that help
Among the screened toxin encoding genes in the studied UPEC isolates (Table 1), the
Analysis of the distribution of toxin encoding genes among the determined phylogenetic groups of studied strains (Table 1) revealed that the tested toxin encoding genes
|107 (97)||28 (26)||5 (5)||53 (50)||21 (20)|
|54 (49)||8 (15)||1 (2)||35 (65)||10 (19)|
|37 (34)||5 (14)||1 (3)||21 (57)||10 (27)|
|14 (13)||0 (0)||0 (0)||14 (100)||0 (0)|
|26 (24)||1 (4)||0 (0)||25 (96)||0 (0)|
|2 (2)||1 (50)||0 (0)||1 (50)||0 (0)|
|28 (25)||1 (4)||0 (0)||26 (93)||1 (4)|
|25 (23)||0 (0)||0 (0)||25 (100)||0 (0)|
|10 (9)||0 (0)||0 (0)||9 (90)||1 (10)|
|48 (44)||1 (2)||0 (0)||42 (88)||5 (10)|
|46 (42)||8 (17)||0 (0)||27 (59)||11 (24)|
|51 (46)||9 (18)||0 (0)||41 (80)||1 (2)|
|22 (20)||4 (18)||0 (0)||12 (55)||6 (27)|
|84 (76)||17 (20)||3 (4)||49 (58)||15 (18)|
|K1||6 (5)||1 (17)||1 (17)||4 (67)||0 (0)|
|K5||11 (10)||2 (18)||1 (9)||8 (73)||0 (0)|
|63 (57)||20 (32)||4 (6)||29 (46)||10 (16)|
|23 (21)||0 (0)||0 (0)||23 (100)||0 (0)|
|Ampicillin||57 (52)||12 (21)||5 (9)||32 (56)||18 (32)|
|Ciprofloxacin||99 (90)||23 (23)||5 (5)||52 (53)||19 (19)|
|Chloramphenicol||50 (45)||7 (14)||3 (6)||32 (64)||8 (16)|
|Kanamycin||95 (86)||20 (21)||5 (5)||51 (54)||19 (20)|
|Mezlocillin||59 (54)||12 (20)||5 (8)||34 (58)||8 (14)|
|Nalidixic acid||64 (58)||12 (19)||3 (5)||38 (59)||11 (17)|
|Norfloxacin||99 (90)||23 (23)||5 (5)||52 (52)||19 (19)|
|Streptomycin||69 (63)||15 (22)||5 (7)||40 (60)||9 (13)|
|Sulfamethoxazole-Trimethoprim||87 (79)||18 (21)||6 (7)||49 (56)||14 (16)|
|Tetracycline||47 (43)||6 (13)||4 (9)||33 (70)||4 (9)|
|Trimethoprim||72 (65)||14 (19)||6 (8)||41 (57)||11 (15)|
|RepFIA||20 (18)||7 (35)||0 (0)||10 (50)||3 (15)|
|RepFIB||57 (52)||18 (32)||2 (4)||26 (46)||11 (19)|
|RepFIIA||24 (22)||7 (29)||0 (0)||15 (63)||2 (8)|
|Integron||34 (31)||12 (35)||1 (3)||12 (35)||9 (26)|
|42 (38)||12 (29)||3 (7)||20 (48)||7 (17)|
4.3. Iron uptake systems
Iron is an essential cofactor for many basic metabolic pathways and bacteria have developed specialized iron uptake systems to capture iron. The most prominent are the siderophores, iron-binding molecules that are taken up by special siderophore receptors and ATP-consuming porin-like transporters in the bacterial outer membrane (Schaible & Kaufmann, 2004). Siderophores can be classified into three groups: (i) the catecholate type (enterobactin, salmochelin = enterochelin), (ii) hydroxamate type (aerobactin) and (iii) a mixed type - a combination of both (yersiniabactin) (Grass, 2006; Schaible & Kaufmann, 2004). In addition to siderophore synthesis strains can use siderophores produced and released into the extracellular medium by other bacteria and even fungi. In the host, bacteria may use iron sources such as heme, hemoglobin, hemopexin, and iron bound to transferrin and lactoferrin (Braun & Braun, 2002). Apart from the siderophores and their receptors, autotransporters, virulence-associated proteins in gram-negative bacteria, can also play a role in obtaining iron for example, the hemoglobin protease Hbp (Otto et al., 2002). All autotransporter proteins are energy-independent secreted via a type 5 secretion system and possess an overall unifying structure, comprising (i) an amino-terminal leader peptide (for secretion across the inner membrane), (ii) the secreted mature protein (or passenger domain), and (iii) a dedicated C-terminal domain, which forms a pore in the outer membrane through which the passenger domain passes to the cell surface (Henderson & Nataro, 2001).
In our study the following iron uptake systems genes were investigated (Table 1):
Analysis of the distribution of iron uptake systems encoding genes among the determined phylogenetic groups of studied strains (Table 1) revealed that all of the studied iron uptake systems were mostly harboured by UPEC strains belonging to the B2 phylogenetic group, as 41 (80%) of the strains harbouring
4.4. Host immunity evading systems
Pathogenic microbes avoid host defences using a wide array of virulence factors, ranging from polysaccharide capsules, serum resistance proteins to immune system modulating agents (Kaper et al., 2004).
Capsules are the discrete structural layers of extracellular polysaccharides that envelope the cell and allow the bacteria to evade or counteract the host immune system (Roberts, 1996). Capsules protect pathogens from assaults such as opsonophagocytosis and complement-mediated killing (Roberts, 1995); and in case of acidic capsules they can act as “sponges” to sequester and neutralize antimicrobial peptides (Llobet et al., 2008). Virtually all UPEC have a K-type polysaccharide capsule. Most UPEC express Group 2 or 3 capsules on their surfaces (Goller and Seed, 2010) and the K-antigens K1, K5, K30 and K92 are the most prevalent among UPEC (Johnson, 1991).
TraT, the surface exclusion protein of the plasmid transfer system, has been implicated in increased serum resistance (Binns et al., 1979). TraT is one of the most prevalent virulence factors in pathogenic
Recently, TcpC, a Toll/interleukin-1 receptor (TIR) domain–containing protein of uropathogenic
Among the 110 studied UPEC isolates (Table 1) 6 (5%) had the K1-capsule and 11 (10%) had the K5-capsule (Starčič Erjavec et al., 2007). The
Analysis of the distribution of the studied immune system evading characteristics among the determined phylogenetic groups (Table 1) showed that, isolates with K1- and K5-capsules were the most prevalent in the B2 group, 4 (67%) and 8 (73%), respectively however, capsule possessing isolates also belonged to the A and B1 group, albeit at low prevalence. One (17%) K1-capsule coated strain was found in the A and one in the B1 group and 2 (18%) K5-capsule coated strains in the A group and one (9%) in the B1 group. The
5. Antimicrobial susceptibility
An important task in clinical microbiology is the performance of antimicrobial susceptibility testing in order to detect possible drug resistance in common pathogens and to assure susceptibility to drugs of choice for particular infections (Jorgensen & Ferraro, 2009). Therefore, several studies on the subject of antimicrobial susceptibility and
In the year 2002, when the studied isolates were collected, treatment of UTI in outpatients in Slovenia was as follows: a course of antibiotics (therapy of choice - trimethoprim 160 mg or trimethoprim/sulfamethoxazole 160 mg/800 mg twice daily for 3 days) and advice to consume sufficient quantities of liquids (2–3 l per day) (Car et al., 2003).
The studied uropathogenic strains were screened for susceptibility to the following antibiotics: ampicillin, chloramphenicol, kanamycin, streptomycin, tetracycline, trimethoprim, trimethoprim-sulfamethoxazole, ciprofloxacin, norfloxacin, nalidixic acid, mezlocillin, amikacin, cefotaxime, cefotiame, cefoxitin, ceftazidime, and gentamicin. Susceptibilities to the tested antibiotics ranged from 109 (99%) susceptible to amikacin, cefotaxime, ceftazidime, to 47 (43%) susceptible to tetracycline. Antibiotics with the highest prevalence of susceptibility, apart from amikacin, cefotaxime, ceftazidime, were to cefotiame, cefoxitin, and gentamicin as 108 (98%), 103 (94%), and 101 (92%) of the studied strains, respectively, were susceptible. Antibiotics with the lowest prevalence of susceptibility, apart from tetracycline, were to chloramphenicol, ampicilin, mezlocillin and nalidixic acid, as 50 (45%), 57 (52%), 59 (54%) and 64 (58%), respectively, were susceptible (Rijavec et al., 2006).
Forty-six (42%) of the studied strains were resistant to more than three classes of the tested antimicrobial agents—beta lactams, quinolone/fluoroquinolone, trimethoprim/ trimethoprim-sulfamethoxazole, tetracycline, chloramphenicol, aminoglycosides (streptomycin, kanamycin)—and were designated as multidrug resistant (MDR). Subsequently, the association between MDR and the phylogenetic group was examined. A statistically significant correlation between non-MDR and the B2 group was determined and a significant correlation between MDR and the D phylogenetic group was found. On the other hand, there were no statistically significant correlations between MDR or non-MDR strains and the A or B1 groups (Rijavec et al., 2006).
6. Mobile genetic elements
The loss and gain of mobile genetic elements has a pivotal role in shaping the genomes of pathogenic bacteria. Horizontal gene transfer is an important mechanism that rapidly disseminates new traits to recipient organisms. Acquiring these new traits is crucial in promoting the fitness and survival of a pathogen while it coevolves with its host (Croxen & Finlay, 2010).
Bacterial plasmids, self-replicating, extrachromosomal elements are key agents of change in microbial populations. They promote the dissemination of a variety of traits, including virulence, enhanced fitness, resistance to antimicrobial agents, and metabolism of rare substances (Johnson & Nolan, 2009).
Integrons are assembly platforms that incorporate exogenous open reading frames by site-specific recombination and convert them to functional genes by ensuring their correct expression. Integrons are composed of three key elements necessary for the capture of exogenous genes: a gene (
The studied UTI strains were screened for replication regions of IncFI and IncFII plasmids. We found (Table 1) a high (62 strains, 56%) incidence of
Since only class 1, 2 and 3 of integrons were shown to be associated with pathogenic
Colicins are bacteriocins produced by
Among the studied UPEC isolates 42 (38%) exhibited colicinogenic activity (Starčič Erjavec et al., 2006). Each of the 110 UPEC strains was resistant to at least 3 colicinogenic strains from Pugsley's collection of colicinogenic strains (Pugsley & Oudega, 1987), 23 UPEC strains (21%) were resistant to all 20 tested Pugsley's strains (our unpublished data). Colicinogenic strains were found in all four phylogenetic groups (Table 1) however, most colicinogenic strains, 20 (48%) belonged to the B2 group (our unpublished data).
Our investigation of UPEC isolates from Slovenia revealed a high prevalence of drug resistance and multidrug resistance. The virulence profile of the examined strains was comparable to that of strains from other geographic regions.
This work was supported by the by grant P1-0198 from the Slovenian Research Agency.