1 Department of Veterinary Microbiology and Preventive Medicine, College of Veterinary Medicine, Iowa State University, Ames, IA 50011, USA
2 Department of Veterinary and Microbiological Sciences, North Dakota State University, Fargo, ND 58105, USA
3 Information Technology Services, North Dakota State University, Fargo, ND 58105, USA
Correspondence
Lisa K. Nolan
lknolan{at}iastate.edu
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ABSTRACT |
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Done in partial fulfillment of the requirements for the MS degree in Microbiology at North Dakota State University.
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INTRODUCTION |
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METHODS |
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Haemolytic reaction.
Test and control organisms were assessed for their haemolytic reaction on 5 % sheep blood agar plates using standard methods (Forbes et al., 1998).
Fermentation of lactose.
Test and control organisms were assessed for lactose utilization by plating on MacConkey agar (Difco) using standard methods (Forbes et al., 1998).
Phylogenetic typing.
Isolates were assigned to phylogenetic groups according to the method of Clermont et al. (2000). Using this method, isolates are assigned to one of four groups (A, B1, B2 or D) based on their possession of two genes (chuA and yjaA) and a DNA fragment (TSPE4.C2) (Table 1
), as determined by PCR. Boiled lysates of overnight cultures were used as a source of template DNA for this study (Johnson & Brown, 1996
). Amplification was performed in a 25 µl reaction mixture containing 18·3 µl double-distilled H2O (ddH2O), 2·5 µl 10x PCR Buffer (Invitrogen), 1·0 µl 50 mM MgCl2, 0·5 µl of a 2·5 mM dNTP mixture (USB), 0·075 µl of 0·1 mM upper and lower primers (Integrated DNA Technologies) (Table 2
), 0·25 µl (5 U µl1) Taq DNA polymerase (Invitrogen) and 2·0 µl template DNA. The reaction mixture was subjected to the following parameters in a Mastercycler Gradient thermocycler (Brinkmann Eppendorf): 4 min at 94 °C, 30 cycles of 5 s at 94 °C and 10 s at 59 °C, and a final extension step of 5 min at 72 °C, followed by a hold at 4 °C.
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Virulence genotyping.
Test and control organisms were examined for the presence of several genes (Table 1) known for their association with ExPEC or APEC virulence, using multiple PCR assays. The APEC genes of interest have been associated with APEC plasmids, such as pTJ100 (Johnson et al., 2002
, 2004
). pTJ100 is a large plasmid known to contain many of the genes associated with APEC virulence, including the iron-acquisition operons, aerobactin (Johnson et al., 2002
), iro (GenBank accession no. AY545598) and sit (GenBank accession no. AY553855); the ColV operon (Johnson et al., 2002
); certain genes associated with serum resistance, iss (Johnson et al., 2002
) and traT (Johnson et al., 2004
); and tsh (Johnson et al., 2002
). Interestingly, pTJ100 also shows similarities in genetic content to a UPEC plasmid (Sorsa et al., 2003
) and PAI (Dobrindt et al., 2001
, 2002
; Oelschlaeger et al., 2002a
,b
).
All primers used in amplification of the virulence genes were obtained from Sigma-Genosys and Integrated DNA Technologies (Table 2). Template DNA for all amplifications was generated as described elsewhere (Johnson & Brown, 1996
). Targeted genes were amplified in multiplex procedures or singly; all amplification procedures have been documented elsewhere. Six different multiplex procedures, targeting different combinations of genes, were used. Five of these were described by Johnson & Stell (2000)
and targeted: 1) a PAI of UPEC CFT073, papA, fimH, kpsMT III, papEF, ireA and ibeA; 2) cnf-1, fyuA, iroN, bmaE, sfa, iutA and papG allele III; 3) hlyD, rfc, ompT, papG allele I', papG allele I, kpsMT II and papC; 4) gafD, cvaC, fliC (H7), cdtB, focG, traT and papG allele II; and 5) papG allele 1, papG alleles 2 and 3, iha, afa, iss, sfaS and kpsMT (K1). Amplification for each of these groups of genes was performed in 25 µl reaction mixtures that included 2 µl template DNA, 12·775 µl ddH20, 2·5 µl 10x PCR buffer (Invitrogen), 4 µl 25 mM MgCl2, 0·25 µl AmpliTaq Gold Taq (5 U µl1) (Roche Molecular Biosystems), 0·625 µl of each 10 mM dNTP (USB) and 0·075 µl 0·1 mM upper and lower primers. The amount of ddH20 varied according to the number of primers used in each group/panel. These reaction mixtures were subjected to the following conditions in a Mastercycler Gradient thermocycler: 12 min at 95 °C to activate the AmpliTaq Gold Taq, 25 cycles of 30 s at 94 °C, 30 s at 63 °C, and 3 min at 68 °C, with a final cycle of 10 min at 72 °C, followed by a hold at 4 °C.
sitA, feoB and irp-2 were detected with another multiplex procedure (Rodriguez-Siek et al., 2005). In this case, the 25 µl reaction mixture included 2 µl template DNA, 18·9 µl ddH2O, 1·5 µl 10x PCR Buffer (Invitrogen), 0·75 µl 50 mM MgCl2, 0·25 µl DNA Taq Polymerase (Invitrogen) 1·0 µl of a dNTP mixture with a concentration of 2·5 mM of each dNTP (USB) and 0·1 µl 0·1 mM upper and lower primers (Table 2
). This mixture was subjected to 5 min at 94 °C, 30 cycles of 30 s at 94 °C, 30 s at 59 °C, and 30 s at 72 °C, with a final 5 min cycle at 72 °C, followed by a 4 °C hold.
tsh and iucC were identified using individual amplification procedures (Rodriguez-Siek et al., 2005). For amplification of tsh, a 25 µl reaction mixture was used that included 15·88 µl ddH2O, 2·5 µl 10x PCR Buffer (Invitrogen), 2·0 µl 50 mM MgCl2, 0·25 µl DNA Taq Polymerase (Invitrogen), 2·0 µl of a dNTP mixture with a concentration of 2·5 mM of each dNTP (USB) and 0·25 µl 0·1 mM upper and lower primers (Table 2
). This mixture was subjected to 5 min at 95 °C, 1 min at 95 °C, 9 cycles of 30 s at 55 °C, 30 s at 72 °C, and 30 s at 94 °C, followed by 25 cycles of 30 s at 55 °C and 1 min at 72 °C, followed by 7 min at 72 °C with a final hold at 4 °C. For amplification of iucC, a 25 µl reaction mixture was used, containing 15·15 µl ddH2O, 2·5 µl 10x PCR Buffer (Invitrogen), 3·0 µl 50 mM MgCl2, 0·15 µl DNA Taq Polymerase (Invitrogen) 2·0 µl of a dNTP mixture with a concentration of 2·5 mM of each dNTP (USB) and 0·25 µl 0·1 mM upper and lower primers (Table 2
). This mixture was subjected to the conditions described by Skyberg et al., 2003
.
All samples were subjected to horizontal gel electrophoresis in 1·5 % agarose, and the size of the amplicons was determined by comparison to the Hi-Lo DNA marker (Minnesota Molecular Inc.). Positive and negative controls were examined with each amplification procedure, and all amplification procedures were repeated three times to reduce the possibility of false negatives. An isolate was considered to contain the gene of interest if it produced an amplicon of the expected size (Table 2).
Biostatistics.
The null hypothesis that the relative proportions of each of the four phylogenetic groups was equal across the APEC and UPEC isolates was tested with the chi-square test of homogeneity (Snedecor & Cochran, 1980). The null hypothesis that the proportion of UPEC containing each virulence gene was equal to the proportion of APEC isolates with that gene was tested by a Z-test on the difference between the proportions (Snedecor & Cochran, 1980
). In a further attempt to discern patterns among all isolates based on their content of virulence genes (papG allele I' was excluded, as it was absent in all isolates), multivariate statistics were used. A linear discriminant analysis (LDA) was used to determine if isolate type (APEC or UPEC) could be predicted, based on the virulence genes present (Huberty, 1994
). Although use of data from binary variables in an LDA, as done here, violates the assumption of multivariate normality, LDA was used, since parametric LDA can be very robust in spite of such violations (McLachlan, 1992
). Additionally, a cluster analysis of the isolates was run using the average linkage method based upon Jaccard's dissimilarity coefficient calculated from the presence of virulence genes (S Institute, Inc., 2004
). In order to better discern patterns among the isolates, results of the cluster and discriminant analyses, along with the isolates' virulence genotypes, phylogenetic groups and states of origin, were used to construct a single figure based on principles of Eisen et al. (1998)
.
Additionally, all isolates were subjected to principal components analysis (PCA), based on their content of virulence genes. Essentially, a PCA allows a reduction of the dimensionality of the dataset through mathematical transformation of the data into a new set of variables [the principal components (PCs)]. As a result of this analysis, the variability of the data can be expressed in fewer dimensions than it would have otherwise (Morrison, 1976). In the present study, PCA was used to reduce 37 dimensions (one per each gene or allele studied) to two PCs. Then, the two PCs, which accounted for much of the variation in the original data, were plotted against one another using different colours to denote whether an isolate was an APEC or UPEC. Results of the cluster and discriminant analyses were also superimposed on the PCA, allowing us to better visualize areas of overlap between APEC and UPEC.
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RESULTS |
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Phylogenetic typing of APEC and UPEC
Some APEC and UPEC isolates were assigned to all four phylogenetic groups (Table 4). The majority of APEC fell into group A, whereas the majority of UPEC were found in group B2. A substantial number of both APEC (29·6 %) and UPEC (18·5 %) were assigned to group D. Analysis of the results demonstrated that there is strong evidence to reject the hypothesis of homogeneity of relative proportions for each of the four phylogenetic groups across isolate source (APEC or UPEC) (
2=151·02, df=3, P<0·0001). That is, APEC and UPEC differed significantly in their assignments to phylogenetic groups.
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The data obtained on virulence genotypes of APEC and UPEC isolates were also subjected to PCA using SAS in an effort to provide a visual assessment of the degree of separation among the clusters of isolates. The two PCs were then plotted against one another (Fig. 2), and the results of the cluster and discriminant analyses were superimposed on this plot. Again, overlap among APEC and UPEC isolates was apparent in all clusters, but cluster 6 isolates, which were predominately UPEC, were found to be quite distinct, as indicated by their distance from the other isolates on this plot. Notice that the greatest numbers of misclassified isolates occurred in the lower left-hand portion of the PCA plot and were primarily from the uppermost mixed cluster of isolates that are typically characterized by low numbers of virulence genes.
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DISCUSSION |
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Further, the data presented in the present study reveal that UPEC and APEC have similarities in their serogroups, virulence genotypes and assignments to phylogenetic groups, supporting the hypothesis that poultry may be a vehicle for E. coli capable of causing human urinary tract disease. For instance, the most commonly occurring serogroups among the UPEC were all shared with the APEC. The O2 serogroup, which was one of the more commonly occurring serogroups among the UPEC, was also among the more commonly occurring serogroups of the APEC. In fact, the O2 serogroup is considered to be among the three most commonly occurring serogroups of APEC (Sojka & Carnaghan, 1961). Also, an interesting finding of the present study was that the O11 serogroup occurred among the APEC. Manges et al. (2001)
documented the emergence of UPEC clonal group A, a previously unrecognized clone of multidrug-resistant UPEC, as a cause of UTIs in women in three different states. That these UPEC emerged in three states and showed a high degree of genetic homogeneity, at least in the California isolates, suggested to the authors that these UPEC might have originated from contaminated food. In their study, two serogroups predominated among the clonal group A isolates from California, one of which was the O11 serogroup that was found among the APEC of the present study. It would be interesting to compare the genotypes of these California UPEC isolates to the APEC of the same serogroup to see if these isolates share other similarities.
In addition, results of the gene prevalence studies revealed some similarities between the two groups of isolates. Several APEC and UPEC strains shared identical genotypes for the virulence genes assayed (Table 6). In fact, when the isolates of this study were subjected to cluster analysis and PCA, and the results plotted (Figs 1 and 2
), overlap between the UPEC and APEC isolates was detectable. So too, assignment of isolates to APEC and UPEC groups, based on a discriminant analysis of the virulence genotypes, resulted in misclassification of some isolates, especially when counts of virulence genes were low. A substantial number of both groups contained genes involved in the formation of different adhesins, including P (various pap genes and alleles) and type 1 fimbriae. APEC and UPEC isolates also contained many genes related to iron acquisition, including fyuA and irp-2, genes of the yersiniabactin operon (Oelschlaeger et al., 2002a
; Pelludat et al., 1998
; Schubert et al., 1998
, 2000
, 2002
); iucC and iutA, genes of the aerobactin operon (de Lorenzo et al., 1986
; de Lorenzo & Neilands, 1986
); iroN of the salmochelin operon (Russo et al., 2002
; Hantke et al., 2003
); and sitA of the sit operon (Runyen-Janecky et al., 2003
). They also had a high prevalence of genes related to complement resistance, such as iss (Binns et al., 1979
, 1982
; Pfaff-McDonough et al., 2000
; Nolan et al., 2003
) and traT (Moll et al., 1980
; Binns et al., 1982
; Pfaff-McDonough et al., 2000
).
Some of the genes that were found to be widely distributed among both UPEC and APEC are known for their contribution to APEC virulence and have been localized to large, transmissible R plasmids, such as pTJ100 (Johnson et al., 2002, 2004
). Similar plasmids have been identified in UPEC (Sorsa et al., 2003
), and several of these pTJ100-associated genes have been found on the UPEC chromosome in PAIs (Dobrindt et al., 2001
, 2002
; Oelschlaeger et al., 2002a
,b
). In the present study, APEC and UPEC isolates were assessed for their possession of several of these pTJ100-related genes. All of the plasmid-related genes studied were found in over 60 % of the APEC examined (Table 5
), suggesting that plasmids such as pTJ100 are widely distributed among APEC, and that APEC might be a reservoir of plasmid-mediated virulence genes transmissible to other bacteria. Although there were significant differences in distribution of the pTJ100-related genes between APEC and UPEC, except for sitA, 60 % or more of the UPEC contained sitA, iss and traT, and a third of the UPEC contained all the plasmid genes assessed except for cvaC. Unlike the situation that exists on pTJ100, where iroN, iucC, iss, traT, tsh and sitA are found in conjunction with cvaC, these genes in UPEC are rarely associated with cvaC. A possible explanation for this discrepancy might lie in the location of these genes in UPEC and APEC. In UPEC, these genes may be more likely found on non-ColV plasmids or within chromosomal PAIs (Dobrindt et al., 2001
, 2002
; Oelschlaeger et al., 2002a
,b
; Sorsa et al., 2003
) than on pTJ100-like plasmids. Further study will be needed to ascertain the location of these genes in UPEC.
There was also discernable overlap in APEC and UPEC isolates in their assignments to phylogenetic group, with some intriguing differences. As predicted by the literature, a majority of the UPEC fell into group B2 (Clermont et al., 2000). In contrast, less than 20 % of the APEC were assigned to this group. However, a substantial number of both fell into group D, which is also known for its content of virulent ExPEC (Clermont et al., 2000
). Interestingly, about half of the APEC and 10·5 % of the UPEC were identified as belonging to group A, a group thought to be composed of commensal E. coli (Clermont et al., 2000
). Perhaps the isolates in group A are commensals, and their association with disease reflects the opportunistic nature of the infections from which they were obtained.
There were also notable differences between the two groups of isolates. For example, the O78 serogroup, which occurred frequently among the APEC of this study and is considered one of the three most commonly occurring serogroups in APEC (Sojka & Carnaghan, 1961), was not found among the UPEC studied here. Only one APEC strain was haemolytic, whereas 16 % of UPEC were. Also, several differences in the prevalence of the virulence genes across the two groups of isolates were found. For instance, few APEC had the cnf-1 gene, but about 28 % of UPEC contained it. Although a substantial number of isolates from both groups contained genes associated with pTJ100, these genes were significantly more likely to occur in APEC than they were in UPEC, except for sitA. Other differences in gene prevalence were also detected between these two groups of isolates (Table 5
). These differences suggest that all the UPEC studied are likely not derived from APEC, but the similarities among several of the isolates suggest that it is possible that some are. Also, interpretation of these results must be tempered by the fact that a limited sample of UPEC, all isolated from the same geographic location, was examined in this study. A larger study, using alternate typing methods and UPEC isolates from widely different areas and types of UTIs, might prove beneficial to the overall interpretation of the data. However, a bias due to their origin from a single state was not readily evident, as the UPEC tested were quite diverse in terms of their serogroups, genotypes and phylogenetic groups. In fact, the UPEC, which all originated from North Dakota, were well represented in several clusters, whereas the bulk of the APEC, originating from multiple states, was found in a single cluster.
In sum, APEC and UPEC cause widely prevalent extraintestinal diseases. Their propensity for causing such diseases relates to their possession of many virulence-associated traits, including certain serogroups, adhesins, iron acquisition systems, toxins, protectins and invasins, that enable them to grow and cause disease in these host environments. Consequently, it seems reasonable that UPEC and APEC might show similar adaptations for an extraintestinal lifestyle, which, in turn, might enable APEC to cause extraintestinal disease in human beings. Further research will be necessary to determine if APEC can cause human UTIs or serve as a reservoir of virulence genes contributing to uropathogenesis.
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ACKNOWLEDGEMENTS |
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Received 20 July 2004;
revised 3 February 2005;
accepted 14 April 2005.
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