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* Department of Large Animal Clinical Sciences, and
Department of Biomedical Sciences and Pathobiology, Virginia Polytechnic Institute and State University, Blacksburg 24061
Department of Animal and Dairy Science, University of Georgia, Athens 30602
1 Corresponding author: elvinger{at}vt.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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Key Words: curli • Escherichia coli • fingerprinting • mastitis
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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Curli were first described on Escherichia coli isolated from bovine mastitis cases (Olsen et al., 1989). Thin, aggregative fimbriae have been described in Salmonella spp. and are similar to curli in structure, function, and regulation (Collinson et al., 1991; Sjobring et al., 1994; Römling et al., 1998). The genes required for curli formation in E. coli are encoded within 2 divergently transcribed operons: csgBAC and csgDEFG (Hammar et al., 1995). The curli structural subunit, known as curlin or CsgA, is secreted extracellularly and polymerized on the surface (Hammar et al., 1996). It has been shown that CsgD serves as an important transcriptional regulator of the csgBAC operon (Hammar et al., 1995; Römling et al., 1998).
In general, curli are produced in low temperature (<30°C) and low osmolarity conditions during stationary phase growth (Olsen et al., 1989, 1993; Maurer et al., 1998). When incubated at 26 to 28°C for 48 h in limited nutrient media containing Congo red dye, curli-producing colonies will usually display a red, dry, and rough morphotype, whereas curli-deficient strains often produce smooth, white, and moist colonies (Hammar et al., 1995, 1996). Altering environmental conditions such as NaCl content or temperature will cause some curli-producing bacteria to revert to the white phenotype (Olsen et al., 1993; Römling et al., 1998), although some strains are capable of expressing curli independent of temperature or other environmental factors (Römling et al., 1998; Bian et al., 2000; Uhlich et al., 2001). Curli expression in E. coli is variable among wild-type, laboratory, and pathogenic strains. Curli production was observed in 55% of bovine mastitis isolates (Olsen et al., 1989), 38% of non-O157 Shiga-toxin–producing E. coli (STEC) isolates, and is rare among O157 strains (Cookson et al., 2002).
Clinical studies in vitro and in vivo indicate that curli-producing bacteria are more pathogenic than curli-negative strains in their ability to adhere to, persist in, and invade host cells. Curli increased colonization and persistence of bacteria in avian colibacillosis, and similar strains demonstrated reduced persistence once their curli production was inhibited (la Ragione et al., 1999, 2000). Mice challenged with curli-producing E. coli variants displayed shorter survival times than those challenged with curli-deficient variants (Uhlich et al., 2002). Curli-producing Salmonella strains also achieved greater growth and yolk invasion in chicken eggs compared with their curli-deficient counterparts (Cogan et al., 2004). Curli facilitated internalization of E. coli by epithelial and HEp-2 cell lines, possibly mediated by fibronectin binding, where they may persist and evade detection by immune cells (Gophna et al., 2001, 2002). More recently, curli have been found to play a role in human sepsis (Nasr et al., 1996; Bian et al., 2000).
Curli may contribute to the pathogenesis of E. coli mastitis through several mechanisms. It has been shown that some bacterial strains can produce curli at increased temperatures, such as those encountered within the in vivo mammary environment. Curli may increase ability of bacteria to adhere to, colonize, or persist within the teat cistern and mammary tissue as well as evade the immune system. As proteins, curli may also activate immunologic pathways, increasing inflammation, recovery time, and clinical severity.
Targeting enterobacterial repetitive intergenic consensus (ERIC) sequences by PCR can be used to produce a unique genetic fingerprint for a bacterial agent. When compiling ERIC fingerprints into a dendrogram, groups or clusters of related isolates can be examined for common traits such as expression of a virulence factor and case outcome (Dopfer et al., 1999; de Moura et al., 2001). Should a particular bacterial group or strain be more likely to cause severe signs or possess a particular virulence trait, efforts to further explore, classify, and combat these strains can be focused to gain better insight into these organisms and their ability to cause disease.
In this study, E. coli isolated from clinical cases of bovine mastitis were characterized using ERIC-PCR and screened for their ability to produce curli by Congo red dye binding assay. Effect of curli production on case recovery, based on a return to precase milk yield, was also investigated in a subset of 19 cows.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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Milk was collected on d 0 and 5 of the clinical mastitis event and shipped to a designated diagnostic laboratory for culture and identification of pathogens using National Mastitis Council methods (Hogan et al., 1999). Isolates identified as E. coli were sent to the Virginia-Maryland Regional College of Veterinary Medicine for inclusion in this study.
All isolates received were cultured on MacConkey agar plates (BD Biosciences, Franklin Lakes, NJ) and on Levine’s eosin-methylene blue agar (Difco, Detroit, MI) for 12 h at 37°C. Isolates that produced dry, pink colonies on MacConkey agar and green, reflective colonies on eosin-methylene blue (i.e., positive reactions for lactose-fermenting coliforms) were classified as E. coli. Spot indole and oxidase tests (BBL, Cockeyville, MD) were performed on isolates with a weak or negative agar test result with indole-positive and oxidase-negative results identifying E. coli isolates. Isolates were cultured overnight in brain heart infusion broth (Remel, Lenexa, KS) combined with 30% glycerol (vol/vol), and maintained at –20°C for long-term storage. Stock cultures for daily use were maintained on modified trypti-case soy agar II deeps (BBL) and maintained at 4°C.
Three diagnostic laboratories sent a total of 55 isolates from 29 cows. The Quality Milk Promotion Services Laboratory (Cornell University, Ithaca, NY) sent 36 E. coli isolates from 16 cows; the Colorado State University Veterinary Diagnostic Laboratory (Fort Collins, CO) sent 11 isolates from 6 cows; and the Kansas Veterinary Diagnostic Laboratory (Kansas State University, Manhattan, KS) sent 5 isolates from 5 cows. We determined that 3 isolates were not E. coli and excluded those from the study. An additional 9 isolates from 9 cows were received through the Production Management Medicine Program, Virginia-Maryland Regional College of Veterinary Medicine (Blacksburg, VA). In total, 61 isolates from 36 cows were included in this study.
Isolates were designated by site of origin (B, C, F, K, or V), cow number, and day cultured (d 0 or d 5). Thirty-three of 36 Cornell isolates were cultured from 14 cows from one farm, designated ‘B’, with the remaining 3 isolates from 2 cows from other farms designated ‘C’. Colorado isolates were designated ‘F’, and were all obtained from 1 farm. Kansas isolates were designated ‘K’ and Virginia isolates ‘V’. Multiple isolates from the same cow were designated as a, b, c, or d. One cow (B1300) presented with 1 case of mastitis affecting 2 different quarters, yielding 2 isolates from each infected quarter. These isolates were differentiated by identifying left rear or right rear quarters. Duplicate isolates from the same quarter and sampling day were obtained from 17 quarter-sampling days in 13 cows.
Milk Yields and Mastitis Case Recovery
Daily milk yield was obtained for 19 cows (6 cows from site F and 13 cows from site B), milked 3 times daily from 5 d before (d –5) to 7 d (d 7) after the clinical mastitis episode. Forty-three of the 61 isolates examined were isolated from these 19 cows. Cows that produced 
75% of premastitis (d –5) daily milk yield by d 7 were considered to have recovered.
Curli Phenotype
All isolates were incubated at 26°C for 48 h (Hammar et al., 1995) on Congo red indicator (CRI) agar, which is YESCA agar supplemented with 20 mg/L of Congo red (Alfa Aesar, Ward Hill, MA) and 10 mg/L of Coomassie Brilliant Blue G dye (Tokyo Kaes Kogyo, Tokyo, Japan). Curli-producing E. coli bind Congo red dye and form red colonies on CRI, whereas curli-negative bacteria form white colonies. Stability of curli expression was determined by directly reculturing isolates onto fresh CRI and reincubating for another 48 h at 26°C.
DNA Extraction
Bacteria were grown in Luria-Bertani broth (BD Biosciences) at 37°C for 16 h in a Thermo Forma orbital shaker-incubator model 345 at 200 rpm (Thermo Forma, Marietta, OH). The DNA was extracted from 4 mL of culture using the Qiagen DNA Mini kit (Qiagen Inc., Valencia, CA) according to manufacturer’s instruction.
csgA and csgD PCR
Genes for the curlin subunit protein, csgA, and the curli transcriptional regulator, csgD, were targeted and amplified in a PCR to determine their presence or absence in each isolate. Primers for csgA were selected as listed in Maurer et al. (1998). The csgD csgDF (5'-ATGTTTAATGAAGTCCATAGT-ATT-3') and csgDR (5'-TTATCGCCTGAGGTTATCGTTTGC-3') primers were manually selected from the csgD listing in Genbank (accession number X90754; www.ncbi.nlm.nih.gov; NIH, Bethesda, MD).
Both the csgA and csgD PCR were performed in a total volume of 50 µL with 300 ng of DNA, 20 pmol of each primer, and 45 µL of Platinum PCR SuperMix High Fidelity (Invitrogen Life Technologies, Carlsbad, CA) in an Eppendorf Mastercycler Gradient thermocycler (Eppendorf AG, Hamburg, Germany). The PCR amplification for csgA was as follows: denaturation at 94°C for 2 min, followed by 35 cycles of 30 s at 94°C, 30 s at 54°C, and 30 s at 68°C with a final extension of 10 min at 72°C. The PCR reaction for csgD was cycled with the following protocol: denaturation at 94°C for 2 min, followed by 35 cycles of 30 s at 94°C, 30 s at 46°C, and 30 s at 68°C, with a final 10-min extension at 72°C.
The csgA and csgD PCR products were electrophoresed in 1% agarose gel buffered with sodium borate at 300 V for 10 min (Brody and Kern, 2004). Expected lengths of csgA and csgD PCR products were 200 and 650 bp, respectively. A randomly selected PCR product from each gene was sequenced at the Virginia Bioinformatics Institute at Virginia Tech (Blacksburg, VA) and confirmed as the target by comparing the result with each gene’s GenBank entry.
ERIC-PCR
All strains were fingerprinted by ERIC-PCR using the ERIC2 primer sequence (Meacham et al., 2003). Reactions were carried out in a final volume of 25 µL consisting of 100 ng of DNA, 25 pmol of ERIC2 primer, 2 U of Platinum Taq DNA polymerase (Invitrogen Life Technologies), 0.4 mM of each dNTP, and 5 mM MgCl2 in a 1xPCR buffer (provided with Platinum Taq). The ERIC-PCR reactions were conducted simultaneously on all isolates in a 96-well PCR plate in an Eppendorf Mastercycler Gradient thermocycler with the following protocol: denaturation for 2 min at 95°C, followed by 35 cycles of 30 s at 95°C, 1 min at 57°C, 5 min at 72°C with a final extension step of 10 min at 72°C.
The ERIC-PCR products were electrophoresed in a 1.5% agarose gel buffered with Tris-borate-EDTA at 90 V for 2 h and 45 min. Gels were stained for 40 min with a 1xsolution of SYBR green (Molecular Probes, Inc., Eugene, OR) in a Tris-borate-EDTA buffer adjusted downward to pH 7.95, using 12 M HCl. Each gel contained 15 wells and was run with 3 lanes of 1 Kb Plus standard DNA ladder (Invitrogen Life Technologies; Figure 1
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Statistical Analyses
The TIFF images of ERIC-PCR results were standardized and analyzed using GelCompar II software (Applied Maths, Inc., Austin, TX). Similarity matrices were constructed using Pearson correlation coefficients based on densitometric readings of the banding patterns. Isolates were clustered based on their similarity coefficients using unweighted pair group method analysis. One dendrogram was constructed using data from all isolates. A second dendrogram was constructed by the same method using data from the 43 isolates from 19 cows for which milk yield records were available.
Association of cluster groups with presence or absence of curli production in cows, and with return to precase milk yields, as well as association of use of antibiotics with return to precase milk yields, were determined by Fisher’s Exact tests using Simple Interactive Statistical Analysis (SISA) online statistical analysis software (Uitenbroek, 1997; http://home.clara.net/sisa). Exact P-values are presented.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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When more than 1 isolate was obtained from a cow, those isolates displayed the same curli phenotype, with 1 exception. Two isolates cultured on d 0 from cow B303 (B303D0a, B303D0b) were curli negative, whereas a third isolate obtained on d 5 (B303D5c) was curli positive. Curli-positive E. coli isolates were cultured from 21 cows, and curli-negative strains were cultured from 15 cows.
Presence of csgA and csgD Detected by PCR
The curlin subunit gene, csgA, and the curli transcriptional regulator gene, csgD, were both present in all isolates, regardless of phenotype expressed.
ERIC-PCR Dendrogram
At a threshold of 52% similarity, 58 E. coli isolates were clustered into 2 clonal groups. Group I contained 23 isolates from 13 cows, and group II contained 35 isolates from 22 cows. Three isolates were genetically distinct from the isolates in the 2 clonal groups: V3758D0a shared 49.9% genetic similarity with isolates in the 2 groups, and isolates B1290D5a and B1290D5b had only a 6.7% similarity with the 2 groups, but were 87.7% similar to each other (Figure 2).
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90% similarity to each other and were clustered closely. In the remaining 7 cases, at least one of the sibling isolates was <90% similar and clustered on a different or slightly more distant branch. In total, 28 of the 39 isolates clustered at 90% similarity to isolates within the same cow. Isolate B1167D0a was 85% similar to isolates B1167D5b and B1167D5c, which were 95% similar to each other. Isolate C1030D0a was 86% similar to C1030D5b, but also was more closely related to, and clustered with, B1090D0a and B1080D5a. Isolates F1300LRD0c and F1300LRD0d were only 83% similar to each other and placed in separate subclusters, and, in addition, were only 66% similar to isolates F1300RRD0a and F1300RRD0b. Isolates F12544D0a and F12544D0b were 81% similar and, as a result, clustered on different branches. Isolates B303D0a and B303D0b, 2 curli-negative isolates, were placed in group I and were 99% similar, whereas B303D5c, a curli-positive isolate, was placed in group II with an average similarity of 52% to group I. Isolate B1450D0a was only 60% similar to its sibling isolates B1450D0b, B1450D5c, and B1450D5d, which shared greater than 96% genetic similarity with each other.
A separate cluster analysis was performed and a dendrogram produced for the 43 isolates from 19 cows for which milk yield records were available. This dendrogram also divided isolates at a threshold of 52% similarity into 2 distinct groups (III and IV). Group III consisted of 18 isolates from 8 cows, and group IV of 23 isolates from 11 cows. Isolates B1290D5a and B1290D5b were located outside of these 2 groups and possessed only 6.7% genetic similarity to these groups (Figure 3).
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and 3). Five of 13 cows (38.5%) in group I and 15 of 22 cows (68.2%) in group II (P = 0.157) had 8 and 26 curli-positive isolates, respectively, with 15 and 9 curli-negative isolates in groups I and II, respectively (Figure 2 and Table 1).
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75% of pre-mastitis (d –5) daily milk yield by d 7. Of the remaining 11 cows, 8 cows regained 36 to 69% of their premastitis daily milk yield by d 7, 2 cows died within 24 h of detection of severe clinical signs, and 1 cow died within 7 d of onset of severe clinical mastitis (Table 2). Recovery was not affected by antibiotic treatment because 5 of 8 cows (62.5%) with 75% return to premastitis milk yield, and 8 of 11 cows (72.7%) with <75% return to premastitis milk yield received antibiotics.
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; Table 2). Three of the 8 cows (37.5%) in the recovered group and 8 of 11 cows in the nonrecovered group (72.7%) were classified as curli positive (Tables 1 and 2), with cow B303, which yielded curli-negative isolates on d 0 and 1 positive isolate on d 5, counted as a curli-positive case. |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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Rate of curli expression in E. coli has been shown to be related to bacterial behavior, host, and environment, and varies from rare to 100% (Cookson et al., 2002; Goldwater and Bettelheim, 2002). Similar rates of expression between fecal and mastitic isolates as shown in our study and the study by Olsen et al. (1989) support the idea that mastitic E. coli are opportunistic, environmental bacteria. These data also indicate that curli expression may be a stable phenotype among the mastitic E. coli investigated, because repeated culture of white (negative) variants did not cause them to express the red (positive) phenotype, and vice versa. It should be noted, however, that curli expression was observed under ideal growth conditions, and such stability may not be the case in the changing, in vivo environment of the udder.
All isolates possessed the genes for the curlin subunit protein, csgA, and the curli transcriptional regulator, csgD, regardless of phenotype expressed. Similar findings have been reported (Maurer et al., 1998; Zogaj et al., 2003), and our findings support the claim that both genes are ubiquitous in all E. coli strains. In this experiment, only presence of the csgA and csgD gene was evaluated, and it is possible that, although present, either gene was not expressed or activated in white variants. Another gene required for curli expression also may be missing, altered by insertions or deletions, or not be expressed. The ubiquitous nature of the csg genes does not allow these genes to be used in screening for curli-producing E. coli. At this time, it seems that physical evaluation of the binding of Congo red dye (or another substrate) is the simplest and fastest method to determine if an E. coli isolate is capable of producing curli.
Curli expression was present in 59% of bovine cases, which is similar to the occurrence reported on a per-isolate basis by Olsen et al. (1989). In the 15 cases for which more than 1 isolate was cultured from the same cow, all isolates cultured from the same case displayed the same phenotype, with one exception. The most likely reason for this observation is that additional isolates cultured from the same case were either very closely related or identical clones. Another possibility is that curli-producing bacteria may have a selective advantage within the mammary environment of some cows, increasing the likelihood of culturing a curli-positive strain.
The E. coli cultured from cow B303 were the only isolates to have mixed curli phenotypes. The d 0 isolates were curli negative, whereas the isolate cultured on d 5 was positive. One possible explanation for this outcome is that the initial infecting population experienced a mutation or phase shift that allowed it to activate curli production during the clinical episode or that curli production was activated as an adaptive response (Jubelin et al., 2005). The genetic distance observed by ERIC-PCR between B303D0a and B303D0b (98% similarity), and B303D5c (52% similarity to B303D0a/b), however, indicates that multiple, unrelated isolates may have been cultured from cow B303.
Indeed, genetically diverse isolates were cultured occasionally from the same cow. Sibling isolates in 7 cases clustered at <90%. In 4 of these cases, sibling isolates clustered at 80% similarity whereas isolates from 3 cows, cows B1450, F1300, and B303, clustered at <70% similarity. Cow B1450 had 3 isolates cultured from d 0 and 5 that clustered closely together (96% similarity), but also contained isolate B1450D0a that was only 60% similar to the others. Cow F1300, which was the only case in this study that presented mastitis in 2 quarters, had isolates from the right rear quarter that were very similar (96%) to each other, but were only about 70% similar to the isolates cultured from the left rear quarter, which were only 83% similar to each other. These data indicate that these cows were simultaneously infected with at least 2 genetically diverse E. coli strains. Mechanisms for the isolation of multiple E. coli strains include infection of the mammary gland by 2 or more strains, a separate infection that occurred after d 0, or contamination of the culture by an environmental E. coli. These strains could all be opportunistic invaders if the mastitis was the consequence of a systemic condition (such as stress, hormonal, or nutritional disorders) that caused temporary immunosuppression.
The ERIC-PCR did not differentiate curli-positive E. coli from those unable to produce curli in this study. Why such differences were not detectable by ERIC-PCR might be because the genetic capabilities required for curli expression are not located between ERIC elements or that they do not greatly alter the length of DNA (such as with the deletion of 1 base pair). In addition, curli production in these isolates may be entirely regulated by RNA, which was not examined, or the diversity of ERIC sequences and locations within the genome may overshadow any differences that contribute to curli production. The ERIC-PCR did allow limited differentiation of strains by origin of isolates. Isolates from cows in different sites were in general <90% similar, with the exception of 5 clusters of isolates from 13 cows from various origins, and which had 90% similarity (Figure 2).
The role of curli in the pathogenesis of bovine mastitis is unknown. When Todhunter et al. (1991) examined the outcome of experimental IMI with a curli-positive strain in cows vaccinated with either a curli-positive or a curli-negative strain of E. coli, all 12 cows developed clinical mastitis, regardless of curli expression of the vaccine strain. Our study evaluated the immediate effects of curli as measured by its effect on milk yields in a limited time frame. Curli expression did not affect the cows’ recovery, when measured by the return to 75% of premastitis milk yield, the only available variable to evaluate severity of the mastitis case. Cows with severe clinical signs requiring aggressive supportive care may still be recovering by d 7, and impact of curli on mastitis may extend well beyond milk yields in that period.
Curli have been reported to enhance bacterial virulence, not only through enhanced adhesion, but also through the stimulation of inflammatory mediators, cytokines, and septic-related clinical signs (Nasr et al., 1996; Bian et al., 2000). Our study did not examine cows for evidence of expression of curli outside the mammary gland, such as for presence of antibodies to the curlin subunit, CsgA. Curli have been reported to facilitate cellular internalization of E. coli. Curli may play a role in chronic and recurrent mastitis by allowing some strains to reside in the mammary gland, serving as a reservoir for future infections. Possible areas of investigation to assess curli effect on bovine mastitis could include evaluation of the effect of curli on local and systemic clinical signs exhibited in mastitic cows, experimental mammary challenges with curli-positive and curli-negative E. coli strains, detection of CsgA antibodies in the serum of cows having severe or septic mastitis, and characterization of the role of curli in chronic, recurring mammary infections.
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CONCLUSIONS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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Generally, clinical severity of E. coli mastitis is more dependent on cow-related factors than on bacterial virulence. Curli production in E. coli isolated from clinical cases of mastitis in this study did not affect return to precase milk yield, and E. coli strains did not cluster based on case recovery when fingerprinted by ERIC-PCR. In addition, genetically diverse E. coli were recovered from clinically infected udders.
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ACKNOWLEDGEMENTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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Received for publication March 27, 2006. Accepted for publication August 14, 2006.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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