Mucosal Immunization with Live Attenuated Francisella novicida U112ΔiglB Protects against Pulmonary F. tularensis SCHU S4 in the Fischer 344 Rat Model

The need for an efficacious vaccine against Francisella tularensis is a consequence of its low infectious dose and high mortality rate if left untreated. This study sought to characterize a live attenuated subspecies novicida-based vaccine strain (U112ΔiglB) in an established second rodent model of pulmonary tularemia, namely the Fischer 344 rat using two distinct routes of vaccination (intratracheal [i.t.] and oral). Attenuation was verified by comparing replication of U112ΔiglB with wild type parental strain U112 in F344 primary alveolar macrophages. U112ΔiglB exhibited an LD₅₀>10⁷ CFU compared to the wild type (LD₅₀ = 5×10⁶ CFU i.t.). Immunization with 10⁷ CFU U112ΔiglB by i.t. and oral routes induced antigen-specific IFN-γ and potent humoral responses both systemically (IgG2a>IgG1 in serum) and at the site of mucosal vaccination (respiratory/intestinal compartment). Importantly, vaccination with U112ΔiglB by either i.t. or oral routes provided equivalent levels of protection (50% survival) in F344 rats against a subsequent pulmonary challenge with ∼25 LD₅₀ (1.25×10⁴ CFU) of the highly human virulent strain SCHU S4. Collectively, these results provide further evidence on the utility of a mucosal vaccination platform with a defined subsp. novicida U112ΔiglB vaccine strain in conferring protective immunity against pulmonary tularemia.     

Rectal Carriage of Enterohemorrhagic Escherichia coli O157 in Slaughtered Cattle

Escherichia coli O157:H7 is an important cause of diarrhea, hemorrhagic colitis, and potentially fatal human illness. Cattle are considered a primary reservoir of infection, and recent experimental evidence has indicated that the terminal rectum is the principal site of bacterial carriage. To test this finding in naturally colonized animals, intact rectum samples from 267 cattle in 24 separate lots were obtained immediately after slaughter, and fecal material and mucosal surfaces were cultured for E. coli O157 by direct and enrichment methods. Two locations, 1 and 15 cm proximal to the recto-anal junction, were tested. In total, 35 animals were positive for E. coli O157 at at least one of the sites and 232 animals were negative as determined by all tests. The frequency of isolation and the numbers of E. coli O157 cells were higher at the site closer to the recto-anal junction, confirming our previous experimental findings. We defined low- and high-level carriers as animals with E. coli O157 levels of <1 × 10³ CFU g⁻¹ or <1 × 10³ CFU ml⁻¹ and animals with E. coli O157 levels of ≥1 × 10³ CFU g⁻¹ or ≥1 × 10³ CFU ml⁻¹ in feces or tissues, respectively. High-level carriage was detected in 3.7% of the animals (95% confidence interval, 1.8 to 6.8%), and carriage on the mucosal surface of the terminal rectum was associated with high-level fecal excretion. In summary, our results support previous work demonstrating that the mucosal epithelium in the bovine terminal rectum is an important site for E. coli O157 carriage in cattle. The data also support the hypothesis that high-level fecal shedding (≥1 × 10³ CFU g of feces⁻¹) of enterohemorrhagic E. coli O157 results from colonization of this site.     

Rapid label-free detection of E. coli using a novel SPR biosensor containing a fragment of tail protein from phage lambda

Abstract

In efforts to speed up the assessment of microorganisms, researchers have sought to use bacteriophages as a biosensing tool, due to their host-specificity, wide abundance, and safety. However, the lytic cycle of the phage has limited its efficacy as a biosensor. Here, we cloned a fragment of tail protein J from phage lambda and characterized its binding with the host, E. coli K-12, and other microorganism. The N-terminus of J was fused with a His-tag (6HN-J), overexpressed, purified, and characterized using anti-His monoclonal antibodies. The purified protein demonstrated a size of ～38?kDa upon SDS-PAGE and bound with the anti-His monoclonal antibodies. ELISA, dot blot, and TEM data revealed that it specifically bound to E. coli K-12, but not to Pseudomonas aeruginosa. The observed protein binding occurred over a concentration range of 0.01–5?μg/ml and was found to inhibit the in vivo adsorption of phage to host cells. This specific binding was exploited by surface plasmon resonance (SPR) to generate a novel 6HN-J-functionalized SPR biosensor. This biosensor showed rapid label-free detection of E. coli K-12 in the range of 2?×?10⁴ ?2?×?10⁹ CFU/ml, and exhibited a lower detection limit of 2?×?10⁴ CFU/ml.     

Importance of the Collagen Adhesin Ace in Pathogenesis and Protection against Enterococcus faecalis Experimental Endocarditis

Ace is an adhesin to collagen from Enterococcus faecalis expressed conditionally after growth in serum or in the presence of collagen. Here, we generated an ace deletion mutant and showed that it was significantly attenuated versus wild-type OG1RF in a mixed infection rat endocarditis model (P<0.0001), while no differences were observed in a peritonitis model. Complemented OG1RFΔace (pAT392::ace) enhanced early (4 h) heart valve colonization versus OG1RFΔace (pAT392) (P = 0.0418), suggesting that Ace expression is important for early attachment. By flow cytometry using specific anti-recombinant Ace (rAce) immunoglobulins (Igs), we showed in vivo expression of Ace by OG1RF cells obtained directly from infected vegetations, consistent with our previous finding of anti-Ace antibodies in E. faecalis endocarditis patient sera. Finally, rats actively immunized against rAce were less susceptible to infection by OG1RF than non-immunized (P = 0.0004) or sham-immunized (P = 0.0475) by CFU counts. Similarly, animals given specific anti-rAce Igs were less likely to develop E. faecalis endocarditis (P = 0.0001) and showed fewer CFU in vegetations (P = 0.0146). In conclusion, we have shown for the first time that Ace is involved in pathogenesis of, and is useful for protection against, E. faecalis experimental endocarditis.     

Panton–Valentine Leukocidin Enhances the Severity of Community-Associated Methicillin-Resistant Staphylococcus aureus Rabbit Osteomyelitis

Background

Extensive spread of community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) in the United States, and the concomitant increase in severe invasive staphylococcal infections, including osteomyelitis, in healthy children, has led to renewed interest in Panton-Valentine leukocidin (PVL). However, the pathogenetic role of PVL in staphylococcal infections remains controversial, possibly because it depends on the site of infection.

Methodology/Principal Findings

We compared the course of experimental rabbit osteomyelitis due to the PVL-positive CA-MRSA strain USA 300 (LAC) and its PVL-negative isogenic derivative (LACΔpvl), using a low and a high inoculum (8×10⁵ and 4×10⁸ CFU). With the low inoculum, bone infection was less frequent on day 7 (D7) and day 28 (D28) with LACΔpvl than with LAC (respectively 12/19 and 18/19 animals, p = 0.042). With the high inoculum of both strains, all the animals were infected on D7 and the infection persisted on D28 in almost every case. However, tibial bacterial counts and the serum CRP concentration fell significantly between D7 and D28 with LACΔpvl but not with LAC. Respectively 67% and 60% of LAC-infected rabbits had bone deformation and muscle/joint involvement on D7, compared to 0% and 7% of LACΔpvl-infected rabbits (p = 0.001 and p = 0.005 respectively). Between D0 and D28, the anti-PVL antibody titer increased significantly only with the high inoculum of LAC.

Conclusions/Significance

PVL appears to play a role in the persistence and rapid local extension of rabbit osteomyelitis, in keeping with the greater severity of human bone infections due to PVL-positive S. aureus. The possible therapeutic implications of these findings are discussed.     

Detection of Streptococcus pneumoniae from Different Types of Nasopharyngeal Swabs in Children

Background

A better understanding of the epidemiology of nasopharyngeal carriage of Streptococcus pneumoniae is important to assess the impact of vaccination and the pathogenesis of pneumococcal disease. We compared the recovery of S. pneumoniae from nylon flocked, Dacron and rayon swabs.

Methods

The recovery of S. pneumoniae from mocked specimens using flocked, Dacron and rayon swabs were compared by culture. The yield from paired nasopharyngeal (NP) samples obtained from healthy children sampled with flocked and Dacron swabs was also determined using culture and lytA-targeted real-time polymerase chain reaction (qPCR).

Results

Using mock specimen, the percentage recovery of S. pneumoniae ATCC 49619 (serotype 19F) strain from the flocked swabs was 100%, while it was 41% from Dacron swabs and 7% from rayon swabs. Similar results were observed for S. pneumoniae serotypes 1 and 5. S. pneumoniae was cultured from 18 of 42 (43%) paired NP samples from the healthy children (median age 8 [interquartile range (IQR) 5–16] months). The median number of colony-forming units (CFU) recovered from flocked swabs was two-fold higher (8.8×10⁴ CFU/mL [IQR, 2.0×10² – 4.0×10⁵ CFU/mL]) than Dacron swabs (3.7×10⁴ CFU/mL [IQR, 4.0×10²–3.2×10⁵ CFU/mL], p = 0.17). Using lytA-targeted qPCR from paired NP samples, the median copy number of S. pneumoniae detected from flocked swabs was significantly higher than from Dacron swabs (3.0×10⁵ genome copies/mL [IQR, 1.3×10²−1.8×10⁶] vs. 9.3×10⁴ genome copies/mL [IQR, 7.0×10¹−1.1×10⁶]; p = 0.005).

Conclusion

Flocked swabs released more S. pneumoniae compared to both Dacron and rayon swabs from mock specimens. Similarly, higher bacterial loads were detected by qPCR from flocked swabs compared with Dacron swabs from healthy children.     

Hemin-incorporated nanoflowers as enzyme mimics for colorimetric detection of foodborne pathogenic bacteria

Rapid, sensitive and point-of-care detection of foodborne pathogenic bacteria is essential for food safety. In this study, we found that hemin-concanavalin A hybrid nanoflowers (HCH nanoflowers), as solid mimic peroxidase, could catalyze oxidation of 2,2′-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) diammonium salt (ABTS) in the presence of H₂O₂ to a green-colored product. HCH nanoflowers, integrating the essential functions of both biological recognition and signal amplification, meet the requirements of signal labels for colorimetric immunoassay of bacteria. In view of the excellent peroxidase mimetic catalytic activity of HCH nanoflowers, a colorimetric biosensing platform was newly constructed and applied for sensitive detection of foodborne Escherichia coli O157:H7 (E. coli O157:H7). The corresponding detection limits was as low as 4.1?CFU/mL with wide linear ranges (10¹–10⁶?CFU/mL).     

Impact of rpoS Deletion on Escherichia coli Biofilms

Slow growth has been hypothesized to be an essential aspect of bacterial physiology within biofilms. In order to test this hypothesis, we employed two strains of Escherichia coli, ZK126 (ΔlacZ rpoS⁺) and its isogenic ΔrpoS derivative, ZK1000. These strains were grown at two rates (0.033 and 0.0083 h⁻¹) in a glucose-limited chemostat which was coupled either to a modified Robbins device containing plugs of silicone rubber urinary catheter material or to a glass flow cell. The presence or absence of rpoS did not significantly affect planktonic growth of E. coli. In contrast, biofilm cell density in the rpoS mutant strain (ZK1000), as measured by determining the number of CFU per square centimeter, was reduced by 50% (P < 0.05). Deletion of rpoS caused differences in biofilm cell arrangement, as seen by scanning confocal laser microscopy. In reporter gene experiments, similar levels of rpoS expression were seen in chemostat-grown planktonic and biofilm populations at a growth rate of 0.033 h⁻¹. Overall, these studies suggest that rpoS is important for biofilm physiology.     

Increased Circulating Endothelial Apoptotic Microparticle to Endothelial Progenitor Cell Ratio Is Associated with Subsequent Decline in Glomerular Filtration Rate in Hypertensive Patients

Background

Recent research indicates hypertensive patients with microalbuminuria have decreased endothelial progenitor cells (EPCs) and increased levels of endothelial apoptotic microparticles (EMP). However, whether these changes are related to a subsequent decline in glomerular filtration rate (GFR) remains unclear.

Methods and Results

We enrolled totally 100 hypertensive out-patients with eGFR ≥30 mL/min/1.73 m². The mean annual rate of GFR decline (△GFR/y) was −1.49±3.26 mL/min/1.73 m² per year during the follow-up period (34±6 months). Flow cytometry was used to assess circulating EPC (CD34⁺/KDR⁺) and EMP levels (CD31⁺/annexin V⁺) in peripheral blood. The △GFR/y was correlated with the EMP to EPC ratio (r = −0.465, p<0.001), microalbuminuria (r = −0.329, p = 0.001), and the Framingham risk score (r = −0.245, p = 0.013). When we divided the patients into 4 groups according to the EMP to EPC ratio, there was an association between the EMP to EPC ratio and the ΔGFR/y (mean ΔGFR/y: 0.08±3.04 vs. −0.50±2.84 vs. −1.25±2.49 vs. −4.42±2.82, p<0.001). Multivariate analysis indicated that increased EMP to EPC ratio is an independent predictor of ΔeGFR/y.

Conclusions

An increased circulating EMP to EPC ratio is associated with subsequent decline in GFR in hypertensive patients, which suggests endothelial damage with reduced vascular repair capacity may contribute to further deterioration of renal function in patients with hypertension.     

Presence of Culturable Bacteria in Cocoons of the Earthworm Eisenia fetida

Viable bacteria were found to coexist with developing embryos in egg capsules (cocoons) of the earthworm Eisenia fetida. Earthworms were reared under standardized conditions, and bacterial densities were measured in distinct batches of cocoons collected weekly for 10 weeks. Cocoons weighing 12 mg contained a mean viable bacterial population of approximately 10⁸ CFU/g of cocoons. No difference was found in viable counts obtained from cocoons incubated at 15°C and cocoons incubated at 24°C. Viable bacterial numbers increased with cocoon age, while acridine orange direct counts of microbial cells were stable at approximately 10⁹ cells per g of cocoons. Bacteria isolated from cocoons were used to develop antisera in rabbits for the production of strain-specific fluorescent antibodies. Fluorescent antibody and selective plating techniques were used to monitor populations of these bacteria in earthworm bedding and to determine whether cocoons acquire bacteria from the environment in which they are formed. Cocoon isolates were readily recovered from cocoons formed in inoculated bedding at densities of 10⁸ CFU/g of cocoons. Bradyrhizobium japonicum USDA 110 and UMR 161 added to bedding were also recovered from cocoons, but at lower densities than cocoon isolates. Escherichia coli K-12(pJP4) inoculum was recovered from bedding but not from cocoons. The bacterial complement of Eisenia fetida cocoons is affected by inoculation of selected bacterial isolates in the worm growth environment.     

Rapid concentration of bacteria using submicron magnetic anion exchangers for improving PCR-based multiplex pathogen detection

Rapid concentration of bacterial targets from dilute solutions to improve subsequent PCR detection is investigated in this study. Submicron (average size 500 nm) superparamagnetic anion-exchangers (SiMAG-DEAE) were used successfully to concentrate target bacteria from very dilute solutions. A mass-balance model predicted that for Escherichia coli, the extent of cell concentrating increases almost linearly with increasing sample/SiMAG volume ratio up to about 2000, accompanied by only a slight decrease in the capture efficiency (< 10%). Our experimental data generally support this analysis in that the SiMAG beads concentrated bacterial targets by two to three orders of magnitude using a sample/bead volume ratio of about 1000, and lowered the PCR detection limit to a level of 10² CFU/mL, from 10⁴ to 10⁵ CFU/mL without concentrating. Several target bacteria can be concentrated concurrently and detected via multiplex PCR, as illustrated using E. coli and Agrobacterium tumefaciens as model bacteria. Finally, concentration and detection of bacteria in fresh produce samples were demonstrated. The integration of submicron magnetic ion exchangers and PCR detection provides an appealing alternative to immunomagnetic separation/PCR in improving pathogen detection.     

Optimization of a Reusable Hollow-Fiber Ultrafilter for Simultaneous Concentration of Enteric Bacteria, Protozoa, and Viruses from Water

The detection and identification of pathogens from water samples remain challenging due to variations in recovery rates and the cost of procedures. Ultrafiltration offers the possibility to concentrate viral, bacterial, and protozoan organisms in a single process by using size-exclusion-based filtration. In this study, two hollow-fiber ultrafilters with 50,000-molecular-weight cutoffs were evaluated to concentrate microorganisms from 2- and 10-liter water samples. When known quantities (10⁵ to 10⁶ CFU/liter) of two species of enteric bacteria were introduced and concentrated from 2 liters of sterile water, the addition of 0.1% Tween 80 increased Escherichia coli strain K-12 recoveries from 70 to 84% and Salmonella enterica serovar Enteritidis recoveries from 36 to 72%. An E. coli antibiotic-resistant strain, XL1-Blue, was recovered at a level (87%) similar to that for strain K-12 (96%) from 10 liters of sterile water. When E. coli XL1-Blue was introduced into 10 liters of nonsterile Rio Grande water with higher turbidity levels (23 to 29 nephelometric turbidity units) at two inoculum levels (9 × 10⁵ and 2.4 × 10³ per liter), the recovery efficiencies were 89 and 92%, respectively. The simultaneous addition of E. coli XL1-Blue (9 × 10⁵ CFU/liter), Cryptosporidium parvum oocysts (10 oocysts/liter), phage T1 (10⁵ PFU/liter), and phage PP7 (10⁵ PFU/liter) to 10 liters of Rio Grande surface water resulted in mean recoveries of 96, 54, 59, and 46%, respectively. Using a variety of surface waters from around the United States, we obtained recovery efficiencies for bacteria and viruses that were similar to those observed with the Rio Grande samples, but recovery of Cryptosporidium oocysts was decreased, averaging 32% (the site of collection of these samples had previously been identified as problematic for oocyst recovery). Results indicate that the use of ultrafiltration for simultaneous recovery of bacterial, viral, and protozoan pathogens from variable surface waters is ready for field deployment.     

PCR-Based DNA Amplification and Presumptive Detection of Escherichia coli O157:H7 with an Internal Fluorogenic Probe and the 5′ Nuclease (TaqMan) Assay

Presumptive identification of Escherichia coli O157:H7 is possible in an individual, nonmultiplexed PCR if the reaction targets the enterohemorrhagic E. coli (EHEC) eaeA gene. In this report, we describe the development and evaluation of the sensitivity and specificity of a PCR-based 5′ nuclease assay for presumptively detecting E. coli O157:H7 DNA. The specificity of the eaeA-based 5′ nuclease assay system was sufficient to correctly identify all E. coli O157:H7 strains evaluated, mirroring the previously described specificity of the PCR primers. The SZ-primed, eaeA-targeted 5′ nuclease detection assay was capable of rapid, semiautomated, presumptive detection of E. coli O157:H7 when ≥10³ CFU/ml was present in modified tryptic soy broth (mTSB) or modified E. coli broth and when ≥10⁴ CFU/ml was present in ground beef-mTSB mixtures. Incorporating an immunomagnetic separation (IMS) step, followed by a secondary enrichment culturing step and DNA recovery with a QIAamp tissue kit (Qiagen), improved the detection threshold to ≥10² CFU/ml. Surprisingly, immediately after IMS, the sensitivity of culturing on sorbitol MacConkey agar containing cefeximine and tellurite (CT-SMAC) was such that identifiable colonies were demonstrated only when ≥10⁴ CFU/ml was present in the sample. Several factors that might be involved in creating these false-negative CT-SMAC culture results are discussed. The SZ-primed, eaeA-targeted 5′ nuclease detection system demonstrated that it can be integrated readily into standard culturing procedures and that the assay can be useful as a rapid, automatable process for the presumptive identification of E. coli O157:H7 in ground beef and potentially in other food and environmental samples.     

Intragenic Suppressor of a Plug Deletion Nonmotility Mutation in PotB,a Chimeric Stator Protein of Sodium-Driven Flagella

The torque of bacterial flagellar motors is generated by interactions between the rotor and the stator and is coupled to the influx of H⁺ or Na⁺ through the stator. A chimeric protein, PotB, in which the N-terminal region of Vibrio alginolyticus PomB was fused to the C-terminal region of Escherichia coli MotB, can function with PomA as a Na⁺-driven stator in E. coli. Here, we constructed a deletion variant of PotB (with a deletion of residues 41 to 91 [Δ41–91], called PotBΔL), which lacks the periplasmic linker region including the segment that works as a “plug” to inhibit premature ion influx. This variant did not confer motile ability, but we isolated a Na⁺-driven, spontaneous suppressor mutant, which has a point mutation (R109P) in the MotB/PomB-specific α-helix that connects the transmembrane and peptidoglycan binding domains of PotBΔL in the region of MotB. Overproduction of the PomA/PotBΔL(R109P) stator inhibited the growth of E. coli cells, suggesting that this stator has high Na⁺-conducting activity. Mutational analyses of Arg109 and nearby residues suggest that the structural alteration in this α-helix optimizes PotBΔL conformation and restores the proper arrangement of transmembrane helices to form a functional channel pore. We speculate that this α-helix plays a key role in assembly-coupled stator activation.     

mhpT Encodes an Active Transporter Involved in 3-(3-Hydroxyphenyl)Propionate Catabolism by Escherichia coli K-12

Escherichia coli K-12 utilizes 3-(3-hydroxyphenyl)propionate (3HPP) as a sole carbon and energy source. Among the genes in its catabolic cluster in the genome, mhpT was proposed to encode a hypothetical transporter. Since no transporter for 3HPP uptake has been identified, we investigated whether MhpT is responsible for 3HPP uptake. MhpT fused with green fluorescent protein was found to be located at the periphery of cells by confocal microscopy, consistent with localization to the cytoplasmic membrane. Gene knockout and complementation studies clearly indicated that mhpT is essential for 3HPP catabolism in E. coli K-12 W3110 at pH 8.2. Uptake assays with ¹⁴C-labeled substrates demonstrated that strain W3110 and strain W3110ΔmhpT containing recombinant MhpT specifically transported 3HPP but not benzoate, 3-hydroxybenzoate, or gentisate into cells. Energy dependence assays suggested that MhpT-mediated 3HPP transport was driven by the proton motive force. The change of Ala-272 of MhpT to a histidine, surprisingly, resulted in enhanced transport activity, and strain W3110ΔmhpT containing the MhpT A272H mutation had a slightly higher growth rate than the wild-type strain at pH 8.2. Hence, we demonstrated that MhpT is a specific 3HPP transporter and vital for E. coli K-12 W3110 growth on this substrate under basic conditions.     

Multiplex Fluorogenic Real-Time PCR for Detection and Quantification of Escherichia coli O157:H7 in Dairy Wastewater Wetlands

Surface water and groundwater are continuously used as sources of drinking water in many metropolitan areas of the United States. The quality of water from these sources may be reduced due to increases in contaminants such as Escherichia coli from urban and agricultural runoffs. In this study, a multiplex fluorogenic PCR assay was used to quantify E. coli O157:H7 in soil, manure, cow and calf feces, and dairy wastewater in an artificial wetland. Primers and probes were designed to amplify and quantify the Shiga-like toxin 1 (stx1) and 2 (stx2) genes and the intimin (eae) gene of E. coli O157:H7 in a single reaction. Primer specificity was confirmed with DNA from 33 E. coli O157:H7 and related strains with and without the three genes. A direct correlation was determined between the fluorescence threshold cycle (C_T) and the starting quantity of E. coli O157:H7 DNA. A similar correlation was observed between the C_T and number of CFU per milliliter used in the PCR assay. A detection limit of 7.9 × 10⁻⁵ pg of E. coli O157:H7 DNA ml⁻¹ equivalent to approximately 6.4 × 10³ CFU of E. coli O157:H7 ml⁻¹ based on plate counts was determined. Quantification of E. coli O157:H7 in soil, manure, feces, and wastewater was possible when cell numbers were ≥3.5 × 10⁴ CFU g⁻¹. E. coli O157:H7 levels detected in wetland samples decreased by about 2 logs between wetland influents and effluents. The detection limit of the assay in soil was improved to less than 10 CFU g⁻¹ with a 16-h enrichment. These results indicate that the developed PCR assay is suitable for quantitative determination of E. coli O157:H7 in environmental samples and represents a considerable advancement in pathogen quantification in different ecosystems.     

Distribution of Escherichia coli O157 in Bovine Fecal Pats and Its Impact on Estimates of the Prevalence of Fecal Shedding

The distribution of Escherichia coli O157 in bovine feces was examined by testing multiple samples from fecal pats and determining the density of E. coli O157 in immunomagnetic separation (IMS)-positive fecal samples. The density of E. coli O157 in bovine feces was highly variable, differing by as much as 76,800 CFU g⁻¹ between samples from the same fecal pat. The density in most positive samples was <100 CFU g⁻¹, the limit of reliable detection by IMS. Testing only one 1-g sample of feces per pat with IMS may result in a sensitivity of detection as low as 20 to 50%. It is therefore probable that most surveys have greatly underestimated the prevalence of E. coli O157 shedding in cattle and the proportion of farms with shedding cattle. The sensitivity of the detection of E. coli O157 in bovine feces can be as much as doubled by testing two 1-g samples per pat rather than one 1-g sample.     

Flocculation of Escherichia coli Cells in Association with Enhanced Production of Outer Membrane Vesicles

Microbial flocculation is a phenomenon of aggregation of dispersed bacterial cells in the form of flocs or flakes. In this study, the mechanism of spontaneous flocculation of Escherichia coli cells by overexpression of the bcsB gene was investigated. The flocculation induced by overexpression of bcsB was consistent among the various E. coli strains examined, including the K-12, B, and O strains, with flocs that resembled paper scraps in structure being about 1 to 2 mm. The distribution of green fluorescent protein-labeled E. coli cells within the floc structure was investigated by three-dimensional confocal laser scanning microscopy. Flocs were sensitive to proteinase K, indicating that the main component of the flocs was proteinous. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and nano-liquid chromatography tandem mass spectrometry analyses of the flocs strongly suggested the involvement of outer membrane vesicles (OMVs) in E. coli flocculation. The involvement of OMVs in flocculation was supported by transmission electron microscopy observation of flocs. Furthermore, bcsB-induced E. coli flocculation was greatly suppressed in strains with hypovesiculation phenotypes (ΔdsbA and ΔdsbB strains). Thus, our results demonstrate the strong correlation between spontaneous flocculation and enhanced OMV production of E. coli cells.