Use of Fluorogenic Assays for the Enumeration of Escherichia coli from Selected Seafoods

Escherichia coli was enumerated and immediately confirmed by incorporating 4-methylumbelliferone glucuronide (MUG) into Lactose Broth (LB), Violet Red Bile Agar (VRB) and M-Endo Broth (M-Endo) in selected seafoods. E. coli in the seafood samples ranged from 1.0 × 10¹– 1.0 × 10³ Most Probable Number (MPN)/g, 1.0 × 10¹– 3.5 × 10² cfu/g and 0.9 × 10¹– 2.8 × 10² cfu/g in LB-MUG, VRB-MUG, and M-Endo-MUG, respectively. Although not statistically significant (α= 0.05), LB-MUG resulted in higher counts in fresh finfish, frozen fish and breaded seafoods. Specificity for E. coli in LB-MUG, VRB-MUG and M-Endo MUG was 96.5%, 92.0%, and 90.0%, respectively. All three fluorogenic assays enabled a rapid and sensitive enumeration of E. coli from seafood products.     

Comparative analysis of a modified rapid presence/absence test and the standard MPN method for detectingEscherichia coli in orange juice

A modified rapid presence/absence test was evaluated and compared to the standard most probable number (MPN) method for detecting Escherichia coli in artificially contaminated orange juice. In each of the four experiments conducted, pasteurized and unpasteurized orange juice samples were seeded with one of the three different strains ofE. coli , at levels ranging from 0·4 to 6·5 cfu ml⁻¹. The samples were also seeded with 360–510 cfu ml⁻¹ of other enteric bacteria to simulate background flora. Samples were analysed by the MPN method for E. coli and by the modified ColiComplete (CC) presence/absence test in E. coli (EC) broth at 44·5°C, after pre-enriching 10 ml of juice samples in Universal Pre-enrichment Broth for 24 h (modified CC method). Of the 12 comparative analyses performed, E. coli was detected in all 12 tests by the modified CC method and, furthermore, showed the presence of E. coli in 59 of the 60 (98·3%) orange juice replicates that were examined. In contrast, the standard MPN method was only able to quantify detectable levels of E. coli in eight of 12 tests. The modified CC procedure was faster, required less media and reagents, enabled analysis of 10 ml samples and was more reliable than the standard MPN method for determining the presence or absence of E. coli in artificially contaminated orange juice.     

Inactivation of Escherichia coli Population on Fruit Surfaces Using Ultraviolet-C Light: Influence of Fruit Surface Characteristics

Ultraviolet-C (UV-C 254 nm) light is a possible alternative for chemical disinfection of fresh fruits. However, studies on the influence of surface characteristics on the kinetics of UV-C inactivation of microorganisms on fruits are limited. In this study, UV-C inactivation of generic Escherichia coli (ATCC 23716), a nonpathogenic surrogate strain for E. coli O157:H7, was inoculated onto the skin surface intact pear, pear with surface wounds, and the skin surface of intact peach. Disc shaped (0.057 m diameter?×?0.01 m height) fruit surface were exposed at room temperature to UV-C light ranging from 0 to 7.56?±?0.52 kJ/m² and microbial inactivation kinetics was determined. Maximum reductions of 3.70?±?0.125 log CFU/g were achieved for E. coli on intact pear surfaces (P?<?0.05), with lesser reduction on wounded pear (3.10?±?0.329 log CFU/g) and peach surfaces (2.91?±?0.284 log CFU/g) after 4 min UV-C exposure at 7.56 kJ/m² UV. The Weibull scale factor (α) values of UV-C inactivation for E. coli on an intact pear surface was 0.001?±?0.0007 min (0.235?±?0.001 kJ/m²), wounded pear surface, 0.003?±?0.001 min (0.240?±?0.002 kJ/m²) and peach surface, 0.004?±?0.0004 (0.241?±?0.0008 kJ/m²). The time required for a 90 % reduction in E. coli cell numbers or the reliable life time (t _R) calculated with the Weibull model for intact pear surfaces (0.019?±?0.009 min, 0.268?±?0.017 kJ/m²) was smaller than for wounded pear (0.062?±?0.013 min, 0.348?±?0.024 kJ/m²) and peach surfaces (0.074?±?0.012, 0.371?±?0.012 kJ/m²), suggesting that the wounds on pear surfaces and trichomes (100–1000 μm) on peach surfaces helped to shield and protect microorganisms from UV-C radiation. There was likely a more uniform distribution of bacterial cells onto pear surfaces due to its smaller surface roughness, spreading coefficient, and hydrophobic nature compared to peach. Fourier transform infrared spectroscopy indicate that bacterial membrane damage (phospholipids, protein secondary structures, and polysaccharides) and changes to DNA/RNA in E. coli resulted from UV-C treatment. UV-C can reduce E. coli populations on fresh fruit surfaces, but the efficacy of UV treatment is dependent upon the morphological and surface properties of the fruit and surface integrity.     

Thermal Inactivation of Escherichia coli O157:H7 in Cooked Turkey Products

Survival of Escherichia coli O157:H7 when heated in commercial-type turkey products was determined. Thermal death times (TDT) were determined at 52–60°C in ground turkey with no additives, 3% fat; ground turkey with no additives, 11% fat; turkey ham batter, 11% fat; turkey frank batter, 17% fat; and turkey sausage batter, 31% fat. Mean D₅₂-values ranged from 44.9 to 116 min; D₅₅-values from 6.63 to 39.4 min; D₅₇-values from 2.20 to 11.7 min; D₆₀-values from 0.68 to 5.86 min. At all temperatures, survival of E. coli O157:H7 was greater in formulated products than in turkey meat with no additives. Greatest survival occurred in the turkey frank batter. Using our z-value data, times to provide a 5 D kill of E. coli O157:H7 in turkey franks cooked at 60°C, 65.6°C, or 71°C would be 26, 3.1, or 0.37 min, respectively.     

Comparing effects of bovine Streptococcus and Escherichia coli mastitis on impaired reproductive performance

In 2 epidemiological studies, we evaluated the effect of mastitis induced by gram-positive Streptococcus and gram-negative Escherichia coli on impaired reproductive performance in lactating Holstein cows. In the first study, 52,202 cows from 178 dairy farms throughout Israel were divided into groups based on infection before first artificial insemination (AI) with Streptococcus or E. coli, 3 groups with elevated somatic cell count (SCC) without infection by those pathogens [low SCC (200–400) × 10³ cell/mL; medium SCC (401–1,000) × 10³ cell/mL; high SCC, >1,000 × 10³ cell/mL], and uninfected controls. Pregnancy per first AI (P/1stAI) and pregnancy rate at 300 d in milk (PREG 300) were analyzed by the GLIMMIX procedure (SAS); number of AI per pregnancy (AI/P), days open, and rest days (calving to first AI) were analyzed by the MIXED procedure (SAS Institute Inc., Cary, NC). Values of P/1stAI were similarly low for Streptococcus and E. coli (27–28%) versus 42% in controls; PREG 300 was lower for Streptococcus (76%) than for E. coli (79%) versus 88% for uninfected controls and a mean 83% for the elevated SCC groups. Days open and number of AI/P were higher than in controls and similar in Streptococcus and E. coli groups. The second study included 778 cows on 6 dairy farms; the cows were infected before first AI by Streptococcus or E. coli or uninfected. Resumption of cyclicity was determined by an automated activity-monitoring system, and data were sorted by time of infection before or after cyclicity resumed. The Streptococcus group had lower P/1stAI before and after cyclicity (26 and 27%, respectively) than the E. coli group (31 and 34%, respectively) and uninfected controls (42%). Notably, PREG 300 in the Streptococcus group before (73%) and after (67%) cyclicity was much lower than for the E. coli group (85 and 93%, respectively) and the controls (95%). A marked rise in day of cyclicity resumption (∼80 d) was observed in cows that were infected early on. Number of AI/P was higher in the mastitic groups than in uninfected controls. Uterine disease postpartum, although more prevalent among Streptococcus cows, did not substantially alter the larger reduction in P/1stAI and PREG 300 in Streptococcus versus E. coli cows. Thus, long-term Streptococcus-induced mastitis disrupted fertility more than short-term acute E. coli–induced mastitis, resulting in a much higher percentage of Streptococcus cows in late lactation that did not conceive due to reproduction failure.     

Comparison of 4 label-based immunochromatographic assays for the detection of Escherichia coli O157:H7 in milk

Immunochromatographic assays (ICA) are widely used to detect pathogens. In this study, we used traditional gold nanoparticles (GNP), quantum dots (QD), fluorescent nanoparticles (FNP), and europium (Eu) (III) chelate nanoparticles (EuNP) as ICA labels. We first compared the ability of the 4 ICA test strips to quantitatively detect Escherichia coli O157:H7 in milk. We then optimized various parameters influencing the ICA. The sensitivity to E. coli O157:H7 of the GNP-ICA, QD-ICA, FNP-ICA, and EuNP-ICA was 2.5 × 10⁴, 5 × 10³, 1.0 × 10³, and 5.0 × 10² cfu mL^?1, respectively. The EuNP-ICA exhibited the highest sensitivity. The amounts of monoclonal antibodies (mAb) per GNP-ICA, QD-ICA, FNP-ICA, and EuNP-ICA test strip were 0.16, 0.37, 0.04, and 0.10 μg, respectively. The corresponding coefficients of variation were 7.4 to 15.8%, 10.4 to 18.6%, 2.7 to 7.8%, and 6.9 to 10.5%, respectively. The FNP-ICA required the least mAb per test strip and had the best coefficient of variation. The linear ranges of GNP-ICA, QD-ICA, FNP-ICA, and EuNP-ICA were 1.0 × 10⁴ to 1.0 × 10⁶, 2.5 × 10³ to 1.0 × 10⁶, 2.5 × 10² to 2.5 × 10⁵, and 2.5 × 10² to 2.5 × 10⁵ cfu mL^?1, respectively. The FNP-ICA and EuNP-ICA had wider linear ranges than GNP-ICA and QD-ICA. Additionally, the FNP-ICA and EuNP-ICA showed better tolerance than GNP-ICA and QD-ICA in the milk samples. The FNP-ICA and EuNP-ICA showed remarkable potential for detection of pathogens in milk.     

Foodborne enterotoxigenic Escherichia coli: Identification and enumeration on nitrocellulose membrane by enzyme immunoassay

A solid-phase enzyme immunoassay capable of direct enumeration of Escherichia coli that produce heat-labile enterotoxin (LT) has been developed. Pure or mixed cultures of bacteria or artificially contaminated foods were plated on nitrocellulose membranes placed on semisynthetic agar containing lincomycin. After overnight growth, the colonies were lysed in situ and were reacted with rabbit antiserum to LT. The presence of rabbit IgG in a colony was detected by goat antirabbit IgG peroxidase conjugate. Results of 71 strains tested by this method were in complete accord with LT assays performed by the mouse Y-1 adrenal cells. Three foods, namely, oysters, raw milk and Brie cheese, containing 2 × 10⁶, 1.3 × 10⁵ and 3.9 × 10⁶ total plate count, respectively, were artificially contaminated with known levels of toxigenic E. coli. Upon analysis of membrane filters on which a range of 1 × 10³ to 10⁴ cells was deposited, 1 to 10 LT⁺E. coli could be enumerated with a recovery greater than 85%. The test on foods can be completed in approximately 30 h. The test is sensitive enough to visualize 1 ng of pure LT contained in 1 mm² area of the membrane.     

Risk assessment modelling of fecal shedding caused by extended-spectrum cephalosporin-resistant Escherichia coli transmitted through waste milk fed to dairy pre-weaned calves

Waste milk feeding is a common practice in dairy operations. Regardless of the benefits of this practice to the dairy farmers, concerns from the potential dissemination of antimicrobial-resistant bacteria through the gut and subsequent shedding by calves into the environment are increasing. In this study, we employed Monte Carlo simulation to assess the risk of shedding extended-spectrum cephalosporin-resistant Escherichia coli (ESC-R E. coli) caused by waste milk feeding in pre-weaned calves using an exponential dose-response model fit to data for E. coli O157:H7 in cattle. Data from pertinent studies were included in our model to predict the risk of shedding. The median (5th and 95th percentiles) for the daily risk of shedding ESC-R E. coli by calves fed only contaminated waste milk was predicted to be 2.9 × 10^?3 (2.1 × 10^?3, 3.7 × 10^?3), representing a median daily risk of 29 out of 10,000 calves shedding ESC-R E. coli due to exclusive feeding of waste milk containing ESC-R E. coli. This median value was reduced by 94% when accounting for the proportion of waste milk that does not contain ESC-R E. coli. The overall risk of shedding ESC-R E. coli through the pre-weaning period for farms that feed waste milk to calves was 5.7 × 10^?3 (2.4 × 10^?3, 1.1 × 10^?2), representing 57 out of 10,000 calves. When accounting for the proportion of farms that do not feed waste milk, the pre-weaning period risk was reduced by 23%. By varying the prevalence of ESC-R E. coli in waste milk using values of 3, 1.5, and 1%, the daily risk of shedding decreased by factors of 50, 65, and 82%, respectively, which supports the reduction of contamination or discontinuation of feeding waste milk containing ESC-R E. coli as major mitigation measures to reduce the risk of shedding caused by ingestion of resistant bacteria. It is anticipated that the effects of antimicrobial residues in waste milk, which was not considered herein due to lack of data, would further increase risks. Although waste milk feeding to calves may be economically beneficial to the dairy farmers, there exists the risk of dissemination of ESC-resistant bacteria into the environment.     

Determination of the effect of sodium lactate on the survival and heat resistance of Escherichia coli O157:H7 in two commercial beef patty formulations

The effect of 4% sodium lactate (NaL) in beefburger patty formulations on the survival and heat resistance of Escherichia coli O157:H7 was investigated. Fresh beef trimmings were inoculated with E. coli O157:H7 to a concentration of 6·0–7·0 log₁₀ cfu g⁻¹ and subjected to the processing stages of beefburger patty production. Two commercial beefburger patty formulations were produced: a ‘quality’ patty (100% beef) and an ‘economy’ patty (70% beef, 30% other ingredients, including onion, water, salt, seasoning, rusk and soya concentrate). Sodium lactate (4% w/v) was added to the beefburger patties during mincing and the formed patties were frozen and stored for 1 month. Beefburger patties without added NaL were used as controls. After frozen storage for 1 month, patties were examined for E. coli O157:H7 counts. There was a synergistic effect between freezing and NaL, which resulted in a small but significant reduction (P<0·05) (approximately 0·5 log₁₀ cfu g⁻¹) in E. coli O157:H7 numbers. The frozen beefburger patties were also heat-treated at 50, 55 and 60°C and the data analysed to derive D -values for E. coli O157:H7 cells. At each temperature treatment, theD -values of the quality and economy beefburger patties with 4% NaL were significantly lower (P<0·001) than the D -values of the patty formulations without NaL. The study demonstrates that the presence of 4% NaL in beefburger patty formulations can reduce the overall risks posed to consumers by the presence ofE. coli O157:H7 by, first; reducing pathogen survival during freezing and frozen storage of the uncooked product; and, second, by increasing the susceptibility of the pathogen to heat during normal cooking processes.     

Changes in heat tolerance of Escherichia coli O157:H7 after exposure to acidic environments

Acid-adapted bacterial cells are known to have enhanced tolerance to various secondary stresses. However, a comparison of heat tolerance of acid-adapted and acid-shocked cells of Escherichia coli O157:H7 has not been reported. D - and z -values of acid-adapted, acid-shocked, and control cells of an unusually heat-resistant strain (E0139) of E. coli O157:H7, as well as two other strains of E. coli O157:H7, were determined based upon the number of cells surviving heat treatment at 52, 54 or 56°C in tryptic soy broth (pH 7·2) for 0, 10, 20 or 30 min. The unusual heat tolerance of E. coli O157:H7 strain E0139 was confirmed. D -values for cells from 24-h cultures were 100·2, 28·3, and 6·1 min at 52, 54 and 56°C, respectively, with a z -value of 3·3°C. The highest D -values of other E. coli O157:H7 strains were 13·6 and 9·2 min at 52 and 54°C, respectively, whereas highest D -values of non-O157:H7 strains were 78·3 and 29·7 min at 52 and 54°C. D -values of acid-adapted cells were significantly higher than those of unadapted and acid-shocked cells at all temperatures tested. In a previous study, we observed that both acid-adapted cells and acid-shocked cells of strain E0139 had enhanced acid tolerance. This suggests that different mechanisms protect acid-adapted and acid-shocked cells against subsequent exposure to heat or an acidic environment. The two types of cells should be considered separately when evaluating survival and growth characteristics upon subsequent exposure to different secondary stress conditions.     

Assessment of Microbiological and Bio-chemical Quality of Fish in a Supply Chain in Negombo,Sri Lanka

This study aimed to investigate quality of fish landed in Negombo area and distributed in suburban areas in Western province of Sri Lanka. Hundred samples of large fish (Katsuwonus pelamis and Euthynnus affinis) and 60 samples of small fish (Amblygaster sirm, Pterocaesio chrysozona, Stolephorus commersoni, and Sardinella albella) were sampled from different stages of a supply chain at five and six sampling visits, respectively. All fish samples (N=160) were analysed for aerobic plate counts (APC) at 37^oC, Coliforms, feacal coliforms, Escherichia coli, Salmonella spp., Listeria monocytogenes, total volatile base nitrogen (TVB-N) while 130 were analysed for histamine. Water from fishery harbor basin, fishery harbour, ice manufcaturing plants and ice used in multiday boats were also analysed for microbiological parameters. Large and small fish contained APC in the range of 2.0 x10² - 2.0 x 10⁶ and 8.0×10³ - 2.0×10⁸ cfu/g, respectively. Faecal coliform counts ranged between not detected (ND) and 90 MPN/g in large fish and between ND and >1100 MPN/g in small fish. 5% of large fish were contaminated with E.coli and ranged from ND to 15 MPN/g. E.coli was present in 70% of small fish samples and ranged from ND to >1100 MPN/g. Of the 160 fish samples, tested Salmonella spp were detected in nine occassions. Of the 160 fish samples, L. monocytogenes was found in eight Katsuwonus pelamis and one Sardinella albella fish. TVB-N of large fish were found at range of 1-67 mgN/100 g and 79% samples contained unaccptable levels. Small fish contained about 25.10-104.30 mgN/100 g while 78% samples exceeded acceptable levels. Histamine level of large and small fish, 26% and 83% of samples exceeded the maximum acceptable levels, respectively. Harbour basin water was heavily contaminated with Salmonella spp. (50%), Faecal streptococci (100%), Faecal coliforms and E.coli (100%). Ice samples (20%) from one ice plant were found contaminated with Salmonella spp.     

Influence of acidulant identity on the effects of pH and acid resistance on the radiation resistance of Escherichia coli O157:H7

The effects of pH (4.0–5.5), acid identity (acetic, citric, lactic, malic, and hydrochloric), and the induction of pH-dependent stationary phase acid resistance on the radiation resistance of E. coli O157:H7 Ent-C9490 was studied using cells grown in Tryptic Soy Broth with and without dextrose (induced and non-induced to acid resistance) and then resuspended in brain–heart infusion broth containing 5 g/l of an organic acid and acidified with concentrated hydrochloric acid. After treatment with gamma radiation, the number of survivors was determined by plating on brain–heart infusion agar (injured and non-injured cells) and MacConkey agar (non-injured cells), and the data used to calculate radiation D-values. The induction of pH-dependent stationary phase acid resistance consistently provided the enterohemorrhagic E. coli strain cross-protection from subsequent irradiation, increasing radiation D-values by 1.2–3.3-fold, depending on the organic acid present. The radiation resistance of E. coli varied with acid identity, but was largely unaffected by pH within the range examined. The results indicate that induction of cross-protection resulting from induction of acid resistance is a factor that should be considered to accurately determine the radiation dose needed to inactivate enterohemorrhagic E. coli in foods.     

Sensitive fluorescence ELISA with streptavidin scaffolded DNA tetrads for the detection of Escherichia coli O157:H7

Escherichia coli O157:H7 poses a threat to humans. Traditional ELISA is not a sensitive method for the detection of E. coli O157:H7. Here, an efficient method was designed for improving the load capacity of alkaline phosphatase (ALP) with streptavidin scaffolded DNA tetrad (SS-DNAt). With more ALP, more ascorbic acid 2-phosphate was catalyzed to ascorbic acid that was used to synthesize fluorescence poly adenine-thymine–templated copper nanoclusters. Based on SS-DNAt, fluorescence ELISA was successfully proposed for improving the sensitivity for detection of E. coli O157:H7 in milk samples. The method showed a linear range of 10⁴ to 10⁶ cfu/mL. The limit of detection of fluorescence ELISA was 3.75 × 10³ cfu/mL and 6.16-fold better than that of traditional ELISA. The recovery of the fluorescence ELISA was 86.7 to 93.6% with the coefficient of variation of 5.6 to 10.5% in milk. This method could be used to detect hazardous material in food.     

A novel smartphone-based colorimetric aptasensor for on-site detection of Escherichia coli O157:H7 in milk

Effective testing tools for Escherichia coli O157:H7 can prevent outbreaks of foodborne illness. In this paper, a smartphone-based colorimetric aptasensor was developed using functionalized gold nanoparticles (GNP) and multi-walled carbon nanotubes (MWCNT) for monitoring E. coli O157:H7 in milk. The maximum absorption peak of GNP bonded with aptamer (Apt) generated evident transformation from 518 to 524 nm. The excess GNP-Apt was removed by functionalized MWCNT magnetized with carbonyl iron powder (CIP) and hybridized with a DNA probe, whereas the GNP-Apt immobilized on E. coli O157:H7 remained in the system. In the presence of a high-salt solution, the GNP-Apt that captured E. coli O157:H7 remained red, but the free GNP-Apt aggregated and appeared blue. The chromogenic results were analyzed by a smartphone-based colorimetric device that was fabricated using acrylic plates, a light-emitting diode, and a mobile power pack. To our knowledge, this was the first attempt to use a smartphone-based colorimetric aptasensor employing the capture of GNP-Apt coupled with separation of MWCNT@CIP probe to detect E. coli O157:H7. The aptasensor exhibited good reproducibility and no cross-reaction for other bacteria. A concentration of 8.43 × 10³ cfu/mL of E. coli O157:H7 could be tested in pure culture, and 5.24 × 10² cfu/mL of E. coli O157:H7 could be detected in artificially contaminated milk after 1 h of incubation. Therefore, the smartphone-based colorimetric aptasensor was an efficient tool for the detection of E. coli O157:H7 in milk.     

Survival and detection of Escherichia coli O157:H7 and Listeria monocytogenes during the manufacture of dry sausage using two different starter cultures

A sausage batter (35% pork, 35% beef, 30% fat) was inoculated with high (5·46–5·68), medium (3·78–4·54) or low (2·30–2·60 log₁₀cfug⁻¹) levels of Escherichia coli O157:H7 and with high (5·05–5·41) or medium (2·92–3·35 log₁₀cfug⁻¹) levels of Listeria monocytogenes serovar 4b and fermented using starter cultures A (Staphylococcus xylosus DD-34 with bacteriocin-producingPediococcusacidilactici PA-2 and Lactobacillus bavaricus MI-401) and B(S. carnosus MIII withLb. curvatus Lb3). Sausages were manufactured (fermented and dried) in a smoke chamber at 17–23°C for 15 days and further stored at 15–17°C for 34 days. The numbers of E. coli O157:H7 decreased more using starter B than starter A (first experiment P<0·0015, second experiment P<0·0002) but the organism was not eliminated. Small numbers of E. coli O157:H7 were more often detected after enrichment for 18–24 h than for 6 h (P=0·0044) when tested after deep freezing. By contrast, L. monocytogenes decreased more rapidly in the high-inoculum sausages produced with starter A (P<0·0001) but no significant difference was detected between the starters in the medium-inoculum sausages. L. monocytogenes was eliminated from the medium-inoculum sausages after 49 days.     

Short communication: Molecular characterization and antimicrobial resistance of pathogenic Escherichia coli isolated from raw milk and Minas Frescal cheeses in Brazil

The aim of this study was to quantify, identify, evaluate antimicrobial resistance, and characterize the virulence factors of enteropathogenic (EPEC), Shiga-toxigenic (STEC), and enterohemorrhagic (EHEC) Escherichia coli in raw milk (RM) and legal (LMFC) and illegal (IMFC) Minas Frescal cheeses in southern and northeast Brazil. Illegal cheeses are those made without official inspection service or sanitary surveillance. We evaluated samples of RM produced in Paraná (southern) and Maranhão (northeast) States, LMFC produced using pasteurized milk in inspected industries, and IMFC potentially produced with raw milk. Mean total coliform counts were 8.4 × 10⁴ cfu/mL for RM, 1.4 × 10⁷ cfu/mL for LMFC, and 2.9 × 10⁷ cfu/mL for IMFC. Mean E. coli counts were 2.4 × 10³ cfu/mL for RM, 1.9 × 10² cfu/mL for LMFC, and 1.1 × 10⁵ cfu/mL for IMFC. Among the 205 E. coli isolates from RM, 9.75% were identified as EPEC, mainly (90%) in samples from Paraná. Of the total isolates from the cheese samples, 97.4% (n = 111) came from IMFC, of which 1.8 and 2.7% were identified as EPEC and STEC, respectively; no EHEC was detected. The phylogenetic group A (60%) and typical EPEC (68%) predominated, which confirms the possible human origin of pathogenic isolates in RM and IMFC. Of these, 50% were resistant to at least one antibiotic, and streptomycin was the antimicrobial with the highest number (8) of EPEC and STEC resistant isolates. This study reports the first isolation of serogroup O28ac in Brazilian milk. We found no predominance of a specific serogroup of EPEC or STEC in milk or cheese or clonal isolates in the same sample, indicating different origins of the contamination in these products, presumably mostly related to poor hygienic handling.     

Development of an immunosensor based on surface plasmon resonance for enumeration of Escherichia coli in water samples

An immunosensor for the rapid, sensitive and selective detection of generic Escherichia coli in water samples was developed on the basis of surface plasmon resonance (SPR). Antibodies against E. coli were immobilized on a sensor surface and water samples with different concentrations of E. coli were injected to the flow cell. The changes in the response unit (RU) due to the binding of different concentrations of bacteria were computed and a linear correlation (R² = 0.976) was obtained between log E. coli concentration and the change in the RU with a working range of 9 × 10¹ and 1.8 × 10⁵ cfu ml⁻¹. The complete regeneration of the sensor surface was realized by using 1% SDS solution. The selectivity of the developed sensor was examined with Enterobacter aerogenes and Enterobacter dissolvens which did not produce any significant response. The ability of the developed sensor to detect E. coli in real water samples was investigated and the results were closed to that obtained from plate counting method. Based on these results, the developed immunosensor can be employed for rapid, label-free, sensitive and selective detection of E. coli in water samples.     

Characterization of mammary pathogenic Escherichia coli reveals the diversity of Escherichia coli isolates associated with bovine clinical mastitis in Brazil

Mammary pathogenic Escherichia coli (MPEC) is one of the most common pathogens associated with clinical mastitis. We analyzed isolates obtained from milk samples of cows with clinical mastitis, collected from 10 farms in Brazil, to verify molecular and phenotypic characteristics. A total of 192 (4.5%) mammary pathogenic E. coli isolates were obtained from 4,275 milk samples analyzed, but we tested 161. We assigned most of these isolates to E. coli phylogroups B1 (52.8%) and A (36.6%), although phylogroups B2, C, D, E, and unknown also occurred. All isolates were assessed for the presence of several genes encoding virulence factors, such as adhesins (sfaDE, papC, afaBC III, ecpA, fimH, papA, and iha), toxins (hlyA, cnf1, sat, vat, and cdt), siderophores (iroN, irp2, iucD, ireA, and sitA), an invasion protein (ibeA), and serum resistance proteins (traT, KpsMTII, and ompT), and isolates from phylogroups B1, B2, and E showed up to 8 genes. Two isolates harbored the locus of enterocyte effacement (escN⁺) and lack the bundle-forming pilus (bfpB^?) operon, which corresponds to a molecular profile of a subgroup of diarrheagenic E. coli (aEPEC), thus being classified as hybrid MPEC/aEPEC isolates. These isolates displayed a localized adherence-like pattern of adherence in HeLa cells and were able to promote F-actin polymerization underneath adherent bacteria. Based on the pulsed-field gel electrophoresis analyses, considerable genetic variability was observed. A low index of antimicrobial resistance was observed and 2 extended-spectrum β-lactamase–producing E. coli were identified, both harboring bla_CTX-M15 gene, and were classified as ST10 and ST993 using multilocus sequence typing. A total of 148 (91.2%) isolates were weak biofilm producers or formed no biofilm. Because raw milk is still frequently consumed in Brazil, the occurrence of virulence factor–encoding genes from extraintestinal or diarrheagenic E. coli added to the presence of extended-spectrum β-lactamase–producing isolates can turn this veterinary medicine problem into a public health concern.     

Microbiological Evaluation of Alaska Shore-based Surimi Production

Two shore-based surimi plants in Alaska were investigated to determine microbial conditions of Alaska pollock flesh during processing. Median aerobic plate count (APC) was 2.0 × 10³/g after mincing, 2.3 × 10³/g after washing/screening, 4.2 × 10⁴/g after refining and 1.6 × 10⁴/g after dewatering. Reprocessing, needed for nonanalog grade surimi, resulted in APC of 1.2 × 10⁵/g after a second refining and 3.0 × 10⁵/g after a second dewatering. The APC for analog grade surimi was 5.5 × 10⁴/g and 2.0 × 10⁶/g for nonanalog grade surimi. Highest total coliform most probable number (MPN) of > 1100/g was determined from a nonanalog grade surimi sample and from a mince that had been refined twice. Highest Escherichia coli MPN of 460/g was determined from two minces.